ENDOTOXIN DETECTION METHOD
The invention relates to a method for detecting endotoxins in a sample.
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This application is a continuation of U.S. Application No. 10/583,415, filed as International Application No. PCT/DE2004/002778 on Dec. 20, 2004, which claims priority to German Application No. DE 103 60 844.3, filed Dec. 20, 2003. The entire text of each of the above-referenced disclosures is specifically incorporated herein by reference.
REFERENCE TO SEQUENCE LISTINGPursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named “DEBEP0067USC1.txt”, created on Feb. 19, 2013 and having a size of ˜38 KB. The content of the aforementioned file is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTIONThe present invention relates to methods for detecting endotoxins in a sample.
Endotoxin (ET) describes a family of lipopolysaccharides which together with proteins and phospholipids form the outer cell wall of Gram-negative bacteria. Endotoxins occur exclusively in this bacterial group and play an important role in the organisation, stability and barrier function of the outer membrane. Numerous bacteriophages use endotoxin or general lipopolysaccharide for specific detection of their host bacteria.
All endotoxin variants comprise a heteropolysaccharide which is bonded covalently to lipid A. Lipid A anchors endotoxin in the outer bacterial membrane. The heteropolysaccharide, which comprises a core oligosaccharide and the O antigen, appears in the surrounding solution and determines the serological identity of the bacterium. The O antigen comprises repetitive oligosaccharide units, the composition of which is strain-specific. Characteristic building blocks of the core oligosaccharide are 2-keto-3-deoxyoctonate (KDO) and L-glycero-D-mannoheptose (Hep).
The most conservative part of endotoxin of different types is the lipid A. The inner core region is preserved similarly to lipid A, the outer core region already has a higher variation. The inner core region, KDO and lipid A itself carry a plurality of phosphate groups as substituents and are therefore responsible for the negative charge of endotoxin. Furthermore, the phosphate groups on the lipid A and on the core region can be substituted variably with arabinose, ethanolamine and phosphate. Individual saccharide building blocks of the O antigen are acetylated, sialated or glycosylated. The O antigen varies in addition with respect to the number of repetitive units, for which reason the endotoxin population of each bacterium has a certain heterogeneity.
Endotoxins are biomolecules which can be found in practically all aqueous solutions without corresponding precautionary measures. Endotoxins in humans and animals can lead to sepsis, to a strong incorrect response of the immune system. Hence, for example when producing pharmaproteins, contamination with endotoxin should be detected precisely and should be removed completely subsequently. Endotoxin represents a problem with genetically engineered pharmaceuticals, gene therapeutics or substances, which are injected into humans or animals (e.g. veterinary treatment or in animal tests). However, not only in medicinal but also in research applications, such as transfection experiments of mammal cells, inhibition or lowering of the transfection efficiency by means of endotoxin can be observed.
In order to be able to use proteins within the framework of clinical studies, the European and American pharmacopoeia demand that the proteins fall below specific boundary values for endotoxin level (e.g. immune serum globulin 0.91 EU/ml, this corresponds to 5 EU/kg bodyweight and hour (dosage=EU/kg*h); EU=endotoxin unit; FDA (Food and Drug Administration): Guideline on Validation of LAL as End Product). If a medicine or proteins contained therein have too high an endotoxin level, this can lead to the death of the experimentee. The misdirected immune defence damages the patient due to overreaction. This can lead to tissue inflammation, drop in blood pressure, heart racing, thrombosis, shock etc. Even a longer enduring endotoxin exposition in picogram quantities can lead to chronic side effects, such as e.g. immune deficiences, septic symptoms etc. Within the framework of substance production, in particular in processes with “good manufacturing practice” (GMP) conditions, it is therefore attempted to deplete endotoxin as far as possible. However, endotoxin removal in proteins, polysaccharides and DNA is problematic. In the case of proteins themselves, there are large problems due to their intrinsic properties, such as charge state or hydrophobicity, which can virtually prevent endotoxin removal or can lead to large product losses in the removal procedure.
At present, four methods for endotoxin detection in biological solutions are described, only the first two methods being permitted by the FDA. 1. “Rabbit Pyrogen Testing”; a method in which a living rabbit is injected with an endotoxin solution and hence an immune reaction is triggered. This endotoxin-induced immune response is detected by the development of fever. 2. The “Limulus Amoebocyte Lysate (LAL)”—Test, the test which is used most frequently at present (Bio Whittacker, Inc., Charles-River, Inc., Associates of Cape Cod, Inc., all USA), can be standardised in a significantly improved way. With this method, the agglomeration of the blood of the horseshoe crab (Limulus polyphemus) is measured after endotoxin contact. 3. The in vitro pyrogen test is based on the detection of interleukin-1β in human blood, which is involved in fever induction. The test consists of an incubation step of human blood with the solution to be examined, and the subsequent detection of the interleukin via antibodies. 4. A further possibility is the use of a special cell culture system (Sterogene Inc., USA) with which activation of monocytes is tracked via the appearance of specific cytokines
The two first-mentioned methods are however very expensive and, due to the large requirement for test animals or for blood of the very rare horseshoe crab, are dubious not least on the grounds of animal protection. The LAL test can in fact also be miniaturised and automated but, due to low stability of the components, has huge disadvantages in application. Once a LAL solution has been opened it must be processed and used up immediately since the components aggregate within a few hours. The in vitro pyrogen test requires preferably fresh human blood and is relatively time consuming, as the production of the interleukin requires about 10 to 24 hours. Aside of endotoxins, other pyrogens may also be recognized with the pyrogen test. However, first of all, this test is used as substitute for the “rabbit pyrogen test”. Skilled personnel are required for all test methods and the methods are very susceptible to interference, because for example the immune system of rabbits can react entirely differently to the same dose of endotoxin. The cell culture method of the Sterogene Company, like all cell culture methods, is likewise very complex and has problems with respect to standardisation.
It can be established overall that there is no easily handled economical method for endotoxin detection and the methods used at present have a series of disadvantages. There is therefore a requirement for a method which avoids these disadvantages.
BRIEF SUMMARY OF THE INVENTIONThe object underlying the invention is therefore to provide a method which can detect endotoxins in solutions and samples faster, easier and more standardized.
The objects are achieved by the subject defined in the patent claims.
The subsequent Figures explain the invention.
The term “sample material” or “sample” as used herein comprises any solutions in which endotoxins are to be detected. Examples for solutions are provided in the following list: aqueous solutions and mixtures of water and organic solvents, blood, blood products, plasma, serum, urine, media. Examples for solutions are further those in which solid low molecular and/or high molecular substances to be examined or to be purified are dissolved, such as sugar, salts, antibiotics, proteins, DNA, RNA, food stuff, medicaments, vaccines, or organic and inorganic chemicals like for example NaCl, MgCl2, purine or pyrimidine.
The term “endotoxin” as used here describes bacterial lipopolysaccharide which is a component of the outer membrane of Gram-negative bacteria. “Endotoxin” not only refers to the complete lipopolysaccharide but also to the single components such as lipid A, the inner and the outer heart region.
The term “bacteriophage tail protein” as used here describes those proteins which occur in bacteriophages and can bind endotoxins. Normally, these proteins are localised in the bacteriophage tail but can also be localised on the bacteriophage head or on the normal bacterial shell in the case of bacteriophages without a tail. The term bacteriophage tail protein comprises short as well as long bacteriophage tail proteins. In this respect, bacteriophages with a basal plate (e.g. myoviridae such as T4 like phages) can have different bacteriophage tail proteins, so called long or short bacteriophage tail proteins, which also have different specificities for structures on bacterial membranes.
The term “homology”, as used herein, refers to a significant similarity of an amino acid sequence to a reference sequence or parts thereof. The homology of a sequence is determined using the similarity algorithm BLAST (Basic Local Alignment Search Tool, Altschul et al., Journal of Molecular Biology 215, 403-410 (1990). Sequences are regarded as “significant similar”, as used herein, which exhibit a homology of at least 60% or which exhibit, for example by using standard parameters in the BLAST-service of NCBI, a significance level (E-value or probability) of P<10−5, if they are compared with the reference sequences.
The term “non-specific immobilisation” or “undirected immobilisation” as used here means that coupling of a protein to a matrix is effected via protein radicals (primary amines) which can be distributed over the entire protein surface. The choice of group used for the coupling of the individual protein molecule is random.
The term “surface” or “carrier”, as used herein, comprises all materials to which a coupling or adhesion of a protein molecule is possible, such as for example glass surfaces, chromatography materials, such as agarose or sepharose, plastic surfaces, such as polystyrene or polypropylene, filter materials, such as cellulose.
The term “directed immobilisation” as used here means that coupling is effected via amino acid radicals or other radicals (e.g. glycosylations of the protein), the position of which in the protein (e.g. N- or C-terminal) is known. The choice of these groups for the coupling is effected by the choice of suitable reaction partners/linkers which react preferably with these radicals (e.g. coupling of sulfhydryl radicals to iodoacetate radicals; iodoacetate reacts a thousand times more quickly with sulfhydryl radicals than with amino radicals).
One aspect of the present invention relates to a method for detecting endotoxin, comprising the steps:
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- a) incubation of a sample with bacteriophage tail proteins, and subsequently
- b) detection of endotoxin bonded to bacteriophage tail proteins by means of spectroscopic methods, ELISA, chemical and enzymatic detection reaction of endotoxins or cleaved-off Endotoxin components, or by means of capacitance measurement.
If necessary, after step a) and before step b), an additional step a′) of separation of bacteriophage tail protein-endotoxin complex from the sample, is introduced.
The detection by means of spectroscopic methods can be implemented by, for example, fluorescence emission, fluorescence polarisation, absorption or circular dichroism, the detection by means of capacitance measurement can be implemented for example by electrical signals. The listed detection methods can further be combined with a competition detection.
The present invention relates preferably to a method, in which after separation of the in step a) formed bacteriophage tail protein endotoxin complex from the sample, the detection of the endotoxins occurs by immunological, chemical or enzymatic reactions. For that purpose the bacteriophage tail proteins can be bound to the respective carrier such as sepharoses or magnetic beads, which are coated with streptavidin or streptactin, by using special ligands such as biotin, strep tag or his tag. Afterwards, if desired, a separation of the coarse-grained carriers from the sample can occur by filtration, centrifugation or magnetic separation. The separation is preferably desired, if the bacteriophage tail protein endotoxin complex is fixed to a surface, which can not be used in the applied detection methods.
The immunological detection is effected for example by binding of endotoxin specific antibodies to the endotoxins, binding of a secondary antibody to the primary antibody and subsequent detection via an enzymatic reaction, which is catalysed by an enzyme fused to the secondary antibody (ELISA).
The endotoxin detection can also occur after chemical cleavage of the endotoxin by acid or base and subsequent detection of single endotoxin components such as 2-keto-desoxyocton acid, the heptoses (Lee C.-H., Tsai C.-M., Analytical Biochemistry, 1999; 267:161-168) or the hydroxyl fatty acids (Lyngby J., Olsen L. H., Eidem T., Lundanes E., Jantzen E., Biologics, 2002; 30:7-13).
In a further aspect the present invention relates to a method for detection of endotoxin, the method comprising the steps of:
a) Contacting a sample containing endotoxins with a carrier, subsequently
b) incubating of bacteriophage tail proteins with the endotoxin immobilised on the carrier, and
c) detection of bacteriophage tail proteins by means of spectroscopic methods, ELISA, chemical or enzymatic detection reaction for endotoxins or cleaved off endotoxin components, or by means of capacitance measurement.
If necessary, after step b) an additional step b′) of separation of bound bacteriophage tail proteins from endotoxin, is performed.
Particularly preferred are the p12 proteins of the phages K3, T2, T4, Ox2, RB32-33, AR1, PP01 or RB69.
A method is preferred, in which after the binding of endotoxin to a surface, which carries endotoxin binding ligands such as polymyxin B, poly L lysin, chitosan or the like, bacteriophage tail proteins bind to the immobilised endotoxins and these bacteriophage tail proteins are detected by means of a subsequent enzymatical reaction. The bacteriophage tail proteins can be detected by an ELISA, which is specific for the bacteriophage tail protein, or by enzymes, which are fused via genetical engineering or bound via chemical reactions to the bacteriophage tail protein. The enzymes can be, for example, alkaline phosphatase, peroxidase or others.
Preferably, the ion composition of the bivalent ions, e.g. Ca2+, Mg2+ and/or the pH value is adjusted prior to the incubation step of the methods according to the invention in order to obtain an optimal endotoxin-bacteriophage tail protein binding. Furthermore, during or after incubation, “demasking” of the bonded endotoxin by addition of detergents and/or salts, e.g. Tween, triton NaCl or ammonium sulphate or other substances, e.g. chitosan, sugar or lipids, which accelerate detachment of the endotoxins from e.g. proteins or nucleic acids, is preferred.
The bacteriophage tail protein used for the detection of endotoxin can be a naturally occurring or be molecular-biologically or biochemically modified one.
Particularly preferred are bacteriophage proteins, which bind to highly conserved regions of endotoxin, more precisely, to the heart region of LPS or to lipid A. Particularly preferred are the short bacteriophage tail proteins preferably of myoviridae phages. However, the endotoxin binding proteins of a bacteriophage head or the normal bacteriophage coat of bacteriophages without tail can be used as well. Exceptionally preferred are bacteriophage tail proteins with a homology of at least 60% on the amino acid level to the p12 protein of T4.
The bacteriophage tail protein can be modified by genetic engineering and/or biochemically for various reasons. For the methods according to the invention, not only the naturally occurring bacteriophage tail proteins can however be used, but also their variants. In the sense of the present invention, variants means that the bacteriophage tail proteins have an altered amino acid sequence. These can be obtained by screening of the naturally occurring variants or by random mutagenesis or targeted mutagenesis, but also by chemical modification. The bacteriophage tail proteins used for the methods according to the invention can be adapted by targeted or random mutagenesis in their specificity or their binding properties to carrier structures. This binding to the carriers can be effected permanently, e.g. covalently or via a specific or non-specific biotinylation, but also can be effected reversibly, e.g. via a reducible disulfide bridge. Furthermore, the stability can be increased by a modification. By means of the molecular-biological or chemical mutagenesis, mutations are introduced which can be amino acid additions, -deletions, -substitutions or chemical modifications. These mutations can effect a change in the amino acid sequence in the binding region of the bacteriophage tail proteins, with the aim of adapting specificity and binding affinity to test requirements, e.g. increasing the binding of the endotoxins to the bacteriophage tail proteins or making them irreversible in order to improve detection. Furthermore, a genetically engineered or biochemical modification of the phage proteins can be implemented with the aim of switching off the possibly present enzymatic activity in order consequently to improve the binding or to make it irreversible. Furthermore, a genetically engineered or chemical modification of the phage proteins can be implemented in order to adapt the present physical properties of the protein, such as solubility, thermal stability etc., in the sense of the method according to the invention.
Furthermore, coupling of the bacteriophage tail proteins with enzymatically active proteins can be performed in order to be able to detect the bacteriophage proteins more sensitively. Enzymatically active proteins like alkaline phosphatase or horseradish peroxidase, for which commercial substrates are available, can be coupled to the bacteriophage tail proteins by means of chemical coupling methods or by means of genetical fusion. The enzymatic reaction introduced via these proteins increases the sensitivity of the detection significantly.
Work to explain the three-dimensional structure of T4 p12 had shown that, at increased temperature, proteolytic fragments of 33 kDa and 45 kDa can be produced, the N- and C-terminal (33 kDa) or only N-terminal (45 kDa) are shortened. In contrast to the 33 kDa fragment, the 45 kDa fragment is still able to bind to bacteria (Thomassen, E., et al., Mol. Biol.; 331: 361-373, 2003). Consequently, the C-terminus is involved in the cell binding. Thus, by a N terminal modification a directed binding to the surfaces can be performed and thus ultimately the binding of endotoxin can be optimised indirectly. Furthermore, a direct optimisation of the endotoxin binding is possible.
The modification can furthermore have the purpose in particular of enabling direct detection, e.g. by means of measurement of the tryptophan fluorescence. For example T4p12 has five tryptophan radicals. The fluorescence spectrum of the native protein indicates that these radicals are extensively solvent-inaccessible. It is known from a multiplicity of scientific works that aromatic amino acids are almost always involved in the binding of sugar radicals, as occur also in endotoxin. The binding of the sugar radicals to proteins can be followed by a quench of the tryptophan fluorescence or if necessary also in addition by changing the fluorescence maximum. It can be supposed from some works that the unfavourable distribution of the fluorophores of natural p12 prevents exploitation of the fluorescent properties of p12 for binding measurement. The fluorescence properties of p12 are dominated by the five tryptophan radicals, the fluorescence of which is altered by the addition of endotoxin in a non-measurable manner. It is expected from these data that rather tyrosine radicals are involved as tryptophan radicals in the binding, the signal alteration of which cannot be made visible in front of the high tryptophan background. On the basis of the proteolysis results, six tyrosines on the C-terminus of p12 are possible for the endotoxin detection kit which can be made correspondingly “visible”. By means of a selective molecular-biological exchange of the five tryptophan radicals for tyrosines, the spectroscopic properties are specifically altered in a first step such that the endotoxin binding by fluorescence signal alteration of a single tryptophan radical is measurable. Subsequently, by means of a specific exchange of respectively one of the six tyrosines in the C-terminal region for a tryptophan radical, the intensity of the measurable signal is significantly increased in order to obtain attractive signal differences for the development of an endotoxin-detection kit. As shown for the p12 protein of T4, other short bacteriophage tail proteins such as for example those from myoviridae phages such as T4, T2, K3, Ox2, RB32-33 or RB69 can also be modified accordingly. However, also in this case the endotoxin binding proteins of a bacteriophage head or the normal bacteriophage coat of bacteriophages without tail can be used as well, in particular of PhiX 174.
The bacteriophage tail proteins which are used depends upon which endotoxins are intended to be detected or drawn off. Even now, a large number of known bacteriophages is available for a large part of the previously described bacteria and can be used for the methods according to the invention. The phages and the corresponding host bacteria are inter alia obtainable in the case of the following strain collections: ATCC (USA), DSMZ (Germany), UKNCC (Great Britain), NCCB (Netherlands) and MAFF (Japan); or can be isolated from environmental samples by means of microbiological standard methods. Bacteriophage proteins can be derived from the family of myoviridae, thus can be the tail proteins, in particular from the group of pseudo T even, schizo T even or the T even phages. Preferably the short bacteriophage tail proteins of the phages K3, T2, T4 Ox2, RB32-33, AR1, PP01 and RB69 or the endotoxin binding proteins of the bacteriophages without tail such as PhiX 174 are used for the detection methods according to the invention.
Preferably, the bacteriophage tail proteins for the methods according to the invention stem from bacteriophages, the host bacteria of which have relevant significance with respect to medicine or biotechnology, such as e.g. E. coli which is used in the production of recombinant proteins or of nucleic acids for gene therapy. The bacteriophage tail proteins which bind highly conserved regions of endotoxin, such as e.g. the core region or lipid A, are particularly preferred. In particular, T4p12 and T4p12-similar bacteriophage tail proteins are preferred such as T2-p12, K3-p12 (Burda-M R, Hindennach-I, Miller-S, Biol. Chem. 2000; 381: 255-258). In a combination of endotoxin impurities from various host bacteria, a combination of the corresponding endotoxin-detecting bacteriophage tail proteins can be used for the detections or depletions according to the invention.
The detection of endotoxin in or from a sample is effected via the binding of endotoxin to the bacteriophage tail proteins. This binding can be detected for example by direct measurement by means of spectroscopic methods, e.g via fluorescence emission, fluorescence polarisation, absorption or circular dichroism. Furthermore, the binding can be made visible by electrical signals, e.g. a capacitance measurement. Furthermore, the binding of endotoxin to the bacteriophage tail proteins can also be detected indirectly via displacement experiments.
In addition, the binding of bacteriophage tail proteins to endotoxin can be detected by means of first mobilising the endotoxins via other endotoxin binding substances or also via a second bacteriophage tail protein on a surface, followed by the binding of another bacteriophage tail protein to endotoxin. After washing away excess bacteriophage tail protein the amount of the bound other bacteriophage tail protein is subsequently quantified. This is achieved either by means of antibodies directed against the other bacteriophage tail protein (a so called ELISA), or by means of an enzymatic reaction catalysed by a protein, which is fused to the other bacteriophage tail protein. For this purpose the surface can be coated with endotoxin binding substances in advance, such as polymyxin B, histidine, histamine, poly L lysine, DEAE, polyethyleneimine, deoxycholic acid, poly γ-aminomethyl L glutamine, polyvinyl alcohol, poly N,N dimethylaminopropylacrylamide, dextran, chitosan, and the like. Futhermore, a bacteriophage tail protein can be used for the immobilisation of endotoxin. The detection of endotoxin is achieved then with a second bacteriophage tail protein, having other endotoxin binding properties than the bacteriophage tail protein used for the immobilisation. The immobilisation of endotoxin binding substances is achieved either by adhesion, covalent coupling, or by binding via particular immobilisation groups, such as biotin, streptavidin, strep tag, his tag and comparable groups. The detection of the bacteriophage tail protein can also occur after displacement of the protein from the surface.
For the detection methods according to the invention, the bacteriophage tail proteins, if separation of the bacteriophage tail protein-endotoxin complexes from the sample is required, can be coupled to suitable surfaces, e.g. magnetic particles, sepharose particles, agarose particles, microtitre plates, filter materials or throughflow cell chambers (indirect detection). The carrier structures can comprise for example polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass, silicon or agarose. The coupling can be achieved for example by adsorption or covalent binding.
Functional coupling is hereby important, i.e. bacteriophage tail proteins, despite binding to the carrier material, have structures or binding sites, respectively, which are accessible for endotoxin. The coupling of the bacteriophage tail proteins can be effected non-specifically or else preferably directed, via for example a selective biotinylation or coupled or via a spacer or linker.
For this purpose, the bacteriophage tail proteins can be cross-linked with low-molecular substances, e.g. biotin, in order to bind via these low-molecular substances to polypeptides, e.g. streptavidin, which for their part were immobilised on the carrier. Instead of biotin, the so-called Strep-tag (Skerra, A. & Schmidt, T. G. M. Biomolecular Engineering 16 (1999), 79-86) can furthermore be used, which is a short amino acid sequence and binds to streptavidin. Furthermore, the His-tag can be used which, via bivalent ions (zinc or nickel) or an antibody specific for it (Qiagen GmbH, Hilden), can bind to a carrier material. The Strep-tag and the His-tag are bonded preferably via DNA recombination technology to the recombinantly produced bacteriophage proteins. This coupling can be effected directed, e.g. on the N- or C-terminus or at other positions in the bacteriophage tail protein. The directed coupling is effected via a suitable, reactive amino acid, such as cysteine, which is of course not frequently surface-exposed in phage proteins and has been introduced specifically at a suitable position. Since phage tail proteins are synthesised in the cytoplasma, disulfide bridges do not need to be taken into account. Preferably, coupling can take place also via other amino acids, directly or as also with cysteine indirectly via a “spacer” or “cross linker” (monofunctional or bifunctional).
In the case of cysteine coupling, all bifunctional crosslinkers with NH- and SH-reactive groups are possible, with and without intermediate spacers, e.g. 11-maleimidoundecanoic acid sulfo-NHS or succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy-[6-amido]caproate. If no spacers are present, 8-12 C-atom-spacers with a terminal NH group can be inserted. Preferably the cysteine coupling is effected via a specific biotinylation of cysteine by for example EZ-link-PEO-maleimide activated biotin (Pierce).
Bivalent ions, such as e.g. Ca2+ or Mg2+ are important for binding endotoxins to bacteriophage tail proteins, such as p12 of T4 or the short tail fiber proteins of the phages K3, T2, Ox2, RB32-33, AR1, PP01 or RB69. By adding suitable chelating agents, such as e.g. EDTA or EGTA, this binding can however be broken. For the binding, Ca2+ concentrations are preferred in the range of approximately 0.1 μM to approximately 100 mM, particularly preferred in the range of approximately 0.1 μM to approximately 10 mM, and especially preferred in the range of approximately 0.1 μM to approximately 1 mM and furthermore particularly preferred in the range of approximately 10 μM to 1 mM. Furthermore, for the binding, Mg2+ concentrations are preferred in the range of approximately 0.1 μM to approximately 10 mM, particularly preferred in the range of approximately 0.1 μM to approximately 1 mM, especially preferred in the range of approximately 10 μM to approximately 1 mM. If the concentration of bivalent ions is lowered by adding 1 mM EDTA under 100 nM, then the binding of endotoxin to p12 is broken. Mg2+ concentrations above 10 mM make the binding of endotoxin to p12 worse, which becomes noticeable in an increase in the dissociation constant. Without addition of Mg2+, a Kd value of 50 nM is produced and, in a buffer with 10 mM Mg2+, a Kd value of 1 μM was measured. Zinc revealed an even higher inhibiting effect. 1 mM Zn increases the Kd value to 10 μM. An adjustment of the concentration of bivalent or other ions (e.g.: Cu2+, Al3+, Zn2−, Fe2+, Ca2+, Ba2+, Mg2+, Cd2+) to a range which is optimal for the binding, can be effected by substances such as HEDTA, NTA or general chelating agents/buffers (ADA: N-[2-acetamido]-2-iminodiacetic acid; 5-AMP: adenosine-5′-monophosphate; ADP: adenosine-5′-diphosphate; ATP: adenosine-5′-triphosphate; Bapta: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetic acid; citrate: citric acid; EDTA: ethylene diamine tetraacetic acid; EGTA: ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid; HEDTA: N-hydroxyethylethylenediaminetriacetic acid; NTA: nitrilotriacetic acid; SO4 sulfate), which can be used as buffers for bivalent ions.
The methods according to the invention can therefore comprise further washing steps. According to whether a direct or indirect detection requires separation of sample and bacteriophage tail protein, washing steps can be incorporated. Since Ca2+ or other metal ions (e.g. Mg2+) are essential for the binding, the binding of endotoxin to e.g. p12 (e.g. the short tail fiber proteins of phages K3, T2, T4, Ox2, RB32-33, AR1, PP01 and RB69) can be broken by suitable washing steps. According to the aim of whether endotoxin is intended to remain bonded on the bacteriophage tail protein, e.g. p12 (e.g. the short tail fiber proteins of phages K3, T2, T4, Ox2, RB32-33, AR1, PP01 and RB69), washing takes place with EDTA-free buffer, if the binding is intended to be broken, with EDTA-containing buffer, the EDTA concentrations being in the range of at least 0.05 mM to more than 10 mM, preferably in the range of 2 mM to 5 mM.
Since ionic interactions can fundamentally always be affected by changes in the ion strength, increases or reductions of other salts in the solution, such as e.g. NaCl or KCl, can also affect the binding of endotoxin to the bacteriophage tail proteins.
In order to make the binding visible directly or indirectly in the detection method, the protein can also be altered molecular-biologically or biochemically in order to enable measurement or to improve it. In order to make binding of endotoxin e.g. to p12 of T4 or to the short tail fiber proteins of phages K3, T2, T4, Ox2, RB32-33, AR1, PP01 or RB69, or to endotoxin binding proteins of bacteriophages without tail, directly visible, a molecular-biological exchange of tyrosine radicals for tryptophan can be implemented. It can thereby be necessary for a reduction in the signal background to exchange the originally contained tryptophans for tyrosines. In order to be able to make measurements also in protein-containing solutions, p12 of T4 or the short tail fiber proteins of phages K3, T2, T4, Ox2, RB32-33, AR1, PP01 or RB69 can be modified chemically in addition after tryptophan introduction. Tryptophan radicals are thereby altered by Koshland reagent (2-hydroxy-5-nitrobenzylbromide) with respect to their spectroscopic properties. In the case of displacement experiments, marked, e.g. fluorescence-marked endotoxin (e.g. Sigma) can be displaced by endotoxin, e.g. by p12 of T4 or by the short tail fiber proteins of phages K3, T2, T4, Ox2, RB32-33, AR1, PP01 or RB69 or by e.g. PhiX 174, which is located in the sample and the concentration of free fluorescent endotoxin can be determined.
With the methods according to the invention, endotoxin can be detected in all aqueous solutions. These solutions can contain: proteins, plasmid-DNA, genomic DNA, RNA, peptidoglycans, polysaccharides, protein-nucleic acid complexes, such as e.g. phages or viruses, saccharides, vaccines, drugs, reaction buffers, buffer solutions in general, media, dialysis buffers (medicine), salts, blood, blood constituents, or other substances contaminated by endotoxin binding.
A further aspect of the invention is bacteriophage proteins, to which the so-called tags, e.g. the Strep- or His-tag, are coupled preferably to the N- or C-terminus of the protein, particularly preferred to the C-terminus. The coupling or cross-linking of the tags with the bacteriophage proteins via DNA recombination technology is preferred. Production of the nucleic acid, comprising the sequence of the bacteriophage protein and of the tag and the production of the expression product are the state of the art and do not require to be explained here separately. A further aspect of the invention is the nucleic acid sequence which encodes a bacteriophage protein together with the Strep- or His-tag. The p12 protein of the phage T4 is a particularly preferred bacteriophage protein which is modified with the Strep- or His-tag but all other bacteriophage proteins, which are involved in detection and binding of bacteria or are responsible for this, are likewise preferred.
For the methods according to the invention preferably bacteriophage proteins with a tag are used, which has a surface-exposed cysteine for specific directed biotinylation, e.g. the tags according to SEQ ID NO: 5, 6 and 7. An example of a p12 with a tag is the amino acid sequence cited in SEQ ID NO: 8. A p12 with a tag is preferred, in particular with a tag with a surface-exposed cysteine, in particular a p12 with the tag according to SEQ ID NO: 6 and 7. This directed biotinylation can be imparted in addition by a suitable spacer or linker.
The methods according to the invention, relative to the prior detection methods for endotoxin, offer advantages in the performance of corresponding applications. Furthermore, the production of antibodies against LPS core oligosaccharides is very difficult, which renders corresponding methods based on antibodies very expensive.
The present invention further relates to a endotoxin detection kit comprising the components required for the method according to the invention. The kit comprises in particular a carrier coated with the bacteriophage tail proteins as mentioned herein, a container containing a reference endotoxin for the preparation and measurement of a standard curve, a further container with at least one further bacteriophage tail protein described herein, which is if necessary, modified for the detection as mentioned herein or which can be coupled to an active protein, or a container with anti lipid A antibody for the detection of endotoxin.
The following examples explain the invention and should not be understood as restrictive. If not otherwise indicated, molecular-biological standard methods were used, such as e.g. described by Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Hereinafter described experiments were performed with p12 proteins of T4, T2 and K3. If not stated explicitly otherwise, the results were only displayed for p12 of T4, as the results of the three proteins are interchangeable due to the high homology (Burda M. R., Hindenach I., Miller S., Biol. Chem. (2000) 381, 225-258; Riede I., Mol Gen Genet. 1987 Jan; 206 (1):110-115). This also applies for the other proteins displayed in
For the endotoxin removal, all the glass vessels were depyrogenated by heating at 200° C. (4 h) and exclusively pyrogene-free plastic materials (e.g. pipette tips, microtitre plates) were used. Other non-heat resistant appliances or vessels were treated either with 3% hydrogen peroxide or washed with 1% sodium deoxycholate. Subsequently, they were rinsed with endotoxin-free water. The buffers were produced from extensively endotoxin-free buffer substances (Sigma) and mixed with endotoxin-free water. Salts, such as e.g. NaCl, which can be heated to 200° C., were heated up (200° C., 4 h). Buffers used for chromatographic purifications were degassed and filtered.
EXAMPLE 2 Endotoxin Detection by Means of LAL TestEndotoxin control tests were implemented with a chromogenic LAL test (Limulus-Amoebocyte-Lysate test, Charles-River Endosafe, Charleston, USA) corresponding to the instructions of the producer. In order to determine the concentrations, endotoxin standards (Charles-River Endosafe, Charleston, USA) in the range of 0.005-50 or 0.02-50 EU/ml were used. The absorption measurement at 405 nm took place in a temperature-controlled microtitre plate reader (Genios, Tecan GmbH).
EXAMPLE 3 Western-Blot for p12 DetectionThe detection of p12 in the residue of samples treated with beads or in the fractions of the affinity chromatography was effected by Western Blots. In part, the proteins were concentrated in advance by NaDOC/TCA precipitation (sodium deoxycholate/tetrachloroacetate). The samples were electrophoretically separated for this purpose on 12% SDS gels and transferred onto PVDF membranes (Immobilon, Millipore). The membranes were washed with PBS for 30 min, blocked with 5% milk powder (1 h) and subsequently incubated with polyclonal anti-p12 antibody (1 h, dilution: 1:1000). After incubation with a secondary antibody (goat-anti-rabbit IgG), conjugated with alkaline phosphatase, the development of the samples was effected with BCIP/NBT (5-bromo-4-chloroindolylphosphate/nitroblue tetrazolium salt).
EXAMPLE 4 Endotoxin PurificationThe purification of endotoxin was implemented according to the specification of Galanos, C., Luderitz, O. & Westphal, O. 1969, Europ. J. Biochem. 9, 245-249.
EXAMPLE 5 Specific Coupling of p12 to Immobilised Iodoacetyl RadicalsIn order to achieve a directed binding of p12 to the surface, the amino acid serin at position 3 of the Strep-tag according to SEQ ID NO:5 was replaced by cysteine as in example 12 and the protein was immobilised via iodoacetyl radicals which bind preferably free sulfydryl radicals. The resulting p12 was called p12S3C.
A 1 ml Sulfolink Coupling Gel (Pierce) was poured out, washed with 6 ml 1% sodium deoxycholate and equilibrated with 6 ml coupling buffer (50 mM tris, 150 mM NaCl, 5 mM EDTA, pH 8.5). Subsequently, 1 ml p12S3C (=N-strepS3Cp12) was injected (1-1.5 mg/ml in coupling buffer), the column was agitated gently for 15 min, incubated for a further 30 min without agitation at room temperature, and 1 ml p12S3C was injected again and the incubation steps were repeated. This coupling of p12S3C was repeated in total 4 times, and subsequently the column was washed with 6 ml coupling buffer. The throughflows were collected and the respective p12S3C concentration was determined by absorption measurement at 280 nm. 2.2-2.8 mg p12S3C per ml gel were bonded. Subsequently, surplus iodoacetyl radicals were blocked by incubation (45 min) with 1 ml cysteine (50 mM in 50 mM tris, 5 mM EDTA, pH 8.5). After washing the column with 16 ml 1M NaCl and 16 ml 20 mM hepes, 150 mM NaCl pH 7.5, the column was ready for use.
The capacity of this gel to remove endotoxin from protein solutions was tested with BSA (2-4 mg/ml), carbonic anhydrase (1-2 mg/ml) and lysozyme (3-4 mg/ml). BSA and lysozyme solutions were spiked with endotoxin from E. coli O55:B5 (Charles-River Endosafe, Charleston, USA) or E. coli HMS 174 (100-1000 EU/ml), whilst the carbonic anhydrase was not mixed with additional endotoxin. Respectively 0.5 ml protein solution was introduced to the column, incubated for 1 hour at room temperature and subsequently the column was washed with buffer. The proteins were collected in fractions and the endotoxin content, prior to and after the column, was determined by means of a chromogenic LAL test (Charles-River Endosafe, Charleston, USA). In addition, the protein retrieval was determined by absorption measurements at 280 nm. The endotoxins were able to be removed almost completely (93-99%) from all 3 protein solutions, as shown in
N-hydroxysuccinimide (NHS) is displaced from compounds by primary amino radicals and therefore is used to couple proteins to surfaces. NHS-activated sepharose columns (HiTrap NHS-activated HP, 1 ml, Amersham-Pharmacia-Biotech) were washed firstly with 6 ml ice cold 1 mM hydrochloric acid. Subsequently, 10-15 ml p12S3C (1.0-3.5 mg/ml) in 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3 were pumped in a circle over the column at room temperature (flow rate 0.8 ml/min). After 60 min, the throughflow was collected in fractions and the column was washed with 6 ml buffer. From these fractions, the NHS was separated by desalting the solution via HiTrap-desalting column (5 ml, Amersham-Pharmacia-Biotech) and subsequently the p12 quantity was determined by absorption measurement at 280 nm. 20-25 mg p12S3C were bonded to the column. The column was rinsed after the coupling corresponding to the instructions of the producer repeatedly with respectively 6 ml blocking buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing buffer (0.1 M acetate, 0.5 M NaCl, pH 4.0). Subsequently, the column was equilibrated with 6 ml usable buffer (20 mM hepes, 150 mM NaCl, pH 7.5 or 20 mM tris, 150 mM NaCl, pH 8.5).
The endotoxin removal via this column was tested with lysozyme solutions (3-4 mg/ml in 20 mM hepes, 150 mM NaCl, pH 7.5 or 20 mM tris, 150 mM NaCl, pH 8.5). The lysozyme solutions were spiked with endotoxin from E. coli HMS 174 (-500 EU/ml). 0.5 ml protein solution were introduced onto the column, incubated for 1 hour at room temperature and subsequently the column was washed with buffer. The lysozyme was collected in fractions and the endotoxin content was determined prior to and after the column by means of a chromogenic LAL test (Charles-River Endosafe, Charleston, USA). In addition, the protein retrieval was determined by absorption measurements at 280 nm. The endotoxins were removed up to 85-90% from the solution, as shown in
In order to achieve a directed binding to the chromatography carrier material, a bifunctional linker was bonded to NHS-activated surface, which linker made a coupling of p12S3C possible via its free cysteine and iodoacetyl radicals of the bifunctional linker.
NHS-activated sepharose columns (HiTrap NHS-activated HP, 1 ml Amersham-Pharmacia-Biotech) were washed firstly with 6 ml ice cold 1 mM hydrochloric acid, thereafter 1 ml ethylene diamine (10 mg/ml in 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) was injected and the column was incubated for 30 min at room temperature. After blocking surplus NHS groups with ethanolamine (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and washing (0.1 M acetate, 0.5 M NaCl, pH 4.0) of the column, the column was equilibrated with 6 ml borate buffer (50 mM sodium borate, 150 mM NaCl, 5 mM EDTA, pH 8.3). Subsequently, 10 ml N-succinimidyl-iodoacetate (SIA, Pierce, 200 μl SIA parent solution in 10 ml borate buffer; SIA parent solution: 1.4 mg SIA in 1 ml DMSO) was rinsed in a circle over the column for 30 min. The column was thereafter washed with 6 ml borate buffer and p12S3C (1 mg/ml, 50 ml in borate buffer) was rinsed over the column for 1 hour. Excess iodoacetyl radicals were neutralised with 1 ml cysteine solution (5 mM cysteine in borate buffer, incubation at room temperature for 15 min), before the column with the usable buffers (20 mM hepes, 150 mM NaCl, pH 7.5 or 50 mM tris, 150 mM NaCl, ph 8.5) were equilibrated. The coupling reactions with SIA were implemented in the dark.
The endotoxin removal over this column was tested with lysozyme solutions (3-4 mg/ml in 20 mM hepes, 150 mM NaCl, pH 7.5 or 20 mM tris, 150 mM NaCl, ph 8.5). The lysozyme solutions were spiked with endotoxin from E. coli HMS 174 (˜500 EU/ml). 0.5 ml protein solution was introduced onto the column, was incubated for 1 hour at room temperature and subsequently the column was washed with buffer. The lysozyme was collected in fractions and the endotoxin content was determined prior to and after the column by means of a chromogenic LAL test (Charles-River Endosafe, Charleston, USA). In addition, the protein retrieval was determined by absorption measurements at 280 nm. The endotoxins were removed up to 90% from the solutionand 75-85% of the lysozyme were able to be eluted again from the column by washing with usable buffer. The column was subsequently washed with 6 ml 5 mM EDTA, 20 mM hepes, 150 mM NaCl, pH 7.5 and 6 ml 1 M NaCl. In order to exclude impurities of the protein fractions after running over the column due to separating p12, the fractions were tested for p12 by means of the Western Blot technique. No p12 was able to be detected in the fractions.
EXAMPLE 8 Removal of Endotoxin from a BSA Solution in the Throughflow MethodHiTrap-NHS activated sepharose (Amersham Biosciences, Uppsala Sweden) was coupled, according to the specification of the producer, non-specifically via primary amino groups with p12. 8 mg p12 per ml gel material were thereby immobilised covalently. The thus obtained 1 ml chromatography column was equilibrated with a flow rate of 1 ml/min with 10 ml buffer A (20 mM hepes, pH 7.5, 150 mM NaCl, 0.1 mM CaCl2). Next, 4 ml of a BSA solution (11.5 mg BSA (Carl Roth GmbH, Germany)/ml buffer A) were applied (injection: I) and the throughflow (E) was collected in 2.5 ml fractions. The column was washed subsequently with 15 ml buffer A and the endotoxin bonded to the column was eluted with 7 ml buffer B (20 mM hepes, pH 7.5, 150 mM NaCl, 2 mM EDTA). During washing and elution, respectively 2 ml fractions were collected. After each experiment, the column was regenerated with 20 ml buffer C (20 mM hepes, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% sodium deoxycholate). The endotoxin concentration was determined by a chromogenic Limulus Amoebocyte Lysate (LAL) (Charles-River Endosafe, Charleston, USA) according to the specification of the producer. Determination of the protein concentration was effected by measurement of the UV absorption. The endotoxin removal efficiency was between 95-99% and the protein loss was approximately 6-10%.
EXAMPLE 9 Removal of Small Endotoxin Quantities from Buffer by Means of Non-Specifically Coupled p1220 ml NHS-activated sepharose 4 FastFlow (Amersham Biosciences) were washed firstly with ice cold hydrochloric acid and subsequently incubated with 292 mg p12 (7 mg/ml in 25 mM citrate pH 7.0) for 4 hours at room temperature with agitation. Subsequently, the sepharose was washed with 7×80 ml 5 mM citrate pH 2.0 and respectively 1 ml of the washing fractions was dialysed against 5 mM citrate pH 2.0. These dialysates were used in order to quantify the excess p12 in the washing fractions by means of absorption measurement at 280 nm. A charge density of 8.7 mg p12 per 1 ml sepharose was determined. Non-reacted NHS radicals were neutralised by 12 h incubation of the sepharose with 1M tris pH 8.0. Columns with 2 ml volume were filled with this column material and this was stored until use at 4° C. in 20% ethanol.
In 3 parallel tests, respectively 4 ml endotoxin solution (S) were applied onto a column. The endotoxin solution comprised endotoxin from E. coli O55:B5 (Charles-River Endosafe, Charleston, USA) in equilibration buffer (20 mM hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5). The endotoxin concentration of this solution was 4.6 EU/ml.
The column was rinsed firstly with 12 ml regeneration buffer (20 mM hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5) and subsequently with 12 ml equilibration buffer. Subsequently, equilibration buffer was introduced once again to the column and 1 ml was fractionated.
The endotoxin solution was applied onto the columns (I) and fractions of 5 ml and 2 ml were collected. Subsequently, the column was regenerated with 4 ml regeneration buffer (B). In the throughflow fractions, no endotoxin could be detected, i.e. the endotoxin impurities were able to be removed completely in all three experiments. This example illustrates clearly, that binding occurs also at low endotoxin levels, which is equivalent to a detection of low endotoxin.
EXAMPLE 10 Non-Specific Coupling of Biotinylated p12 to Magnetic Streptavidin Beadsp12 (3 mg/ml in PBS, 0.05% Tween 20) was incubated with sulfo-NHS-LC-LC-biotin (Pierce), in the ratio 1:10 to 1:20 for 1 hour at RT and subsequently was dialysed against buffer (e.g. PBS or 20 mM hepes, 150 mM NaCl, 5 mM EDTA, pH 7.5). NHS-activated biotin binds thereby to primary amino radicals of p12. Subsequently 50 μl biotinylated p12 (1 mg/m1) were added to 1 ml streptavidin beads (MagPrep streptavidin beads, Merck), were agitated at room temperature for 2 h and subsequently excess p12 was removed by washing four times with 1.5 ml 20 mM tris, 10 mM EDTA, pH 7.5.
The endotoxin removal was tested with buffer (20 mM hepes, 150 mM NaCl, pH 7.5) and protein solutions (0.1 mg/ml BSA, 0.1 mg/ml lysozyme, 0.1 mg/ml carbonic anhydrase in 20 mM hepes, 150 mM NaCl, pH 7.5). The buffer and the BSA and lysozyme solution was spiked with 5 EU/ml (endotoxin from E. coli O55:B5, Charles-River Endosafe, Charleston, USA). The carbonic anhydrase solution contained approximately 1 EU/ml. 25 μl magnetic beads with immobilised p12 were added to 200 μl buffer or protein solution, mixed by pipetting up and down and were incubated for 30 min at room temperature. The beads were removed from the solution by means of a magnet, the residue was pipetted off. The endotoxin content of untreated samples and samples incubated with beads was subsequently determined with the LAL test and the protein retrieval was determined by absorption measurement at 280 nm. The endotoxin could be practically completely removed from the buffer (99.9% endotoxin removal) and the endotoxin was depleted also from the protein solution by 70-92%. The protein retrieval was between 57% and 99% (BSA: 87%, carbonic anhydrase: 99%, lysozyme: 57%;
p12 (3 mg/ml in PBS, 0.05% Tween20) was incubated with sulfo-NHS-LC-LC-biotin (Pierce), in the ratio 1:10 to 1:20 for one hour at RT and subsequently dialysed against buffer (e.g. PBS or 20 mM hepes, 150 mM NaCl, 5 mM EDTA, pH 7.5). NHS-activated biotin thereby binds to primary amino radicals of p12. The biotinylated p12 is subsequently incubated for 1 h at room temperature with chromatography material laden with streptavidin (ImmunoPure immobilised streptavidin: 6% cross-linked agarose beads) and excess p12 is removed by washing with PBS.
The endotoxin removal was tested with buffer (20 mM tris, 150 mM NaCl, pH 8.0) and BSA (0.5 mg/ml in 20 mM tris, 150 mM NaCl, pH 8.0). Respectively 1 ml buffer or BSA solution was spiked with 10 EU/ml, 50 μl p12 agarose was added, agitation took place for 1 hour at room temperature. The p12 agarose was centrifuged off subsequently and the endotoxin- and protein concentration in the residue was measured. 99% endotoxin could be removed from the buffer and 86% from the BSA solution. BSA was retrieved up to 90%.
EXAMPLE 12 Tests via p12 Endotoxin Binding by Means of Surface Plasmon Resonance MeasurementsBinding of p12 to endotoxin or to bacteria via the liposaccharides in the outer cell membrane was tested by means of surface plasmon resonance measurements (Biacore J). In order to determine the dissociation constant (Kd), endotoxin from E. coli O55:B5 (Sigma) was immobilised on a hydrophobic HPA chip corresponding to the instructions of the producer and p12 was injected in various concentrations (
In order to test the binding of bacteria to p12, biotinylated p12 was immobilised on streptavidin chips and various E. coli strains were injected. The bacteria were absorbed in PBS for the measurements. E. coli strains were used which have lipopolysaccharides with different polysaccharide components. The polysaccharide part comprises a “core” region which is cross-linked to the lipid A and to the so-called O antigen. The O antigen varies very greatly between different types of bacteria and also strains of bacteria, whilst the “core” region is highly preserved. Strains, which have the “core” region and Oantigen (e.g. E. coli), and strains which have a complete “core” region (E. coli D21), were bonded by p12, whilst strains with a greatly shortened “core” region (e.g. E. coli D21f2) were no longer detected by p12 (
Construction of p12 with N-terminal Strep-tag (N-strep-p12): by means of PCR, the nucleotide sequence for the Strep-tag (U.S. Pat. No. 5,506,121) was introduced to the 5′ end of the T4p12 gene. A primer was constructed for this purpose for the 5′ end of the p12 gene (5′ -GAA GGA ACT AGT CAT ATG GCT AGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT AAT AAT ACA TAT CAA CAC GTT-3′ (SEQ ID NO:1), which comprises the nucleotide sequence of the Strep-tag at its 5′ end (italicised in the sequence) and has a restriction interface (NdeI, underlined in the sequence) such that the gene in the right-hand reading grid can be inserted into the expression plasmid. For the 3′ end of the p12 gene, a primer was constructed which introduces, behind the p12 gene, a BamH I restriction interface (italicised in the sequence) (5′-ACG CGC AAA GCT TGT CGA CGG ATC CTA TCA TTC TTT TAC CTT AAT TAT GTA GTT-3′), (SEQ ID NO:2). The PCR was implemented with 40 cycles (1 min 95° C., 1 min 45° C. and 1 min 72° C.). The PCR batch was cut with the restriction endonucleases NdeI and BamHI and the desired fragment was inserted after size fractionation via an agarose gel and elution from the gel into the NdeI and BamHI site of the expression plasmid pET21a. The sequence of the N-strep-p12 gene was checked for its correctness via DNA sequencing. The further steps for the plasmid pNS-T4p12p57 were implemented as described by Burda, M. R. & Miller, S. (Eur J Biochem.1999 265 (2), 771-778) for T4p12p57. The plasmid pNS-T4p12p57 was then transformed into the expression strain BL21(DE3).
2. Insertion of an N-terminal cysteine radical in N-strep-p12 (N-strep-S3C-p12 and N-strep-S14C-p12): the insertion of an N-terminal cysteine radical was implemented as described under 1, two new primers for the 5′ end being constructed for this purpose. There was used for the N-strep-S3C-p12, the primer 5′-GAA GGA ACT AGT CAT ATG GCT TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC AGT AAT AAT ACA TAT CAA CAC GTT-3′ (SEQ ID NO:3), there was used for the N-strep-S14C-p12, the primer 5′-GAA GGA ACT AGT CAT ATG GCT AGC TGG AGC CAC CCG CAG TTC GAA AAA GGC GCC TGT AAT AAT ACA TAT CAA CAC GTT-3′ (SEQ ID NO:4).
3. Purification of N-strep-p12 protein: the E. coli strain BL21(DE3) with the plasmid pNS-T4p12p57 was drawn in 2 1 shaker cultures (LB medium with ampicillin 100 μg/ml) up to a OD600 of 0.5-0.7 at 37° C. and the expression of the N-strep-p12-protein was induced by addition of 1 mM IPTG (isopropyl-β-thio-galactopyranoside). After incubation at 37° C. for 4 h, the cells were collected. Collected cells from 10 1 culture were taken up in 50 ml sodium phosphate, 20 mM pH 7.2, 2 mM MgSO4, 0.1 M NaCl, broken up by French press treatment (20,000 psi) three times and subsequently centrifuged off for 30 min at 15,000 rpm (SS34). After washing twice in the same buffer, the N-strep-p12 protein was extracted from the pellet, the pellet was extracted three times by agitation for 30 min in 40 mM trisHCl pH 8.0, 10 mM EDTA, the batch was centrifuged for 30 min at 15,000 rpm (SS34) and the dissolved NS-p12 was stored in the residue at 4° C. The extraction was repeated twice and the combined residues were applied (IBA GmbH Gottingen) onto a StrepTactin affinity column (15 ml), equilibrated with buffer “W” (100 mM trisHCl pH 8, 1 mM EDTA, 150 mM NaCl). After washing with 5 column volumes of buffer “W”, elution took place with three volumes of buffer “W” with 2.5 mM dethiobiotin in buffer “W”. After multiple dialysis against buffer “W” and concentration, the concentration and purity of N-strep-T4p12 was determined via SDS-PAGE and UV spectroscopy (Burda et al. 1999). From 10 litres culture, approximately 100 mg N-strep-T4p12 were thus purified.
Four columns with 0.5 ml volume each were cast with polymyxin B sepharose (detoxi gel, Pierce). Each column was washed with 3 ml sodium phosphate buffer (20 mM sodium phosphate, pH 12.0) and 3 ml regeneration buffer (20 mM Hepes, 150 mM NaCl, 2 mM EDTA, pH 7.5). Subsequently, on two of these columns 1 ml LPS per column of E. coli O55:B5 was loaded (0.1 mg/ml in Hepes buffer, 106 EU/ml). The two other columns were each rinsed with 1 ml regeneration buffer. After that all columns were each washed with 3 ml equilibration buffer (20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5) and subsequently 1 ml of this buffer was loaded once more and eluted as fraction 1. Afterwards 0.5 ml of a solution with the bacteriophage tail protein p12 (0.844 mg/ml in 20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2) was loaded onto the columns and washed with 2.5 ml equilibration buffer and 2 ml regeneration buffer. The flow through was collected in fractions of three times 1 ml and once 2 ml, and the concentration of the bacteriophage tail protein p12 in these fractions was determined by means of absorption measurement at 280 nm (
In the used T4p12 mutant the amino acid tryptophan at positions 359 and 283 is substituted by tyrosine For the fluorescence analysis a polysaccharide (MW=2 kDa) derived from the endotoxin of Salmonella typhimurium, was used. The p12 mutant (40 or 200 μg/ml) as well as the endotoxin polysaccharide were dissolved in 5 mM citrate pH 2. The fluorescence in the range of 305-450 nm was measured at an excitation of 295 nm. Of 120 μl of a solution with the p12 mutant W359Y W283Y provided in a fluorescence cuvette, the fluorescence was measured and subsequently polysaccharide was added stepwise (final concentration: 0.5-120 000 nM) and after mixing the sample the fluorescence was measured anew. In control experiments the same experiment was carried out without the p12 mutant and the measured curves were corrected with this data.
The binding of endotoxin or of an endotoxin polysaccharide reduces the fluorescence of this mutant (
For the detection lipopolysaccharides are bound first from a sample solution to a surface and subsequently these immobilised lipopolysaccharides are detected by means of a second protein binding to the lipopolysaccharides. To illustrate the feasibility of this detection method, such a “sandwich” was constructed on the surface of a Biacore chip and the binding of lipopolysaccharide and a lipid A antibody was monitored by means of surface plasmon resonance measurement.
For this purpose first of all T4p12 was covalently coupled to the surface of a CM-5 chip (Biacore). The carboxyclic residues on the surface were activated by a mixture of EDC (N-(3-dimethylaminopropyl-)carbodiimide hydrochloride, 75 mg/ml) and NHS (N-hydroxysuccinimide, 11.5 mg/ml), and subsequently T4p12 was injected, the primary amino residues of which reacted with the activated surface. The surface of a second reference chamber remained untreated. The binding of 100 μl lipopolysaccharide (E. coli O55:B5, 0.1 mg/ml) to T4p12 could be monitored by the increase of the resonance signal (see
The experiments were performed in the following buffer: 20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5).
EXAMPLE 17 Immobilisation of Lipopolysaccharide via T2p12 and Detection of the Binding via an Anti Lipid A AntibodyT2p12 was cloned, expressed and purified in analogy to T4p12. For the detection lipopolysaccharides of a sample solution are bound first onto a surface and subsequently these immobilised lipopolysaccharides are detected by means of a second protein binding to lipopolysaccharides. To illustrate the feasibility of this detection method, such a “sandwich” was constructed on the surface of a Biacore chip and the binding of lipopolysaccharide and a lipid A antibody was monitored by means of surface plasmon resonance measurement.
For this purpose first of all T2p12 was covalently coupled to the surface of a CM-5 chip (Biacore). The carboxyclic residues on the surface were activated by a mixture of EDC (N-(3-dimethylaminopropyl-)carbodiimide hydrochloride, 75 mg/ml) and NHS (N-hydroxysuccinimide, 11.5 mg/ml), and subsequently T2p12 was injected, the primary amino residues of which reacted with the activated surface. The surface of a second reference chamber remained untreated. The binding of 100 μl lipopolysaccharide (E. coli O55:B5, 0.1 mg/ml) to T2p12 could be monitored by the increase of the resonance signal. Even after the end of the injection the resonance signal remained on its level thereby indicating a stable binding. Subsequent injections of an anti lipid A antibody (2.1 μg/ml, polyclonal antibody against goat lipid A from Accurate Chemical & Scientific Corporation, product number YVS6921) also indicated an increase of the resonance signal thereby indicating the binding of the antibody, as well. The resonance signal could be lowered to its initial level by injection of EDTA (2 mM), which abrogates the binding of lipopolysaccharide to T2p12. This means, that the signal increase, induced by the injection of the antibody, was due to the binding to lipopolysaccharide. The experiments were performed in the following buffer: 20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5).
EXAMPLE 18 Immobilisation of Lipopolysaccharide via RB69p12 and Detection of the Binding via an Anti Lipid A AntibodyRB69p12 was cloned, expressed and purified in analogy to RB69p12. For the detection lipopolysaccharides of a sample solution are bound first onto a surface and subsequently these immobilised lipopolysaccharides are detected by means of a second protein binding to lipopolysaccharides. To illustrate the feasibility of this detection method such a “sandwich” was constructed on the surface of a Biacore chip and the binding of lipopolysaccharide and a lipid A antibody was monitored by means of surface plasmon resonance measurement.
For this purpose first of all RB69p12 was covalently coupled to the surface of a CM-5 chip (Biacore). The carboxyclic residues on the surface were activated by a mixture of EDC (N-(3-dimethylaminopropyl-) carbodiimide hydrochloride, 75 mg/ml) and NHS (N-hydroxysuccinimide, 11.5 mg/ml), and subsequently RB69p12 was injected, the primary amino residues of which reacted with the activated surface. The surface of a second reference chamber remained untreated. The binding of 100 μl lipopolysaccharide (E. coli O55:B5, 0.1 mg/ml) to RB69p12 could be treated by the increase of the resonance signal. Even after the end of the injection the resonance signal remained on its level thereby indicatingA stable binding. Subsequent injections of an anti lipid A antibody (2.1 μg/ml, polyclonal antibody against goat lipid A from Accurate Chemical & Scientific Corporation, product number YVS6921) also indicated an increase of the resonance signal thereby indicating the binding of the antibody, as well. The resonance signal could be lowered to its initial level by injection of EDTA (2 mM), which abrogates the binding of lipopolysaccharide to RB69p12. This means, that the signal increase, induced by the injection of the antibody, was due to the binding to lipopolysaccharide. The experiments were performed in the following buffer: 20 mM Hepes, 150 mM NaCl, 0.1 mM CaCl2, pH 7.5).
Claims
1. A method for detection of endotoxin, comprising the steps:
- a) incubation of a sample with a bacteriophage tail protein, and subsequently
- b) detection of endotoxin bound to bacteriophage tail proteins by means of spectroscopic methods, ELISA, chemical or enzymatic detection reaction of endotoxins or cleaved-off endotoxin components, or by means of capacitance measurements.
2. A method according to claim 1, further comprising after step a) and prior to step b) the additional step
- a′) separation of the bacteriophage tail protein-endotoxin complexes from the sample.
3. A method for detection of endotoxin, the method comprising the steps of:
- a) contacting a sample containing endotoxins with a surface, subsequently
- b) incubating of bacteriophage tail proteins with the endotoxin immobilised on the surface, and
- c) detection of bacteriophage tail proteins by means of spectroscopic methods, ELISA, chemical or enzymatic detection reaction of endotoxins or cleaved-off endotoxin components, or by means of capacitance measurements.
4. A method according to claim 3 further comprising after step b) and before step c) an additional step
- b′) separation of said bound bacteriophage tail proteins from endotoxin.
5. A method according to claim 1, wherein the bacteriophage tail protein is a protein of the short bacteriophage tail fiber or a coat protein of bacteriophages without tail.
6. A method according to claim 5, wherein the protein of the short bacteriophage tail fiber is selected from K3, T2, T4, Ox2, RB32-33, AR1, PP01 and RB69.
7. A method according to claim 6, wherein the bacteriophage tail protein has a homology of at least 60% to T4p12 protein on the amino acid level.
8. A method according to claim 1, wherein the bacteriophage tail proteins are modified.
9. A method according to claim 1, wherein the bacteriophage tail proteins are covalently linked to enzymatically active proteins.
10. A method according to claim 1, wherein the bacteriophage tail protein comprises a strep-tag or a his-tag.
11. A method according to claim 10, wherein the tag comprises an amino acid sequence according to SEQ ID NOS 5, 6 or 7.
12. A method according to claim 11, wherein the p12-protein of phage T4, K3, T2, Ox2, RB32-33, AR1, PP01 or RB69 is used as bacteriophage tail protein.
13. A method according to claim 1, wherein the Ca2+ concentration is in the incubation 0.1 μM to 10 mM and/or the Mg2+ concentration is 0.1 μM to 10 mM.
14. A method according to claim 1, wherein marked endotoxin is displaced from the binding with a bacteriophage tail protein and wherein the marked endotoxin is detected subsequently.
15. An endotoxin detection kit comprising a carrier coated with bacteriophage tail proteins, a container containing a reference endotoxin for measurement of a standard curve, a container with at least one further bacteriophage tail protein or an anti lipid A antibody.
16. An endotoxin detection kit comprising a carrier coated with an endotoxin binding substance, a container containing a reference endotoxin for measurement of a standard curve, and a container with at least one bacteriophage tail protein.
Type: Application
Filed: Feb 20, 2013
Publication Date: Sep 19, 2013
Applicant: HYGLOS INVEST GMBH (Bernried)
Inventors: Roman MEYER (Schmidmuhlen), Michael SCHÜTZ (Kareth-Lappersdorf), Holger GRALLERT (Schriesheim), Renate GRASSL (Regensburg), Stefan MILLER (Regensburg)
Application Number: 13/772,117
International Classification: G01N 33/92 (20060101);
