Methods and reagents for diagnosing hantavirus infection
Novel methods and immunodiagnostic test kits for the detection of hantavirus infection are disclosed. The methods and kits employ combinations of recombinant N and/or G1 antigens from at least six different hantavirus serotypes, including Hantann (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV). Additional hantavirus antigens from these and other hantavirus types may also be present. The methods provide for highly accurate results and allow the detection of infection so that treatment can be administered and death avoided.
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This application is a continuation application of U.S. patent application Ser. No. 11/629,799 filed Dec. 17, 2007, which is a 371 application of PCT/US05/18066, filed on May 24, 2005 and claims benefit under 35 U.S.C. §119(e) of provisional application 60/581,027 filed on Jun. 18, 2004, all of which applications are incorporated herein by reference in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with support under NIH Grant U01 AIO54779, from the National Institute of Allergy and Infectious Diseases. Accordingly, the United States Government may have certain rights in this invention
TECHNICAL FIELDThe present invention pertains generally to hantaviruses. In particular, the invention relates to immunogenic reagents derived from multiple hantavirus serotypes, including immunogenic nucleocapsid and glycoprotein polypeptides, for use in compositions for diagnosis of hantavirus infection.
BACKGROUNDHantaviruses (Bunyaviridae: Hantavirus) are lipid-enveloped, minus-sense RNA viruses. The RNA of the viral genome is tripartite, consisting of three fragments generally designated as S, M and L for small, medium and large genome fragments, respectively. The M segment encodes a precursor protein that is processed to form the two envelope glycoproteins, termed G1 and G2. The S segment encodes a nucleocapsid protein, termed N which forms the filamentous helical nucleocapsid of the virus and elicits the humoral response. The L segment of the genome encodes an RNA-dependent RNA polymerase (RDRP). Over 20 distinct hantaviruses are found in association with specific rodent or insectivore hosts worldwide. Their modes of transmission to humans, natural reservoirs, and the clinical features of human infection have been reviewed (see, e.g., Mertz et al., Adv. Internal Med. (1997) 42:369-421).
Hantavirus pulmonary syndrome (HPS), also known as hantavirus cardiopulmonary syndrome (HCPS) is an acute febrile illness with up to a 50% mortality rate. Patients present with nonspecific symptoms and progress rapidly to fulminant pulmonary edema and cardiovascular collapse. The predominant agent of HPS in North America is Sin Nombre virus (SNV; also known as Four Corners virus and Muerto Canyon virus). (Song et al., Lancet (1994) 344:1637; Khan et al., J. Med. Virol (1995) 46:281-286; Kahn et al., Am. J. Med. (1996) 100:46-48; Morzunov et al., J. Virol (1995) 69:1980-1983; Rollin et al., J. Med. Virol (1995) 46:35-39; Centers for Disease Control and Prevention, Morbid Mortal. Weekly Rep. (1993) 43:45-48). At least three other hantaviruses have been associated with HPS in North America, including New York virus (NYV), Black Creek Canal virus (BCCV) and Bayou virus (BAYV).
Worldwide, a larger toll of illness is caused by the Eurasian hantaviruses that cause hemorrhagic fever with renal syndrome (HFRS). HFRS-associated viruses include Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), and Dobrava-Belgrade (DOBV) viruses (Lee et al., J. Infect. Dis. (1978) 137:298-308; Lee, et al., J. Infect. Dis. (1982) 146:638-644; Lee et al., J. Infect. Dis. (1982) 146:645-651; Brummer-Korvenkontio et al., J. Infect. Dis. (1980) 141:131-134). Clinical manifestations of HFRS are generally most severe for HTNV and DOBV infections, whereas PUUV infection is associated with a milder form of HFRS, nephropathia epidemica (NE), occurring in Scandinavia, Finland, Western Russia and Central Europe. Mortality rates of up to 20% have been reported from the most severe forms of HFRS.
Among the HFRS-associated hantaviruses, HTNV, SEOV and DOBV are antigenically similar. The HPS-associated viruses are also closely related to one another, and cross-react with PUUV. Antigenic cross-reactivity is most pronounced among the viral N proteins. In recombinant antigen diagnostic assays, the viral N antigen is dominant over the viral glycoproteins. Antibodies to the N antigen arise early in the course of infection and are universally detectable in convalescence. All persons with acute SNV infection have detectable antibodies against the SNV N antigen of the IgM class by the onset of clinical symptoms, and almost all have IgG antibodies directed against the N and G1 antigens (Bharadwaj et al., J. Infect. Dis. (2000) 182:43-48). SNV G1 antibodies are not reactive with the G1 antigens of other hantaviruses (Jenison et al., J. Virol. (1994) 68:3000-3006; Hjelle et al., J. Gen. Virol. (1994) 75:2881-2888).
Hantaviruses are transmitted to humans via inhalation of virus-contaminated aerosols of rodent saliva, urine and feces. A worker can contract hantavirus infection merely by entering into a room with infected rodents, which strongly supports the prevailing view that hantaviruses are transmitted through the air. This observation is also supported by a recent epidemiologic investigation showing that indoor exposures are extremely common. Person-to-person transmission has been demonstrated for the Andes virus (ANDV) in Argentina and is likely to be responsible for two family clusters in Chile. The virus may also be transmitted after rodent bites and possibly through ingestion of contaminated food or water.
All species of hantavirus appear to be primarily associated with a specific rodent host. There are three broad groups of hantaviruses and they are associated with the rodent subfamilies of Murinae, Arvicolinae and Sigmondontinae. The phylogenetic relations among rodents in these various subfamilies parallel, for the most part, the phylogenetic and antigenic relations of viruses associated with each particular reservoir. Each of these groups of hantaviruses contains one or more species or types that are known human pathogens. Information concerning the various hantaviruses is presented in Table 1.
Significant strides have been made in the management of hantavirus infection, but successful management requires that patients be diagnosed before the immediate preagonal stage of illness. Advances in tertiary care management have occurred that may reduce mortality due to hantavirus infection. However, since infection progresses very rapidly, these advances are likely to affect the prognosis only of those patients for whom a diagnosis can be made in a timely manner. A method for rapid detection of hantavirus antibodies appropriate for a rural setting and that could be applied during the early stages of illness could improve the prospects for early intervention.
Several assays have been attempted for the timely diagnosis of hantavirus infection. For example, a variety of formats for serologic diagnosis of hantavirus infection have been employed including bead agglutination, immunofluorescence and immunoprecipitation assays using laboratory-cultivated viruses, hemagglutination inhibition, plaque- and focus-reduction neutralization assays, and ELISA formats (Lee et al., J. Infect. Dis. (1978) 137:298-308; Lee et al., J. Infect. Dis. (1982) 146:638-644; Lee et al., J. Infect. Dis. (1982) 146:645-651; Lundkvist et al., Clin. Diagnos. Lab. Immunol. (1995) 2:82-86; Chu et al., Virology (1994) 198:196-204; Elgh et al., J. Med. Virol (1995) 45:146-150).
Strip immunoblot assays have also been used in an attempt at efficient diagnosis of hantavirus infection (see, e.g., Hjelle et al., J. Clin. Microbiol. (1997) 35:600-608; Bharadwaj et al., J. Infect. Dis. (2000) 182:43-48; Yee, et al., J. Wildl. Dis. (2003) 39:271-277). However, a universal assay for identifying multiple strains of hantaviruses is not currently available.
The wide-spread availability of an accurate, efficient and rapid assay for hantavirus infection would be highly desirable and could save a considerable number of lives.
SUMMARY OF THE INVENTIONThe present invention provides a simple, accurate and efficient method for diagnosing hantavirus infection, as well as for determining the type of hantavirus present. The methods allow the rapid detection, e.g., in less than one hour, of hantavirus infection caused by several hantaviruses, such as hantaviruses from more than one hantavirus serotype. If infection is detected, the individual can be given appropriate treatment in adequate time to prevent death. The method utilizes N and/or G1 antigens and/or antibodies directed against these antigens from at least six different hantavirus serotypes, including HTNV, PUUV, SEOV, DOBV, SNV and ANDV. The assays provide a rapid and reliable diagnostic test and are suitable for use in facilities with relatively unsophisticated laboratory capabilities. Moreover, the assays can be used to screen wild rodents for hantavirus infection in order to determine if a particular rodent population is infected with the virus, thereby preventing infection in laboratory workers, field crews and others who work with and encounter wild rodents.
Recombinant techniques can be used to produce the products described herein to provide protein preparations devoid of other molecules normally present, such as other viral contaminants and harmful proteins.
Accordingly, in one embodiment, the invention is directed to a method of detecting hantavirus antibodies in a biological sample. The method comprises:
(a) contacting the biological sample with at least six hantavirus recombinant antigens, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV) wherein at least one recombinant antigen from each of the serotypes is present, the contacting performed under conditions which allow hantavirus antibodies, when present in the biological sample, to bind to at least one of the G1 or N antigens to form an antibody/antigen complex; and
(b) detecting the presence or absence of the antibody/antigen complex, thereby detecting the presence or absence of hantavirus antibodies in the sample. The at least six hantavirus recombinant antigens can be any combination of G1 and N antigens as long as at least one antigen from at least six hantavirus serotypes, viz. Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV), is present. In a preferred embodiment, the at least six hantavirus recombinant antigens are all N antigens.
In additional embodiments, the invention is directed to an immunodiagnostic test kit for detecting hantavirus infection. The test kit comprises:
(a) at least six hantavirus recombinant antigens, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV) wherein at least one antigen from each of the serotypes is present;
(b) and instructions for conducting the immunodiagnostic test.
In a preferred embodiment, the at least six hantavirus recombinant antigens are all N antigens.
In further embodiments, the invention is directed to a method of detecting hantavirus antigens in a biological sample. The method comprises:
(a) contacting the biological sample with at least six different antibodies, wherein each of said antibodies is specific for at least one of six hantavirus antigens, wherein the six hantavirus antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV), wherein at least one antibody specific for at least one antigen from each of the serotypes is present, wherein the contacting is done under conditions which allow hantavirus antigens, when present in the biological sample, to bind to the antibodies to form an antibody/antigen complex; and
(b) detecting the presence or absence of the antibody/antigen complex, thereby detecting the presence or absence of hantavirus antigens in the sample.
In certain embodiments of the above method, the antibodies are monoclonal antibodies.
In yet an additional embodiment, the invention is directed to an immunodiagnostic test kit for detecting hantavirus infection. The test kit comprises:
(a) at least six different antibodies, wherein each of the antibodies is specific for at least one of six hantavirus antigens, wherein the six hantavirus antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV), wherein at least one antibody specific for at least one antigen from each of the serotypes is present; and
(b) instructions for conducting the immunodiagnostic test.
In certain embodiments of the above immunodiagnostic test kit, the antibodies are monoclonal antibodies.
In additional embodiments, the invention is directed to a solid support comprising at least six hantavirus recombinant antigens, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV) wherein at least one antigen from each of the serotypes is present. The solid support can be a nitrocellulose strip.
In certain embodiments, the solid support further comprises at least one anti-human immunoglobulin antibody, such one or more antibodies selected from the group consisting of an anti-human IgM antibody, an anti-human IgG antibody and an anti-human IgA antibody, wherein the hantavirus antigens and the anti-human immunoglobulin antibody are immobilized in discrete positions on the solid support.
In additional embodiments, the solid support further comprises at least two internal controls, wherein one of the controls defines the lower detection limit for a positive result in an immunoassay using the solid support and the other control defines a highly positive result in an immunoassay using the solid support.
In further embodiments, the invention is directed to an immunodiagnostic test kit for detecting hantavirus. The test kit comprises:
(a) a solid support as described above; and
(b) instructions for conducting the immunodiagnostic test.
In additional embodiments, the invention is directed to a method of detecting the presence of hantavirus antibodies in a biological sample. The method comprises:
(a) providing a biological sample;
(b) providing a solid support as described above;
(c) contacting the biological sample with the solid support, under conditions which allow hantavirus antibodies, if present in the biological sample, to bind with at least one of the hantavirus antigens to form an antibody/antigen complex; and
(d) detecting the presence of the antibody/antigen complex, thereby detecting the presence of hantavirus antibodies in the biological sample.
In certain embodiments, the method above further comprises:
(e) removing unbound hantavirus antibodies;
(f) providing one or more moieties capable of associating with the antibody/antigen complex; and
(g) detecting the presence of the one or more moieties, thereby detecting the presence of hantavirus antibodies in the biological sample.
In certain embodiments, the one or more moieties comprises a detectably labeled hantavirus antigen. In these and other embodiments, the detectable label can be an enzyme.
In yet an additional embodiment, the invention is directed to a method of preparing a blood supply comprising whole blood, platelets, plasma or serum, substantially free of hantavirus. The method comprises:
(a) screening aliquots of whole blood, platelets, plasma or serum from collected blood samples by the method of any one of the methods described above;
(b) eliminating any samples in which a hantavirus antigen or a hantavirus antibody is detected; and
(c) combining samples in which neither a hantavirus antigen or a hantavirus antibody is detected to provide a blood supply substantially free of hantavirus.
In any of the methods above, the biological sample can be from a human blood sample. Additionally, In any of the embodiments above, the G1 antigen(s) can comprise an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40. The G1 antigen(s) can be one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47.
Additionally, the N antigen(s) can comprise an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39. In other embodiments, the N antigen(s) can be one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
The G1 antigen(s) can comprise an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40 and the N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39. In additional embodiments, the G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, and the N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34 and SEQ ID NO:36.
In additional embodiments, at least one G1 antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV, and at least one N antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV.
In further embodiments, at least one N antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV.
These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Fundamental Virology, 3rd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.
The following amino acid abbreviations are used throughout the text:
-
- Alanine: Ala (A) Arginine: Arg (R)
- Asparagine: Asn (N) Aspartic acid: Asp (D)
- Cysteine: Cys (C) Glutamine: Gln (Q)
- Glutamic acid: Glu (E) Glycine: Gly (G)
- Histidine: His (H) Isoleucine: Ile (I)
- Leucine: Leu (L) Lysine: Lys (K)
- Methionine: Met (M) Phenylalanine: Phe (F)
- Proline: Pro (P) Serine: Ser (S)
- Threonine: Thr (T) Tryptophan: Trp (W)
- Tyrosine: Tyr (Y) Valine: Val (V)
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a G1 polypeptide” includes a mixture of two or more such polypeptides, and the like.
The terms “polypeptide” and “protein” refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
The term “antigen” when used with reference to the various hantavirus polypeptides for use with the present invention, refers to an N, G1, etc., polypeptide, whether native, recombinant or synthetic, which includes one or more epitopes that recognize hantavirus antibodies and which are derived from any of the various isolates of the hantavirus strain in question. Accordingly, a “hantavirus antigen” is an antigen derived from any of the hantavirus serotypes, strains and isolates, including, without limitation, any of the various isolates of the Murinae, Sigmodontinae and Arvicolinae subfamilies. For example, an SNV antigen will be derived from any of the various Sin Nombre virus isolates, such as the prototype New Mexico SNV isolate, 3H226, California isolates, British Columbia isolates, etc. Similarly, an SEOV antigen will be derived from any of the various Seoul virus isolates, and so on. Additional antigens for use in the present invention can be derived from other HPS- and HFRS-associated hantaviruses, including, but not limited to, Sin Nombre virus (SNV; also known as Four Corners virus and Muerto Canyon virus); Andes virus (ANDV); New York virus (NYV); Black Creek Canal virus (BCCV); Bayou virus (BAYV); Hantaan virus (HTNV), Puumala virus (PUUV), Seoul virus (SEOV), and Dobrava-Belgrade (DOBV) virus. The genomic sequences for these viruses are known and several antigenic sequences for use with the present invention are detailed further below.
The antigen in question need not include the full-length amino acid sequence of the reference molecule but can include only so much of the molecule as necessary in order for the polypeptide to react with the appropriate hantavirus antibodies. Thus, only one or few epitopes of the reference molecule need be present. Furthermore, the antigen may comprise a fusion protein between the full-length reference molecule or a fragment of the reference molecule, and another protein such as another hantavirus antigen and/or a protein that does not disrupt the reactivity of the hantavirus antigen. It is readily apparent that the antigen may therefore comprise the full-length sequence, fragments, truncated and partial sequences, as well as analogs, muteins and precursor forms of the reference molecule. The term also intends deletions, additions and substitutions to the reference sequence, so long as the antigen retains the ability to react with hantavirus antibodies.
In this regard, natural variation will occur from isolate to isolate within a particular hantavirus strain. Thus, the term is intended to encompass such variation and, in particular, an antigen that varies in its amino acid composition by not more than about 20 number percent, more preferably by not more than about 10 to 15 number percent, and most preferably, by not more than about 5 number percent, from the reference antigen. Proteins having substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that do not substantially affect the antibody binding capabilities of the antigen, are therefore within the definition of the reference polypeptide.
An antigen “derived from” a hantavirus serotype, strain or isolate intends an antigen which comprises a sequence of an antigen encoded by the reference hantavirus genome. Typically, the antigen includes one or more epitopes, and will generally have an amino acid sequence substantially homologous to the reference polypeptide, as defined below. Thus, the term “derived from” is used to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.
The terms “analog” and “mutein” refer to biologically active derivatives of the reference molecule, that retain desired activity, such as immunoreactivity in assays described herein. In general, the term “analog” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy immunogenic activity and which are “substantially homologous” to the reference molecule as defined below. A number of conserved and variable regions are known between the various isolates and, in general, the amino acid sequences of epitopes derived from these regions will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 50%, generally more than 60%-70%, when the two sequences are aligned. The term “mutein” refers to peptides having one or more peptide mimics (“peptoids”). Preferably, the analog or mutein has at least the same immunoreactivity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.
The terms “analog” and “mutein” also encompasses purposeful mutations that are made to the reference molecule. Particularly preferred analogs include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non-polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the antigen of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25, 50 or 75 conservative or non-conservative amino acid substitutions, or any integer between 5-75, so long as the desired function of the molecule remains intact. One of skill in the art can readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.
By “fragment” is intended an antigen consisting of only a part of the intact full-length polypeptide sequence and structure. The fragment can include a C-terminal deletion an N-terminal deletion, and/or an internal deletion of the native polypeptide. Representative G1 fragments for use in the present assays are shown in
By “N antigen” is meant an antigen, as defined above, that is derived from the nucleocapsid protein of the hantavirus in question. The DNA and corresponding amino acid sequences for various hantavirus N proteins are known. For example, the nucleotide sequence and corresponding amino acid sequence for representative N antigens from HTNV, PUUV, SEOV, DOBV, SNV and ANDV are shown in
As explained above, N antigens for use in the present assays include the full-length or substantially full-length proteins, as well as fragments, fusions or mutants of the proteins, which include one or more epitopes such that reactivity with antibodies present in a biological sample from an individual with the particular hantavirus infection in question is retained. For example, N antigens for use in the assays described herein may be a recombinant fusion between the full-length SNV N sequence with another protein, such as another hantavirus antigen, and/or a fusion with a protein that aids in recombinant expression, such as with a 50 kDa E. coli maltose binding protein, or a human or yeast superoxide dismutase (SOD) protein. A representative immunoreactive fragment of the N protein useful in the present assays is a 43 amino acid amino terminal-proximal segment corresponding to the sequence of amino acids found at positions 17-59 of the N polypeptide, numbered relative to SNV NM 10 and having the amino acid sequence QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG (SEQ ID NO:39). This fragment includes an immunodominant epitope of the N protein and antibodies directed against this epitope are cross-reactive with N proteins from PUUV, SEOV and HTNV (see, e.g., Yamada et al., J. Virol. (1995) 69:1939-1943; and International Publication No. WO 95/06250, published Mar. 2, 1995). Thus, this fragment, as well as larger fragments including this sequence, will find use in the assays herein. For descriptions of epitopes of the N protein see, also, Lundkvist et al., Clin. Diag. Lab. Immunol. (1995) 2:82-86; and Gott et al., Virus Res. (1991) 19:1-16.
By “G1 antigen” is meant an antigen, as defined above, that is derived from the envelope glycoprotein known as G1, of the hantavirus in question. The DNA and corresponding amino acid sequences for various hantavirus G1 proteins are known. Representative G1 regions are shown in
By “immunogenic” sequence of a hantavirus antigen is meant a molecule that includes an amino acid sequence with at least one hantavirus epitope such that the molecule is capable of reacting with antibodies directed against the hantavirus in question, as well as stimulating the production of antibodies in an appropriate host. By “epitope” is meant a site on an antigen to which specific B cells and/or T cells respond, rendering the hantavirus epitope in question capable of reacting with hantavirus antibodies present in a biological sample, as well as stimulating antibody production. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.” An epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids or more.
Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1985) Proc. Natl. Acad. Sci. USA 82:178-182; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132 for hydropathy plots.
An “immunogenic composition” is a composition that comprises at least one immunogenic polypeptide (e.g., an N and/or G1 hantavirus antigen).
“Substantially purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
By “isolated” is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
By “equivalent antigenic determinant” is meant an antigenic determinant from different isolates or strains of a hantavirus which antigenic determinants are not necessarily identical due to sequence variation, but which occur in equivalent positions in the hantavirus sequence in question. In general the amino acid sequences of equivalent antigenic determinants will have a high degree of sequence homology, e.g., amino acid sequence homology of more than 30%, usually more than 40%, such as more than 60%, and even more than 80-90% homology, when the two sequences are aligned.
“Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.
In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, D.C., which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs are readily available.
Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
An “antibody” intends a molecule that specifically binds to an epitope of interest present in an antigen. By “specifically binds” is meant that the antibody recognizes and interacts with the epitope in a “lock and key” type of interaction to form a complex between the antigen and antibody, as opposed to non-specific binding that might occur between the antibody and, for instance, the test substrate with which the antibody is reacted. Thus, an anti-hantavirus G1 antibody is a molecule that specifically binds to an epitope of a hantavirus G1 protein. The term “antibody” as used herein includes antibodies obtained from both polyclonal and monoclonal preparations, as well as, the following: hybrid (chimeric) antibody molecules (see, for example, Winter et al., Nature (1991) 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2 and F(ab) fragments; Fv molecules (non-covalent heterodimers, see, for example, Inbar et al., Proc Natl Acad Sci USA (1972) 69:2659-2662; and Ehrlich et al., Biochem (1980) 19:4091-4096); single-chain Fv molecules (sFv) (see, for example, Huston et al., Proc Natl Acad Sci USA (1988) 85:5879-5883); dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al., Biochem (1992) 31:1579-1584; Cumber et al., J Immunology (1992) 149B:120-126); humanized antibody molecules (see, for example, Riechmann et al., Nature (1988) 332:323-327; Verhoeyan et al., Science (1988) 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain immunological binding properties of the parent antibody molecule.
As used herein, the term “monoclonal antibody” refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. The term encompasses whole immunoglobulins as well as fragments such as Fab, F(ab′)2, Fv, and other fragments, as well as chimeric and humanized homogeneous antibody populations, that exhibit immunological binding properties of the parent monoclonal antibody molecule.
As used herein, a “solid support” refers to a solid surface to which a macromolecule, e.g., protein, polypeptide, peptide, polynucleotide can be attached, such as a magnetic bead, latex bead, microtiter plate well, glass plate, nylon, agarose, polyacrylamide, silica particle, nitrocellulose membrane, and the like.
“Immunologically reactive” means that the antigen in question will react specifically with anti-hantavirus antibodies present in a biological sample from a hantavirus-infected individual.
“Immune complex” intends the combination formed when an antibody binds to an epitope on an antigen.
As used herein, a “biological sample” refers to a sample of tissue or fluid isolated from a subject such as, but not limited to, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, cerebrospinal fluid, samples of the skin, secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components. The samples detailed above need not necessarily be in the form obtained directly from the source. For example, the sample can be treated prior to use, such as, for example, by heating, centrifuging, etc. prior to analysis.
As used herein, the terms “label” and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, semiconductor nanocrystals, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, strepavidin or haptens) and the like. The term “fluorescer” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range. Particular examples of labels which may be used under the invention include, but are not limited to, horse radish peroxidase (HRP), fluorescein, FITC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and α-β-galactosidase.
II. MODES OF CARRYING OUT THE INVENTIONBefore describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
The present invention is based on the discovery of novel diagnostic methods for accurately detecting hantavirus infection. The methods utilize hantavirus recombinant N and G1 antigens comprising immunodominant epitopes, and/or antibodies directed against these antigens, from at least six different hantavirus serotypes, including HTNV, PUUV, SEOV, DOBV, SNV and ANDV. In certain embodiments, the assays include, for example, six recombinant G1 antigens (and/or antibodies directed against six G1 antigens), one from each of the above serotypes, and/or six recombinant N antigens, one from each of the above serotypes. In alternative embodiments, the assay can include, for example, at least one recombinant N antigen derived from any one of these six serotypes, that produces antibodies that cross-react with one or more of the other five serotypes, and six recombinant G1 antigens, one from each of the six serotypes. For example, the assays can include an N antigen (and/or antibodies directed against the N antigen) from SNV. The assays will also include G1 antigens (and/or antibodies directed against G1 antigens), preferably from all six of the serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV. Alternatively, the assay can include a G1 antigen (and/or antibodies thereto) from any one of these six serotypes, and six N antigens (and/or antibodies thereto) from each of these six serotypes. In another embodiment, the assay can include, for example, three G1 antigens (and/or antibodies thereto) from three of the six serotypes, and three N antigens (and/or antibodies thereto) from the other three of the six serotypes. Alternatively, the assay can include two G1 antigens (and/or antibodies thereto) and four N antigens (and/or antibodies thereto), or four G1 antigens (and/or antibodies thereto) and two N antigens (and/or antibodies thereto), or one G1 antigen (and/or antibodies thereto) and five N antigens (and/or antibodies thereto), or five G1 antigens (and/or antibodies thereto) and one N antigen (and/or antibodies thereto), or four G1 antigens (and/or antibodies thereto) and four N antigens (and/or antibodies thereto), or five G1 antigens (and/or antibodies thereto and five N antigens (and/or antibodies thereto), and so on. It is readily apparent that any combination of recombinant G1 and N antigens (and/or antibodies thereto) can be used so long as the six serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV are represented.
The assays of the present invention can also utilize additional hantavirus antigens, derived from any of the various hantavirus strains described in Table 1 or others subsequently identified.
The use of hantavirus N and/or G1 antigens (and/or antibodies thereto) from the six serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV, optionally in combination with other hantavirus proteins and/or antibodies, allows for the diagnosis of infection caused by any of the various known hantavirus serotypes. Moreover, the assays can be adapted such that the particular hantavirus serotype causing infection can be identified. For example, biological samples from patients infected with a variety of HPS- and HFRS-associated hantaviruses react with SNV N antigens, but not necessarily with SNV G1 antigens. Thus, the presence of reactivity with an SNV N antigen and the absence of reactivity with an SNV G1 antigen, indicates infection with a hantavirus other than SNV, and the like. It is readily apparent that a wide variety of antigens and combinations of antigens (or antibodies directed against these antigens) can be used in the present diagnostic assays.
The methods are useful for detecting hantavirus infection in humans, as well as in rodent populations. The methods can detect hantavirus infection in blood samples, including without limitation, in whole blood, serum and plasma. Thus, the methods can be used to diagnose hantavirus infection in a subject, such as a human or rodent subject, as well as to detect hantavirus contamination in donated blood samples. Aliquots from individual donated samples or pooled samples can be screened for the presence of hantavirus and those samples or pooled samples contaminated with hantavirus can be eliminated before they are combined. In this way, a blood supply substantially free of hantavirus contamination can be provided.
In order to further an understanding of the invention, a more detailed discussion is provided below regarding hantaviruses, as well as various hantavirus antigens and antibodies for use in the subject compositions and methods.
Hantavirus Antigens
As explained above, the hantavirus family of viruses belongs to the Bunyavirus genus and the viruses are enveloped, minus-sense RNA viruses. The RNA of the viral genome is tripartite, consisting of three fragments generally designated as S, M and L for small, medium and large genome fragments, respectively. The M segment encodes two envelope glycoproteins, termed G1 and G2, in a single open reading frame. The S segment encodes the nucleocapsid protein, termed N and the L segment of the genome encodes an RNA-dependent RNA polymerase.
Several distinct hantaviruses are found in association with specific rodent hosts worldwide (see, Table 1). As explained above, N and/or G1 antigens derived from at least six major hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV, will find use herein. Sequences for N and G1 antigens from numerous isolates identified as belonging to these serotypes, and the nucleic acid sequences encoding these antigens, are known. Representative sequences from which G1 antigens can be derived are depicted in
Immunogenic G1 antigens comprising a sequence of amino acids corresponding to the sequence of amino acids shown in SEQ ID NO:40 can include, for example, 31 amino acids, up to the full-length of the G1 molecule, such as 31-500 amino acids, preferably 31-250 amino acids, even more preferably 31-150 amino acids, such as 31 to 50 . . . 60 . . . 70 . . . 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 . . . 90 . . . 100 . . . 200 . . . 300 . . . 400 . . . 500, up to the full-length G1 molecule, or any integer within these ranges. Moreover, the G1 antigens for use herein may lack all or a portion of the transmembrane binding domain and/or the cytoplasmic tail found in the C-terminus of the envelope. Thus, the present invention contemplates the use of envelope polypeptides which retain the transmembrane binding domain and cytoplasmic tail, as well as antigens which lack all or a portion of the transmembrane binding domain and/or the cytoplasmic tail, as well as adjacent portions of the G1 protein. The location of such domains can be readily determined using computer programs and algorithms well known in the art, such as the Kyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132.
Additional sequences from which the G1 antigens can be derived are well known. For example, G1 sequences can be derived from any of the various HTNV sequences such as those sequences deposited with NCBI under accession numbers NC—005219, AF345636, D25532, D25529, D00377, D00376, AF288645, AF366569, AF035831, Y00386, U38177, U37729, M14627 and L08753. G1 sequences can be derived from any of the various SNV sequences such as those sequences deposited with NCBI under accession numbers L37903, L25783, AF030552, AF030551 and U02471. G1 sequences can be derived from any of the various ANDV sequences such as those sequences deposited with NCBI under accession numbers NC—003467 and AF291703. G1 sequences can be derived from any of the various SEOV sequences such as those sequences deposited with NCBI under accession numbers AF458104, AB027521, AF288654, AF288652, AF288650 and S47716. G1 sequences can be derived from any of the various PUUV sequences such as those sequences deposited with NCBI under accession numbers NC—005223, AY526218, U14136, U22418, L08754, L08755, X61034, X55129 and M29979. G1 sequences can be derived from any of the various DOBV sequences such as those sequences deposited with NCBI under accession numbers NC—005234, AJ410616, AY168578, AY168577 and L33685.
Representative sequences from which N antigens can be derived are depicted in
This sequence is not the same in the six hantavirus serotypes of interest herein. Thus, it is to be understood that an amino acid sequence from a non-SNV hantavirus that “corresponds” to this sequence is an amino acid segment from the non-SNV hantavirus that falls in the same region and displays homology to this sequence but does not necessarily show 100% identity to this sequence. The corresponding region in the other five hantavirus serotypes is readily identifiable from a review of
Immunogenic N antigens comprising a sequence of amino acids corresponding to the sequence of amino acids shown in SEQ ID NO:39 can include, for example, 43 amino acids, up to the full-length of the N molecule, such as 43-350 amino acids, preferably 43-300 amino acids, more preferably 43-200 amino acids, even more preferably 43-100 amino acids, such as 43 to 60 . . . 70 . . . 80 . . . 90 . . . 100 . . . 200 . . . 300 . . . 400, up to the full-length N molecule, or any integer within these ranges.
Additional sequences from which the N antigens can be derived are well known. For example, N sequences can be derived from any of the various HTNV sequences such as those sequences deposited with NCBI under accession numbers AB127998, AB027101, AB027523, AB027097, D25533, AF288646, AF288644, AF427324, AF427323, AF427322, AF427320, AF427319, AF427318, AF366568, AY017064, AF321095, AF321094, AF288296, U37768 and M14626. N sequences can be derived from any of the various SNV sequences such as those sequences deposited with NCBI under accession numbers NC—005216, L37904, L25784, L33816, L33683 and U02474. N sequences can be derived from any of the various ANDV sequences such as those sequences deposited with NCBI under accession numbers NC—003466, AF325966, AF0044660 and AF291702. N sequences can be derived from any of the various SEOV sequences such as those sequences deposited with NCBI under accession numbers NC—005236, AF488708, AF488707, AY273791, AB027522, AF329390, AF288655, AF288653, AF288643, AF406965, AY006465. N sequences can be derived from any of the various PUUV sequences such as those sequences deposited with NCBI under accession numbers NC—005224, AY526219, AJ314601, AJ314600, AJ314599, AJ314598, AJ314597, AF442613, AF367071, AF367070, AF367068, AF367067, AF367066, AF367065, AF367064, AJ277030, AJ277076, AJ277075, AJ277034, AJ277033, AJ277032, AJ277031, AJ238791, AJ238790, AJ238789, AJ238788, X61035, AB010731, AB010730, U14137, AF294652, U22423, L08804, L11347 and M32750. N sequences can be derived from any of the various DOBV sequences such as those sequences deposited with NCBI under accession numbers NC—005233, AJ616854, AJ410619, AJ410615, AJ131673, AJ131672, AJ269550, AJ269549, AJ009775, AJ009773, AY168576 and L41916.
The recombinant N and G1 antigens can be provided as discrete products or as fusions of the various N and G1 antigens, with or without other hantavirus antigens from one of more of these six serotypes, as well as with antigens derived from serotypes in addition to HTNV, PUUV, SEOV, DOBV, SNV and ANDV. By way of example, fusions can comprise G1 antigens from the six serotypes above. This fusion can be used alone or in combination with a second fusion including one or more N antigens from one or more of these six serotypes. Similarly, fusions can comprise N antigens from the six serotypes above and this fusion can be used alone or in combination with a second fusion including one or more G1 antigens from one or more of these six serotypes. Alternatively, all N and G1 antigens can be provided in a single fusion, or multiple fusions. It is readily apparent that fusion proteins of the subject invention can take any number of forms so long as N and/or G1 antigens from HTNV, PUUV, SEOV, DOBV, SNV and ANDV are present. If a fusion is produced, the polypeptides need not be organized in the same order as found in the native virus. Thus, for example, a G1 polypeptide can be fused to the N-terminus or C-terminus of an N polypeptide, etc. Other possible fusion proteins include a fusion of a human or yeast superoxide dismutase (SOD) protein, or fragment of the SOD protein, with a hantavirus G1 and/or N polypeptide. For examples of recombinant proteins expressed as human SOD fusion antigens, see Barr et al., Vaccine (1987) 5:90-101; Pichuantes et al., Proteins Struct. Fuct. Genet. (1989) 6:324-327; Pichuantes et al., J. Biol. Chem. (1990) 23:13890-13898.
Antigens for use with the present invention can be obtained using standard techniques. For example, the hantavirus antigens are conveniently generated using recombinant methods, well known in the art. See, e.g., International Publication No. WO 95/00648, published Jan. 5, 1995; International Publication No. WO 95/06240, published Mar. 2, 1995; and Hjelle, et al., J. Virol (1994) 68:592-596, for descriptions of the recombinant production of hantavirus antigens.
Oligonucleotide probes can be devised based on the known sequences of the hantavirus genome and used to probe genomic or cDNA libraries for hantavirus genes encoding for the antigens useful in the present invention. The genes can then be further isolated using standard techniques and, if desired, restriction enzymes employed to mutate the gene at desired portions of the full-length sequence.
Similarly, hantavirus genes can be isolated directly from infected tissue using known techniques, such as by phenol extraction (see, e.g., International Publication No. WO 95/00648, published Jan. 5, 1995), and the sequence can be further manipulated to produce any desired alterations. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA. Finally, the genes encoding the hantavirus antigens can be produced synthetically, based on the known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed. The complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem. (1984) 259:6311.
Polynucleotides can comprise coding sequences for the various polypeptides which occur naturally or can include artificial sequences which do not occur in nature. These polynucleotides can be ligated to form a coding sequence for a fusion protein, if desired, using standard molecular biology techniques.
Once coding sequences have been prepared or isolated, such sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Suitable vectors include, but are not limited to, plasmids, phages, transposons, cosmids, chromosomes or viruses which are capable of replication when associated with the proper control elements. The coding sequence is then placed under the control of suitable control elements, depending on the system to be used for expression. Thus, the coding sequence can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence of interest is transcribed into RNA by a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence which can later be removed by the host in post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.
If present, the signal sequence can be the native leader found in association with the hantavirus polypeptide of interest. For example, if the hantavirus polypeptide being expressed is a G1 antigen, all or a portion of the native G1 leader sequence can be included. Alternatively, a heterologous signal sequence can be present which can increase the efficiency of secretion. A number of representative leader sequences are known in the art and include, without limitation, the yeast α-factor leader, the TPA signal peptide, the Ig signal peptide, and the like. Sequences for these and other leader sequences are well known in the art.
In addition to control sequences, it may be desirable to add regulatory sequences which allow for regulation of the expression of the sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector. For example, enhancer elements may be used herein to increase expression levels of the constructs. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4:761), the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777) and elements derived from human CMV (Boshart et al. (1985) Cell 41:521), such as elements included in the CMV intron A sequence (U.S. Pat. No. 5,688,688). The expression cassette may further include an origin of replication for autonomous replication in a suitable host cell, one or more selectable markers, one or more restriction sites, a potential for high copy number and a strong promoter.
An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the “control” of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence). Modification of the sequences encoding the molecule of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it can be attached to the control sequences in the appropriate orientation; i.e., to maintain the reading frame. The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
As explained above, it may also be desirable to produce mutants or analogs of the polypeptide of interest. Mutants or analogs of hantavirus polypeptides for use in the subject compositions may be prepared by the deletion of a portion of the sequence encoding the molecule of interest, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, and the like, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al. (1987) BioTechniques 5:786; Zoller and Smith (1983) Methods Enzymol. 100:468; Dalbie-McFarland et al. (1982) Proc. Natl. Acad. Sci USA 79:6409.
In order to facilitate recombinant expression, the molecule of interest can be expressed as a fusion protein, such as a fusion with, e.g., a 50 kDa E. coli maltose binding protein, a fusion with a human or yeast superoxide dismutase (SOD) or fragment thereof, or as a ubiquitin fusion protein.
The molecules can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all well known in the art. For example, insect cell expression systems, such as baculovirus systems, are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). Similarly, bacterial and mammalian cell expression systems are well known in the art and described in, e.g., Sambrook et al., supra. Yeast expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.
A number of appropriate host cells for use with the above systems are also known. For example, mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney cells, human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
Nucleic acid molecules comprising nucleotide sequences of interest can be stably integrated into a host cell genome or maintained on a stable episomal element in a suitable host cell using various gene delivery techniques well known in the art. See, e.g., U.S. Pat. No. 5,399,346.
Depending on the expression system and host selected, the molecules are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein is expressed. The expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
The hantavirus antigens are used herein as diagnostics to detect the presence of reactive antibodies directed against the virus in a biological sample. Furthermore, the antigens can be used to determine which hantavirus type is responsible for infection. The antigens may also be used to produce antibodies for use in diagnostics.
Hantavirus Antibodies
The hantavirus antigens can be used to produce hantavirus-specific polyclonal and monoclonal antibodies. Hantavirus-specific polyclonal and monoclonal antibodies specifically bind to hantavirus antigens. Polyclonal antibodies can be produced by administering the hantavirus antigen to a mammal, such as a mouse, a rabbit, a goat, or a horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, preferably affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
Monoclonal antibodies directed against hantavirus-specific epitopes present in the proteins can also be readily produced. Normal B cells from a mammal, such as a mouse (see, e.g., Kohler and Milstein, Nature (1975) 256:495-497), or a rabbit (see, e.g., U.S. Pat. No. 5,675,063 incorporated herein by reference in its entirety), immunized with a hantavirus antigen, can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing hantavirus-specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing hantavirus-specific antibodies are isolated by another round of screening.
It may be desirable to provide chimeric antibodies. Chimeric antibodies composed of human and non-human amino acid sequences may be formed from the mouse monoclonal antibody molecules to reduce their immunogenicity in humans (Winter et al. (1991) Nature 349:293; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220; Shaw et al. (1987) J Immunol. 138:4534; and Brown et al. (1987) Cancer Res. 47:3577; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239:1534; and Jones et al. (1986) Nature 321:522; EP Publication No. 519,596, published 23 Dec. 1992; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994).
Antibody molecule fragments, e.g., F(ab′)2, Fv, and sFv molecules, that are capable of exhibiting immunological binding properties of the parent monoclonal antibody molecule can be produced using known techniques. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659; Hochman et al. (1976) Biochem 15:2706; Ehrlich et al. (1980) Biochem 19:4091; Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879; and U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.
In the alternative, a phage-display system can be used to expand monoclonal antibody molecule populations in vitro. Saiki, et al. (1986) Nature 324:163; Scharf et al. (1986) Science 233:1076; U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al. (1995) J Mol Biol 254:392; Barbas, III et al. (1995) Methods: Comp. Meth Enzymol 8:94; Barbas, III et al. (1991) Proc Natl Acad Sci USA 88:7978.
Once generated, the phage display library can be used to improve the immunological binding affinity of the Fab molecules using known techniques. See, e.g., Figini et al. (1994) J. Mol. Biol. 239:68. The coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized, and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used, including those described above.
Antibodies which are directed against hantavirus epitopes, are particularly useful for detecting the presence of hantavirus or hantavirus antigens in a sample, such as a serum sample from a hantavirus-infected human. An immunoassay for a hantavirus antigen may utilize one antibody or several antibodies either alone or in combination with hantavirus antigens. An immunoassay for a hantavirus antigen may use, for example, a monoclonal antibody directed towards a hantavirus epitope, a combination of monoclonal antibodies directed towards epitopes of one hantavirus polypeptide, monoclonal antibodies directed towards epitopes of different hantavirus polypeptides, polyclonal antibodies directed towards the same hantavirus antigen, polyclonal antibodies directed towards different hantavirus antigens, or a combination of monoclonal and polyclonal antibodies. Immunoassay protocols may be based, for example, upon competition, direct reaction, or sandwich type assays using, for example, labeled antibody and are described further below. The labels may be, for example, fluorescent, chemiluminescent, or radioactive.
The hantavirus antibodies may further be used to isolate hantavirus particles or antigens by immunoaffinity columns. The antibodies can be affixed to a solid support by, for example, adsorption or by covalent linkage so that the antibodies retain their immunoselective activity. Optionally, spacer groups may be included so that the antigen binding site of the antibody remains accessible. The immobilized antibodies can then be used to bind hantavirus particles or antigens from a biological sample, such as blood or plasma. The bound hantavirus particles or antigens are recovered from the column matrix by, for example, a change in pH.
Hantavirus Diagnostic Assays
As explained above, the immunogenic hantavirus antigens and antibodies to the antigens can be used in assays to identify hantavirus infection. Typically, the presence of hantavirus in a biological sample will be determined by the presence of antibodies to hantavirus in the sample, although in appropriate cases the presence of the viral proteins may be detected and used as an indicator of hantavirus in the sample. The reagents can be used for detecting hantavirus in blood samples, including without limitation, in whole blood, serum and plasma. The antigens and antibodies can be used to detect hantavirus infection in a subject, such as a human or rodent subject, as well as to detect hantavirus contamination in donated blood samples. Thus, aliquots from individual donated samples or pooled samples can be screened for the presence of hantavirus and those samples or pooled samples contaminated with hantavirus can be eliminated before they are combined. In this way, a blood supply substantially free of hantavirus contamination can be provided. By “substantially free of hantavirus” is meant that the presence of hantavirus is not detected using the assays described herein, preferably using the strip immunoblot assay described more fully below.
Assays for use herein include Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, strip immunoblot assays, and the like. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
The aforementioned assays generally involve separation of unbound antibody or antigen in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
In one aspect of the invention, the hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV, are used for capture or detection or both of anti-hantavirus antibodies in a sample. In another aspect of the invention, antibodies to the hantavirus antigens can be used for the capture or detection or both of hantavirus antigens in a sample. By “capture” of an analyte (i.e., anti-hantavirus antibodies or hantavirus antigens in a sample) is meant that the analyte can be separated from other components of the sample by virtue of the binding of the capture molecule. Typically, the capture molecule is associated with a solid support, either directly or indirectly. Typically, the detection molecule is associated with a detectable label, either directly or indirectly.
Typically, a solid support is first reacted with a solid phase component (e.g., the hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV and/or anti-hantavirus antibodies) under suitable binding conditions such that the component is sufficiently immobilized to the support. Sometimes, immobilization to the support can be enhanced by first coupling to a protein with better binding properties. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art. Other molecules that can be used to bind the antigen or antibody to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules are well known to those of ordinary skill in the art. See, e.g., Brinkley, M. A. Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl. Biochem. (1984) 6:56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30:117-124.
After reacting the solid support with the solid phase component, any non-immobilized solid-phase components are removed from the support by washing, and the support-bound component is then contacted with a biological sample suspected of containing the ligand component (i.e., hantavirus antigens or antibodies) under suitable binding conditions. After washing to remove any non-bound ligand, a secondary binder moiety can be added under suitable binding conditions, wherein the secondary binder is capable of associating selectively with the bound ligand. The presence of the secondary binder can then be detected using techniques well known in the art.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with the hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV, and/or antibodies according to the present invention. A biological sample containing or suspected of containing either anti-hantavirus immunoglobulin molecules or hantavirus antigens is then added to the coated wells. After a period of incubation sufficient to allow antigen-antibody binding, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.
In one particular format, an ELISA antigen sandwich format is used. In this case, the solid support is coated with the hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV. The sample is then contacted with the support under conditions that allow anti-hantavirus antibodies, if present, to bind one or more or the hantavirus antigens to form an antigen/antibody complex. Unbound reagents are removed and an enzymatically labeled antigen that reacts with the bound antigen/antibody complex, such as a labeled hantavirus N and/or G1 antigen, is added. An enzyme substrate is used to generate a signal.
In another format, the solid support is coated with species-specific anti-isotypic antibodies (e.g., anti-human IgM antibodies, anti-human IgG antibodies, anti-human IgA antibodies, etc). The support is then contacted with the sample under conditions that allow binding of antibodies present in the sample to the anti-isotypic antibodies. Unbound antibodies can be removed and the presence of bound anti-hantavirus antibodies is detected using labeled hantavirus antigens of the present invention. The label will typically be an enzyme label, e.g., a HRP, AP.
In another embodiment, the presence of bound hantavirus ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands. A number of anti-human immunoglobulin (Ig) molecules are known in the art which can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, alkaline phosphatase or urease, using methods known to those of skill in the art. An appropriate enzyme substrate is then used to generate a detectable signal. In other related embodiments, competitive-type ELISA techniques can be practiced using methods known to those skilled in the art.
Other formats for detection of anti-hantavirus antibodies in a sample are known and the combination of hantavirus antigens of the present invention can be used with any known format that employs a hantavirus antigen. See, e.g., Lee et al., J. Infect. Dis. (1978) 137:298-308; Lee et al., J. Infect. Dis. (1982) 146:638-644; Lee et al., J. Infect. Dis. (1982) 146:645-651; Lundkvist et al., Clin. Diagnos. Lab. Immunol. (1995) 2:82-86; Chu et al., Virology (1994) 198:196-204; Elgh et al., J. Med. Virol (1995) 45:146-150; Hjelle et al., J. Clin. Microbiol. (1997) 35:600-608; Bharadwaj et al., J. Infect. Dis. (2000) 182:43-48; Yee, et al., J. Wildl. Dis. (2003) 39:271-277).
The hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV, can be used in an IgM capture ELISA as follows. Anti-human IgM antibodies (e.g., goat anti-human IgM antibodies) are attached to a solid support, the support is contacted with a sample to be tested for the presence of human IgM to hantavirus, under conditions that would allow the binding of the anti-hantavirus IgM, if present, to one or more of the anti-human IgM antibodies attached to the solid support, to form antibody/antibody complexes. The hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV are added under conditions that would allow binding to the anti-hantavirus IgM in the antibody/antibody complexes forming an antibody/antibody/antigen complex. Unbound antigens are removed and detectably labeled anti-hantavirus antibodies are added under conditions that would allow binding to the bound antigens. The presence of IgM to hantavirus in the sample is determined by the presence of detectably labeled anti-hantavirus antibodies to the bound anti-human IgM Ab/human anti-hantavirus IgM/antigen complexes attached to the solid support. Alternatively, the hantavirus antigens themselves may be detectably labeled, this dispensing with the need for detectably labeled anti-hantavirus antibodies.
The hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV can also be used in an indirect IgG ELISA as follows. Antibodies specific for antigens are attached to a solid support, the support is contacted with the hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV under conditions that would allow binding to the anti-hantavirus antibodies bound to the support to form antibody/antigen complexes. Unbound antigens are removed and the support is contacted with a sample to be tested for the presence of human IgG to hantavirus under conditions that would allow binding of human anti-hantavirus IgG, if present, to the antigens in the antibody/antigen complexes. The presence of bound anti-hantavirus IgG can be detected using a detectably labeled anti-human IgG antibody.
While some of the foregoing assay formats are termed “ELISA” (Enzyme Linked ImmunoSorbant Assay) assays, it will be apparent to one of skill in the art that the use of a detectable label other than an “enzyme linked” binding moiety is possible and may be desirable in many situations. Other suitable detectable labels are described herein and are well known in the art.
Assays can also be conducted in solution, such that the hantavirus antigens or antibodies and ligands specific for these molecules form complexes under precipitating conditions. In one particular embodiment, the molecules can be attached to a solid phase particle (e.g., an agarose bead or the like) using coupling techniques known in the art, such as by direct chemical or indirect coupling. The coated particle is then contacted under suitable binding conditions with a biological sample suspected of containing hantavirus antibodies or antigens. Cross-linking between bound antibodies causes the formation of complex aggregates which can be precipitated and separated from the sample using washing and/or centrifugation. The reaction mixture can be analyzed to determine the presence or absence of complexes using any of a number of standard methods, such as those immunodiagnostic methods described above.
In yet a further embodiment, an immunoaffinity matrix can be provided, wherein, for example, a polyclonal population of antibodies from a biological sample suspected of containing hantavirus antibodies is immobilized to a substrate. An initial affinity purification of the sample can be carried out using immobilized antigens. The resultant sample preparation will thus only contain anti-hantavirus moieties, avoiding potential nonspecific binding properties in the affinity support. A number of methods of immobilizing immunoglobulins (either intact or in specific fragments) at high yield and good retention of antigen binding activity are known in the art. Once the immunoglobulin molecules have been immobilized to provide an immunoaffinity matrix, labeled molecules are contacted with the bound antibodies under suitable binding conditions. After any non-specifically bound hantavirus antigen has been washed from the immunoaffinity support, the presence of bound antigen can be determined by assaying for label using methods known in the art.
In a particularly preferred embodiment of the invention, a strip immunoblot assay (SIA) is used to detect hantavirus antibodies in a biological sample using the hantavirus G1 and/or N antigens from at least the six hantavirus serotypes, HTNV, PUUV, SEOV, DOBV, SNV and ANDV, immobilized on the test strip. SIA techniques are well known in the art and combine traditional western and dot blotting techniques, e.g., the RIBA® (Chiron Corp., Emeryville, Calif.) SIA. In these assays, the hantavirus antigens are immobilized as individual, discrete portions, e.g., as bands or dots, on a membranous support. Thus, by “discretely immobilized” on a membrane support is meant that the antigens are present as separate components and not mixed, such that reactivity or lack thereof with each of the antigens present can be assessed. A biological sample suspected of containing antibodies to hantavirus is then reacted with the test membrane. Visualization of anti-hantavirus reactivity in the biological sample is accomplished using anti-human immunoglobulin enzyme-conjugates in conjunction with a colorimetric enzyme substrate. Internal controls, such as anti-human IgM antibody and anti-human IgG antibody, can also be present on the test membrane. The assay can be performed manually or used in an automated format.
Solid supports which can be used in the practice of the strip immunoblot assays include, but are not limited to, membrane supports derived from a number of primary polymers including cellulose, polyamide (nylon), polyacrylonitrile, polyvinylidene difluoride, polysulfone, polypropylene, polyester, polyethylene and composite resins consisting of combinations or derivatives of the above. Particularly preferred are supports derived from cellulose, such as nitrocellulose membranes, as well as nylon membranes. The substrate generally includes the desired membrane with an inert plastic backing as a support.
The amount of antigen applied to the membrane varies, depending on the antigen in question. Generally, the antigen will be applied to the strip in an amount of about 20-500 ng/strip, preferably 50-250 ng/strip, more preferably 75-150 ng/strip. One of skill in the art can readily determine the amount of antigen necessary to produce a usable result. Alternatively, two concentrations of the antigens can be present, such as a low concentration and a high concentration. Thus, for example, an SNV N antigen can be provided in a concentration as specified above, as well as in one or more additional bands, in a concentration of about, e.g., 25-200 ng, such as 50-150 ng, e.g., 100 ng/strip. The high level control will be present in an amount sufficiently higher to give a highly positive result, such as at 200-500 ng, particularly 250-350 ng, e.g., 300 ng/strip. It is apparent that the concentration of antigen to be applied to the test strip will vary depending on the specific antigen used and can be readily determined by one of skill in the art.
The anti-immunoglobulin antibodies, such as anti-human IgM antibody, anti-human IgG antibody and/or anti-human IgA antibody, can be present in a single concentration, or in two concentrations, one low and one high. For example, anti-IgG antibody can be present in a concentration of about 50-250 ng/ml, more preferably about 75-200 ng/ml and most preferably about 100-185 ng/ml. A higher concentration of anti-IgG antibody can also be present along with the low concentration of anti-IgG, to provide another internal control, such as at a concentration of about 400-1200 ng/ml, more preferably about 450-1000 ng/ml and most preferably about 500-950 ng/ml.
After reacting the membrane support with the desired antigens and Ig molecules, any non-immobilized solid-phase components are removed from the membrane by washing, and the membrane-bound components are then contacted with a biological sample suspected of containing antibodies to hantavirus, under suitable binding conditions. After washing to remove any non-bound antibodies, a secondary binder moiety is added under suitable binding conditions, where the secondary binder is capable of associating selectively with bound antibodies. The presence of the secondary binder can then be detected using techniques well known in the art.
In a particularly preferred embodiment, the presence of bound anti-hantavirus antigen ligands from a biological sample can be readily detected using a secondary binder comprising an antibody directed against the antibody ligands. A number of anti-human immunoglobulin (Ig) molecules are known in the art such as commercially available goat anti-human Ig or rabbit anti-human Ig. Ig molecules for use herein will be of the IgG, IgA or IgM type. The Ig molecules can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, glucose oxidase, β-galactosidase, alkaline phosphatase and urease, among others, using methods known to those of skill in the art. An appropriate enzyme substrate is then used to generate a detectable signal.
Furthermore, a conjugate against human heavy-and light-chain antibodies can be used to render the SIA capable of detecting both IgG and IgM responses. This design may serve to increase sensitivity of detection of antibody responses in the early stages of infection.
The above-described assay reagents, including hantavirus antigens and/or antibodies thereto, the solid supports with bound reagents, as well as other detection reagents, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct the assays as described above. The kit may also include control formulations (positive and/or negative), labeled reagents when the assay format requires same and signal generating reagents (e.g., enzyme substrate) if the label does not generate a signal directly. Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay usually will be included in the kit. The kit can also contain, depending on the particular assay used, other packaged reagents and materials (i.e. wash buffers and the like). Standard assays, such as those described above, can be conducted using these kits.
III. EXPERIMENTALBelow are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Example 1 Cloning and Expression of Hantavirus Recombinant G1 ProteinsThe hantavirus G1 antigens shown in Table 2 were prepared as follows. Nucleotide fragments encoding recombinant G1 antigens including 80-83 amino acids of the G1 protein of HTNV, PUUV, SEOV, DOBV, SNV and ANDV were synthesized using a combination of oligonucleotides corresponding to the region of interest for each strain. The portion of the G1 protein sequence in the recombinant antigen is indicated in Table 2. These fragments were annealed, ligated and cloned into a subcloning vector and the correct nucleotide sequence was confirmed by DNA sequencing. The region of interest was excised by restriction endonuclease treatment and cloned along with the ADH2/GAPDH promoter fragment into pBS24.1. Plasmid pBS24.1 is a high copy number expression vector that has been extensively used to express a great variety of recombinant proteins (Pichuantes et al., J. Biol. Chem. (1990) 23:13890-13898; Pichuantes et al., “Expression of heterologous gene products in yeast.” In: Cleland J L, Craik C, editors. Protein engineering: a guide to design and production (1996) New York, N.Y., Wiley-Liss, Inc., pp 129-161). It contains 2μ and inverted repeats (IR) sequences for autonomous replication, the α-factor terminator to ensure transcription termination, and the leu2-d and URA3, for selection. The β-lactamase gene for ampicillin resistance and the ColE1 origin of replication are also present in this vector for selection and autonomous replication in Escherichia coli.
For SOD fusion constructs, the region of interest was cloned into a genetically engineered pBS24.1 vector that already contained the ADH2/GAPDH promoter along with the human SOD gene. The nucleotide sequence and amino acid sequences of the human SOD used in the cloning of the hantavirus recombinant antigens is described in Hallewell et al., Bio/Technology (1987) 5:363-366. The resulting plasmids were transformed into S. cerevisiae strain AD3 and expression of the recombinant proteins was monitored by SDS-PAGE and immunoblot analysis. The various SOD/G1 fusions produced include the G1 sequences shown in
The following protocol was followed to produce the SOD fusions. Although this discussion details the production of the SNV G1/SOD fusion, the remaining fusions were produced in the same way. Since expression of extensive portions of the full-length G1 was hampered by instability and variable expression, a smaller (82-aa) portion of SNV G1 (residues 35-117 relative to the initiating methionine of isolate NM H10) was used in a fusion with the human superoxide dismutase (SOD) sequence shown in
The hantavirus N antigens shown in Table 3 were prepared as follows. The genes for the Hantavirus nucleocapsid proteins of PUUV, SEOV, SNV and ANDV were amplified by PCR using DNA from hantavirus recombinant plasmids as templates. The portions of the hantavirus N protein sequence in the recombinant proteins is indicated in Table 3. The nucleocapsid genes of HTNV and DOBV were chemically synthesized. Cloning and expression was performed essentially as described above. The various N antigens produced are shown in
The hantavirus proteins, recombinant produced as described above, were purified by lysing the transformed S. cerevisiae cells in lysis buffer (50 mM Tris, 0.15 M NaCl, 1 mM EDTA, pH 8) using a Dino-Mill apparatus. The lysate was washed several times with 1-3 M urea in lysis buffer and protein was solubilized by increasing the pH to 11.5 and then subjected to gel filtration chromatography. The purification of recombinant ANDV G and DOBV G proteins also included ammonium sulfate precipitation and solubilization with PBS, 0.1% SDS, 1 mM EDTA.
Example 4 Polyclonal Antibody Production Using the Hantavirus Recombinant ProteinsThe purified recombinant SOD fused G1 antigens of HTNV, PUUV, SEOV, DOBV, SNV and ANDV and the recombinant non-fused N proteins of SEOV, DOBV, SNV and ANDV (see Tables 2 and 3 above) were used to produce rabbit polyclonal antibodies. Two rabbits were immunized and 50 ml of antiserum were produced. The polyclonal antibodies were then tested against the recombinant proteins using Western Blot analysis on a 4-20% Tris Glycine gel. The antibodies produced against each of the six G1 antigens were shown to be cross-reactive with the G1 antigens of the different subtypes. Similarly, the antibodies produced against each of the four N proteins were shown to be cross-reactive with the N antigens of the different subtypes.
Example 5 Strip Immunoblot Assay (SIA)The recombinant G1 antigens and N antigens, with or without the SOD sequence, from the six serotypes, as described above are used in an SIA, such as the RIBA7 test (Chiron Corp., Emeryville, Calif.). The membrane consists of nitrocellulose with an inert plastic backing as support. Six of the G1 antigens, one from each HTNV, PUUV, SEOV, DOBV, SNV and ANDV, and six of the N antigens, one from each of HTNV, PUUV, SEOV, DOBV, SNV and ANDV, are applied in discrete bands to nitrocellulose strips at concentrations of 75-150 ng/strip. As internal controls, additional bands contain purified human IgG at a low level, Level I (50-150 ng/strip) and high level, Level II (250-350 ng/strip).
The assay procedure is performed according to the manufacturer's instructions. All steps are performed at room temperature. Each strip is numbered and then placed in a separate tube to which is added a 1:50 dilution of human serum in a specimen diluent buffer (phosphate-buffered saline (PBS) with bovine protein stabilizers and detergents, 0.1% sodium azide and 0.05% gentamicin sulfate as preservatives). The tubes are rocked gently for 4 to 4.5 h, the solution removed by aspiration, and 1 ml of fresh diluent is added to each tube. The tubes are rocked for 30 minutes, the solution removed by aspiration and 1 ml of wash buffer made from wash buffer concentrate (50×) (phosphate-buffered detergent solution with 0.01% thimerosal as a preservative) is added to each tube. The contents of each tube are emptied into a single wash vessel and the strips are washed by swirling for 20 seconds. The wash buffer is decanted and 30 ml of fresh buffer added and the process repeated. Residual solution is removed by aspiration and 20 ml of conjugate solution (peroxidase-labeled goat anti-human IgG (heavy and light chains), with bovine protein stabilizers, containing 0.01% thimerosal as a preservative) is added. The vessel is rotated at 110 rpm for 9-11 minutes, the conjugate solution is decanted and the wash step is repeated three times. Residual solution is again removed by aspiration and 20 ml of substrate/developer (4-chloro-1-napthol in methanol/phosphate-buffered hydrogen peroxide) added, followed by rotation for 15-20 minutes at 110 rpm. The solution is decanted and the strips are washed twice in distilled water. Developed strips are placed face up on absorbent paper and allowed to dry for 30 minutes in the dark.
A serum is considered reactive against a given antigen only if reactivity is greater than or equal to the level I IgG control band, which is defined to represent a 1+ reactivity. A reactivity equivalent to the level II IgG control band is considered to represent a reactivity of 3+. Reactivity intensity intermediate between the level I and level II IgG control bands is considered to be 2+, and reactivity stronger than the level II band is considered to be 4+.
Thus, novel methods for detecting hantavirus infection are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as described herein.
Claims
1. A method of detecting hantavirus antibodies in a biological sample, comprising:
- (a) contacting said biological sample with at least six hantavirus recombinant antigens, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV) wherein at least one recombinant antigen from each of the serotypes is present, said contacting performed under conditions which allow hantavirus antibodies, when present in the biological sample, to bind to at least one of said G1 or N antigens to form an antibody/antigen complex; and
- (b) detecting the presence or absence of said antibody/antigen complex, thereby detecting the presence or absence of hantavirus antibodies in said sample.
2. The method of claim 1, wherein said G1 antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40.
3. The method of claim 2, wherein said G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47.
4. The method of claim 1, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and N antigens, and wherein said N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39.
5. The method of claim 4, wherein said N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
6. The method of claim 1, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and N antigens, and wherein said G1 antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40 and said N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39.
7. The method of claim 6, wherein said G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, and said N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
8. The method of claim 1, wherein at least one G1 antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV, and at least one N antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV.
9. An immunodiagnostic test kit for detecting hantavirus infection, said test kit comprising:
- (a) at least six hantavirus recombinant antigens, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV) wherein at least one antigen from each of the serotypes is present;
- (b) and instructions for conducting the immunodiagnostic test.
10. The immunodiagnostic test kit of claim 9, wherein said G1 antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40.
11. The immunodiagnostic test kit of claim 10, wherein said G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47.
12. The immunodiagnostic test kit of claim 9, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and N antigens, and wherein said N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39.
13. The immunodiagnostic test kit of claim 12, wherein said N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
14. The immunodiagnostic test kit of claim 9, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and N antigens, and wherein said G1 antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40 and said N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39.
15. The immunodiagnostic test kit of claim 14, wherein said G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, and said N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
16. The immunodiagnostic test kit of claim 9, wherein at least one G1 antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV, and at least one N antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV.
17. A method of detecting hantavirus antigens in a biological sample, comprising:
- (a) contacting said biological sample with at least six different antibodies, wherein each of said antibodies is specific for at least one of six hantavirus antigens, wherein the six hantavirus antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV), wherein at least one antibody specific for at least one antigen from each of the serotypes is present, wherein said contacting is done under conditions which allow hantavirus antigens, when present in the biological sample, to bind to said antibodies to form an antibody/antigen complex; and
- (b) detecting the presence or absence of said antibody/antigen complex, thereby detecting the presence or absence of hantavirus antigens in said sample.
18. The method of claim 17, wherein said antibodies are monoclonal antibodies.
19. An immunodiagnostic test kit for detecting hantavirus infection, said test kit comprising:
- (a) at least six different antibodies, wherein each of said antibodies is specific for at least one of six hantavirus antigens, wherein the six hantavirus antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV), wherein at least one antibody specific for at least one antigen from each of the serotypes is present; and
- (b) instructions for conducting the immunodiagnostic test.
20. The immunodiagnostic test kit of claim 19, wherein said antibodies are monoclonal antibodies.
21. A solid support comprising at least six hantavirus recombinant antigens, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and/or N antigens from hantavirus serotypes Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Dobrava (DOBV), Sin Nombre (SNV) and Andes (ANDV) wherein at least one antigen from each of the serotypes is present.
22. The solid support of claim 21, wherein said G1 antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40.
23. The solid support of claim 22, wherein said G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47.
24. The solid support of claim 21, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and N antigens, and wherein said N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39.
25. The solid support of claim 24, wherein said N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
26. The solid support of claim 21, wherein the at least six hantavirus recombinant antigens comprise a combination of G1 and N antigens, and wherein said G1 antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:40 and said N antigen(s) comprises an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:39.
27. The solid support of claim 26, wherein said G1 antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47, and said N antigen(s) is one or more antigens comprising an amino acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.
28. The solid support of claim 21, wherein at least one G1 antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV, and at least one N antigen is present from each of hantavirus serotypes HTNV, PUUV, SEOV, DOBV, SNV and ANDV.
29. The solid support of claim 21, wherein the solid support is a nitrocellulose strip.
30. An immunodiagnostic test kit for detecting hantavirus, said test kit comprising:
- (a) a solid support according to claim 21; and
- (b) instructions for conducting the immunodiagnostic test.
31. A method of detecting the presence of hantavirus antibodies in a biological sample, said method comprising:
- (a) providing a biological sample;
- (b) providing a solid support according to claim 21;
- (c) contacting said biological sample with said solid support, under conditions which allow hantavirus antibodies, if present in the biological sample, to bind with at least one of the hantavirus antigens to form an antibody/antigen complex; and
- (d) detecting the presence of the antibody/antigen complex, thereby detecting the presence of hantavirus antibodies in the biological sample.
32. The method of claim 31, further comprising:
- (e) removing unbound hantavirus antibodies;
- (f) providing one or more moieties capable of associating with said antibody/antigen complex; and
- (g) detecting the presence of said one or more moieties, thereby detecting the presence of hantavirus antibodies in the biological sample.
33. The method of claim 32, wherein said one or more moieties comprises a detectably labeled hantavirus antigen.
34. The method of claim 33, wherein the detectable label is an enzyme.
35. The method of any one of claims 1, 17, and 31, wherein said biological sample is from a human blood sample.
Type: Application
Filed: Jun 17, 2010
Publication Date: Mar 3, 2011
Applicant:
Inventors: Steve H. Nguyen (Emeryville, CA), Sergio Pichuantes (Emeryville, CA)
Application Number: 12/803,104
International Classification: C12Q 1/70 (20060101); G01N 33/53 (20060101);
