Methods, Compositions, and Kits for Quantitating Antibodies

Abstract

The present teachings provide methods, compositions, and kits for quantitating antibodies. In some embodiments, the method comprises binding two proximity probes to two binding sites on the target antibody, wherein the proximity probes are comprised of a binding moiety and a thereto coupled oligonucleotide, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody. The binding moieties are allowed to bind to the target antibody and the oligonucleotides interact with each other if they are in close proximity to each other. Quantitating the degree of interaction between the oligonucleotides allows for quantitation of the target antibody.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims a priority benefit under 35 U.S.C. § 119(e) from U.S. Application No. 60/797,460, filed May 3, 2006, the contents of which are incorporated herein by reference.

FIELD

The present teachings relate to methods, compositions, and kits for quantitating antibodies.

INTRODUCTION

Proximity ligation assay (PLA) is an approach for protein quantitation that can use two different binder molecules (proximity probes) to bind to a specific detection target (See for example Fredriksson, S. et al., Nat Biotechnol. 2002; 20(5): 473-77, Gullberg, M., et. al., Proc Natl Acad Sci USA. 2004; 101(22): 8420-24, Gullberg, M., et. al., Curr Opin Biotechnol. 2003; 14: 1-5, Pai, S., Ellington, A. D. and Levy, M., Nuc Acids Res. Oct. 19 2005; 33(18): e162, Landegren, U. and Fredriksson, S., US Patent Application 20020064779, May 30, 2002, Fredriksson, S., US Patent Application 20050003361. Typical binders include polyclonal or monoclonal antibody pairs. Each binder molecule can be conjugated to a specific oligonucleotide. One binder's oligonucleotide can form the “left” side of a real-time PCR amplicon, while the other binder can form the “right” side. When the two binders find and attach to the same target, the left and right oligomers are brought into close proximity. With the addition of a connector oligonucleotide (splint) and ligase enzyme, the left and right oligomers can become ligated and thereby allow for the formation of a complete target for a real-time PCR. Further addition of Taqman reaction components followed by thermocycling generates real-time sequence detection data output.

The present teachings include new methods, compositions, and kits for quantitating antibodies that improve upon and expand the applications for the proximity ligation assay.

SUMMARY

In some embodiments, the present teachings provide a method for quantitating a target antibody in solution, the method comprising; binding two proximity probes to two binding sites on the target antibody, wherein the proximity probes are comprised of a binding moiety and a thereto coupled oligonucleotide, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody; allowing the binding moieties to bind to the target antibody and allowing the oligonucleotide to interact with each other if they are in close proximity to each other; and, quantitating the degree of interaction between the oligonucleotides.

Additional methods, as well as compositions and kits are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts some embodiments of the present teachings for quantitating an antibody.

FIG. 2 depicts some embodiments of the present teachings for quantitating an antibody.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the word “a” or “an” means “at least one” unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents defines a term that contradicts that term's definition in this application, this application controls.

PLA for Antibody Quantitation

In a first aspect, the present teachings provide for the PLA process to quantitate antibodies in test samples. The present teachings provide for the attachment of the left and right oligonucleotides to a proximity probe, such as an antibody or aptamer. Following binding of binding regions of the proximity probes to the antibody of interest, an interaction can occur between the left oligonucleotide and the right oligonucleotide. Detection of this interaction, or a surrogate thereof, allows for the quantitation of the antibody of interest in the test sample.

Thus, the present teachings provide a novel application for the proximity ligation assay in which the quantity of an antibody is obtained. As depicted in FIGS. 1 and 2, the proximity probes can be directed to bind to particular regions of an antibody. Upon interaction of the proximity probes with the antibody, their proximity allows for a splint oligonucleotide to hybridize to an oligonucleotide on each of the proximity probes, thus allowing for their ligation. Amplification of the resulting ligation product, for example in a real-time PCR, allows for the quantitation of the antibody.

Specifically, by using the known epitopes of relevant antibodies proximity ligation can be used to quantify the concentration of the antibody of interest. The protein ligands that interact with specific antibodies can be labeled in two separate groups with single stranded DNA that will be used for proximity ligation as shown in FIG. 1. Because of the structure of antibodies, there are two identical binding regions within close proximity to one another. This would allow for three types of antibody ligand configurations as shown in FIG. 2. Statistically half of the antibodies should interact with the ligands in such a way as to allow for proximity ligation to occur. This will allow antibody concentrations to be measured using the proximity ligation assay. FIG. 2A shows antibody binding only one type of labeled ligand. FIG. 2B shows antibody binding the other type of labeled ligand. FIG. 2C shows antibody binding both types of labeled ligand and the ligation event can take place. The solid line depicts a 3^rd single stranded DNA fragment that has complimentary regions to both fragments (also termed a splint). This splint can bring together the 5′ and 3′ end of the DNA to allow for the ligation event to occur with increased frequency. The ligation product can be amplified, for example by the use of a real-time PCR comprising primers that correspond to sequence encoded in the oligonucleotides that are attached to the proximity probes. Read-out of this reaction can occur for example, on a real-time PCR machine such as the Applied Biosystems 7700.

In some embodiments, the splint oligonucleotide can hybridize to an oligonucleotide, thus allowing for an extension reaction or a digestion reaction, as discussed for example in published US Patent Application US20070026430.

Most relevant antibodies have been characterized and their protein ligands have been mapped. This allows for a large, known database for detection reagents to be used without needing to discover new binding molecules. Thus, one of ordinary skill in the art of molecular biology is empowered by the present teachings to rapidly design assays for the quantitation of a wide variety of antibodies of interest.

Thus, in some embodiments, the present teachings provide a method for quantitating a target antibody in solution, the method comprising; binding two proximity probes to two binding sites on the target antibody, wherein the proximity probes are comprised of a binding moiety and a thereto coupled oligonucleotide, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody; allowing the binding moieties to bind to the target antibody and allowing the oligonucleotide to interact with each other if they are in close proximity to each other; and, quantitating the degree of interaction between the oligonucleotides. In some embodiments, the method further comprises amplifying the interacted oligonucleotides to form an amplification product, and quantitating of amplification product. In some embodiments, the binding moieties of the proximity probes are selected from a protein, such as a monoclonal or polyclonal antibody, lectin, soluble cell surface receptor, combinatorially derived protein from phage display or ribosome display, peptide, carbohydrate, nucleic acid, such as an aptamer, or combinations thereof. In some embodiments, the interaction of the oligonucleotides coupled to the binding moieties is through hybridization to a common splint template and ligation of the oligonucleotides.

In some embodiments, the binding moieties are antibodies and said antibodies each bind to the target antibody via a further antibody having binding specificity for the target antibody, and wherein the binding moieties are directed against the Fc portion of the further antibody.

The present teachings also provide a reaction composition comprising; two proximity probes bound to two binding sites on a target antibody, wherein the proximity probes are comprised of a binding moiety and a thereto coupled oligonucleotide, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody. In some embodiments, the reaction composition further comprises a splint oligonucleotide, wherein the splint oligonucleotide is hybridized to the oligonucleotides of the proximity probes.

Illustrative Application Areas

The methods of the present teachings can be applied in any number of contexts. For example, the detection and quantitation of antibodies arising from various cancers is one area where the present teachings will be appropriate. Numerous proteins, upon the mutation of their underlying DNA sequence, have been found to result in the body's production of antibodies directed thereto. Thus, the present teachings provide a facile method of quantitating such antibodies, and thus detecting cancer of various stages. For example, p53 has been found to result in the production of serum-located antibodies directed to the mutated version. Further, measuring the antibodies to the mutated p53 has been shown to be one effective way of assessing cancer relapse following treatment. Lechpammer et al., Int J Colorectal Dis (2004) 19:114-120. Thus, in one embodiment of the present teachings, a method for detecting a cancer is provided, comprising employing a proximity detection assay to quantitate antibodies to mutated p53 in serum.

It is also known that panel of antigens can provide a more informative data set for assessing various cancers. That is, instead of just measuring a p53, mutated p53, and/or antibody directed to a mutated p53, it can be desirable to measure a plurality of different tumor-associated antigens. Such a panel could include, for example, any of c-myc, p53, cyclin B1, p62, Koc, IMP1, and survivin. See for example Zhang et al., Vol. 12, 136-143, February 2003 Cancer Epidemiology, Biomarkers & Prevention. Thus, in some embodiments, the present teachings can provide a multiplexed approach to querying the antibodies resulting from the body's response to mutated versions of these antigens. For example, the oligonucleotides attached to a particular proximity probe can further comprise a unique identifying zipcode sequence. Thus, a multiplexed proximity detection assay can be performed. Thereafter, aliquots of the resulting ligation product can be placed in distinct wells of a microtitre plate. PCR primer, and/or real-time PCR probes, can be placed in individual wells that correspond to the various antibody-specific zipcodes employed in the proximity detection assay. Thus, the signal resulting from each well can provide a measure of the amount of the corresponding antibody. Illustrative multiplexed PLA approaches are further discussed in WO07005649A2.

Additional Embodiments

More generally, approaches for performing ligation, and cycling ligation reactions, coupled to PCR, can be found in U.S. Pat. No. 6,797,470. Also generally, methods employing ligation following a gap-fill by a polymerase, as well as flap repair, are also contemplated by the present teachings, as can be found described in U.S. Pat. No. 6,511,810 (flaps) and U.S. Pat. No. 6,004,826 (gaps).

According to the present teachings, ligation refers to any number of enzymatic or non-enzymatic reagents capable of joining a linker probe to a target polynucleotide. For example, ligase is an enzymatic ligation reagent that, under appropriate conditions, forms phosphodiester bonds between the 3′-OH and the 5′-phosphate of adjacent nucleotides in DNA molecules, RNA molecules, or hybrids. Temperature sensitive ligases, include, but are not limited to, bacteriophage T4 ligase and E. coli ligase. Thermostable ligases include, but are not limited to, Afu ligase, Taq ligase, Tfl ligase, Tth ligase, Tth HB8 ligase, Thermus species AK16D ligase and Pfu ligase (see for example Published P.C.T. Application WO00/26381, Wu et al., Gene, 76(2):245-254, (1989), Luo et al., Nucleic Acids Research, 24(15): 3071-3078 (1996). The skilled artisan will appreciate that any number of thermostable ligases, including DNA ligases and RNA ligases, can be obtained from thermophilic or hyperthermophilic organisms, for example, certain species of eubacteria and archaea; and that such ligases can be employed in the disclosed methods and kits. Chemical ligation agents include, without limitation, activating, condensing, and reducing agents, such as carbodiimide, cyanogen bromide (BrCN), N-cyanoimidazole, imidazole, 1-methylimidazole/carbodiimide/cystamine, dithiothreitol (DTT) and ultraviolet light. Autoligation, i.e., spontaneous ligation in the absence of a ligating agent, is also within the scope of the teachings herein. Detailed protocols for chemical ligation methods and descriptions of appropriate reactive groups can be found in, among other places, Xu et al., Nucleic Acid Res., 27:875-81 (1999); Gryaznov and Letsinger, Nucleic Acid Res. 21:1403-08 (1993); Gryaznov et al., Nucleic Acid Res. 22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry 25:7423-30 (1986); Luebke and Dervan, Nucleic Acids Res. 20:3005-09 (1992); Sievers and von Kiedrowski, Nature 369:221-24 (1994); Liu and Taylor, Nucleic Acids Res. 26:3300-04 (1999); Wang and Kool, Nucleic Acids Res. 22:2326-33 (1994); Purmal et al., Nucleic Acids Res. 20:3713-19 (1992); Ashley and Kushlan, Biochemistry 30:2927-33 (1991); Chu and Orgel, Nucleic Acids Res. 16:3671-91 (1988); Sokolova et al., FEBS Letters 232:153-55 (1988); Naylor and Gilham, Biochemistry 5:2722-28 (1966); and U.S. Pat. No. 5,476,930. Photoligation using light of an appropriate wavelength as a ligation agent is also within the scope of the teachings. In some embodiments, photoligation comprises oligonucleotides comprising nucleotide analogs, including but not limited to, 4-thiothymidine (s⁴T), 5-vinyluracil and its derivatives, or combinations thereof. In some embodiments, the ligation agent comprises: (a) light in the UV-A range (about 320 nm to about 400 nm), the UV-B range (about 290 nm to about 320 nm), or combinations thereof, (b) light with a wavelength between about 300 nm and about 375 nm, (c) light with a wavelength of about 360 nm to about 370 nm; (d) light with a wavelength of about 364 nm to about 368 nm, or (e) light with a wavelength of about 366 nm. In some embodiments, photoligation is reversible. Descriptions of photoligation can be found in, among other places, Fujimoto et al., Nucl. Acid Symp. Ser. 42:39-40 (1999); Fujimoto et al., Nucl. Acid Res. Suppl. 1:185-86 (2001); Fujimoto et al., Nucl. Acid Suppl., 2:155-56 (2002); Liu and Taylor, Nucl. Acid Res. 26:3300-04 (1998) and on the world wide web at: sbchem.kyoto-u.ac.jp/saito-lab.

Illustrative zipcode teachings can be found in PCT Publication Nos. WO 96/12014 and WO 96/41011 and in European Publication No. EP 799,897; and the algorithm and parameters of SantaLucia (Proc. Natl. Acad. Sci. 95:1460-65 (1998)), U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S. Pat. No. 6,451,525 (referred to as “tag segment” therein); U.S. Pat. No. 6,309,829 (referred to as “tag segment” therein); U.S. Pat. No. 5,981,176 (referred to as “grid oligonucleotides” therein); U.S. Pat. No 5,935,793, (referred to as “identifier tags” therein); and PCT Publication No. WO 01/92579 (referred to as “addressable support-specific sequences” therein). In some embodiments the oligonucleotides of the proximity probes can comprise a zipcode, and the detector probe in a real-time PCR can hybridize to the corresponding zipcode during the reaction.

It will be appreciated that the present teachings contemplate any of a variety of ways of quantitating the interaction of the oligonucleotide attached to the proximity probes. In some embodiments employing a donor moiety and signal moiety, one may use certain energy-transfer fluorescent dyes, for example in real-time PCR approaches. Certain non-limiting exemplary pairs of donors (donor moieties) and acceptors (signal moieties) are illustrated, e.g., in U.S. Pat. No. 5,863,727, U.S. Pat. No. 5,800,996, and U.S. Pat. No. 5,945,526. Use of some combinations of a donor and an acceptor have been called FRET (Fluorescent Resonance Energy Transfer). In some embodiments, fluorophores that can be used as detector probes include, but are not limited to, rhodamine, cyanine 3 (Cy 3), cyanine 5 (Cy 5), fluorescein, Vic™, Liz™, Tamra™, 5-Fam™, 6-Fam™, and Texas Red (Molecular Probes). (Vic™, Liz™, Tamra™, 5-Fam™, and 6-Fam™ (all available from Applied Biosystems, Foster City, Calif.). In some embodiments, the amount of detector probe that gives a fluorescent signal in response to an excited light typically relates to the amount of nucleic acid produced in the amplification reaction, and correspondingly the quantity of target antibody. Thus, in some embodiments, the amount of fluorescent signal is related to the amount of product created in the amplification reaction. In such embodiments, one can therefore measure the amount of amplification product by measuring the intensity of the fluorescent signal from the fluorescent indicator. According to some embodiments, one can employ an internal standard to quantify the amplification product indicated by the fluorescent signal, as for example in U.S. Pat. No. 5,736,333. Devices have been developed that can perform a thermal cycling reaction with compositions containing a fluorescent indicator, emit a light beam of a specified wavelength, read the intensity of the fluorescent dye, and display the intensity of fluorescence after each cycle. Devices comprising a thermal cycler, light beam emitter, and a fluorescent signal detector, have been described, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670, and include, but are not limited to the ABI Prism® 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), the ABI GeneAmp® 5700 Sequence Detection System (Applied Biosystems, Foster City, Calif.), the ABI GeneAmp® 7300 Sequence Detection System (Applied Biosystems, Foster City, Calif.), and the ABI GeneAmp® 7500 Sequence Detection System (Applied Biosystems). In some embodiments, each of these functions can be performed by separate devices. For example, if one employs a Q-beta replicase reaction for amplification, the reaction may not take place in a thermal cycler, but could include a light beam emitted at a specific wavelength, detection of the fluorescent signal, and calculation and display of the amount of amplification product. In some embodiments, combined thermal cycling and fluorescence detecting devices can be used for precise quantification of target nucleic acid sequences in samples. In some embodiments, fluorescent signals can be detected and displayed during and/or after one or more thermal cycles, thus permitting monitoring of amplification products as the reactions occur in “real time.” In some embodiments, one can use the amount of amplification product and number of amplification cycles to calculate how much of the target nucleic acid sequence was in the sample prior to amplification. In some embodiments, one could simply monitor the amount of amplification product after a predetermined number of cycles sufficient to indicate the presence of the target nucleic acid sequence in the sample. One skilled in the art can easily determine, for any given sample type, primer sequence, and reaction condition, how many cycles are sufficient to determine the presence of a given target polynucleotide. As used herein, determining the presence of a target antibody can comprise identifying it, as well as optionally quantitating it. In some embodiments, the amplification products can be scored as positive or negative as soon as a given number of cycles is complete. In some embodiments, the results may be transmitted electronically directly to a database and tabulated. Thus, in some embodiments, large numbers of samples can be processed and analyzed with less time and labor when such an instrument is used. In some embodiments, different detector probes may distinguish between different oligonucleotides, and hence different target antibodies. A non-limiting example of such a probe is a 5′-nuclease fluorescent probe, such as a TaqMan® probe molecule, wherein a fluorescent molecule is attached to a fluorescence-quenching molecule through an oligonucleotide link element. In some embodiments, the oligonucleotide link element of the 5′-nuclease fluorescent probe binds to a specific sequence of an identifying portion or its complement. In some embodiments, different 5′-nuclease fluorescent probes, each fluorescing at different wavelengths, can distinguish between different amplification products within the same amplification reaction. For example, in some embodiments, one could use two different 5′-nuclease fluorescent probes that fluoresce at two different wavelengths (WL_A and WL_B) and that are specific to two different target-identifying portions of two different extension reaction products (A′ and B′, respectively). Amplification product A′ is formed if target nucleic acid sequence A is in the sample, and amplification product B′ is formed if target nucleic acid sequence B is in the sample. After amplification, one can determine which specific ligated oligonucleotides are present in the sample based on the wavelength of signal detected and their intensity. Thus, if an appropriate detectable signal value of only wavelength WL_A is detected, one would know that the sample includes ligated nucleotide A, and hence target antibody A, but not ligated oligonucleotide B, and hence not target antibody B. If an appropriate detectable signal value of both wavelengths WL_A and WL_B are detected, one would know that the sample includes both target antibody A and target antibody B. In some embodiments, detection can occur through any of a variety of mobility dependent analytical techniques based on differential rates of migration between different analyte species. Such mobility dependant analysis techniques can be employed for example when mobility modifiers, and/or labels, are included on at least one of the primers in the PCR. Exemplary mobility-dependent analysis techniques include electrophoresis, chromatography, mass spectroscopy, sedimentation, e.g., gradient centrifugation, field-flow fractionation, multi-stage extraction techniques, and the like. In some embodiments, mobility probes can be hybridized to amplification products, and the identity of the target polynucleotide determined via a mobility dependent analysis technique of the eluted mobility probes, as described for example in Published P.C.T. Application WO04/46344 to Rosenblum et al., and WO01/92579 to Wenz et al., and U.S. Pat. No. 6,759,202. In some embodiments, detection can be achieved by various microarrays and related software such as the Applied Biosystems Array System with the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer and other commercially available array systems available from Affymetrix, Agilent, Illumina, and Amersham Biosciences, among others (see also Gerry et al., J. Mol. Biol. 292:251-62, 1999; De Bellis et al., Minerva Biotec 14:247-52, 2002; and Stears et al., Nat. Med. 9:140-45, including supplements, 2003). It will also be appreciated that detection can comprise reporter groups that are incorporated into the reaction products, either as part of labeled primers or due to the incorporation of labeled dNTPs during an amplification, or attached to reaction products, for example but not limited to, via hybridization tag complements comprising reporter groups or via linker arms that are integral or attached to reaction products. Detection of unlabeled reaction products, for example using mass spectrometry, is also within the scope of the current teachings. Further, it will be appreciated that detection of a target polynucleotide includes detecting surrogates of the ligation product. Some examples of a surrogate include but are not limited to, a reporter group that was cleaved from a TaqMan® probe during a nuclease assay can be detected and thus indicates that ligation product is present, a labeled amplified ligation product can be detected on an array, and, a mobility probe can be hybridized to a target-identifying portion, eluted, and detected by a mobility dependent analysis technique (see for example U.S. Pat. No. 6,759,202).

Illustrative Experiment

Preparation of PLA Probes-Oligonucleotide Conjugation of Antigen (Proximity Probe)

Two distinct conjugates of the antigen, for example p53 (Calbiochem, #506147), are constructed by reacting different oligonucleotides, oligo A or oligo B, by coupling a thiol modified oligonucleotide to two separate aliquot of purified maleimide-derivatised antigen (Pierce, #22322). The preferred oligonucleotide conjugated products are purified away from free oligonucleotide and unconjugated protein by precipitation and gel filtration. Each is diluted to 1 nM in buffer C (1×PBS, pH7.4, 5 nM EDTA, 0.1% BSA) and stored at 4° C.

Measuring Serum Antibodies Against p53, a Tumor-Associated Antigen-Preparation of Serum

Prior to PLA, serum is diluted 1:100 by adding 1 uL of serum into 99 uL of PBS. Serial dilutions of 1:10 are subsequently prepared for PLA analysis.

PLA Procedure for Antibody Quantitation

PLA is carried out by first adding 1 uL of each diluted serum sample to 4 uL antigen probe mix (A and B), which is prepared in buffer (for a final probe concentration of 30 pM final for each probe), and is incubated at 37 C for 5 hours. The buffer contains 1×PBS, pH7.4, 1% BSA, 16 ug/ml poly A, 1 mM biotin. 45 uL of ligation/PCR mix containing 50 mM KCl, 20 mM Tris, pH8.3, 2.5 mM MgCl₂, 200 uM dNTPs, 80 uM ATP, 400 nM connector (splint) oligo, 500 nM forward primer, 500 nM reverse primer, 200 nM probe, 0.4 units T4 DNA ligase (New England Biolabs), 1.5 units Platinum Taq (Invitrogen), and passive reference ROX (Applied Biosystems) is then added and the mixture is incubated at 37 C for 5 minutes before being cooled to 4 C for 5 minutes. 10 uL aliquots of the proximity ligation product are transferred to individual wells of a 384-well PCR plate and real-time quantitative PCR is performed according to the following cycling parameters: 95 C 2 min, 40 cycles of 95 C 15 sec, 60 C 60 sec, with a 7900HT instrument (Applied Biosystems). Lower C_T values when higher amounts of antibody are present in serum is observed, showing successful antibody quantitation.

Kits

The instant teachings also provide kits designed to expedite performing certain of the disclosed methods. Kits may serve to expedite the performance of certain disclosed methods by assembling two or more components required for carrying out the methods. In certain embodiments, kits contain components in pre-measured unit amounts to minimize the need for measurements by end-users. In some embodiments, kits include instructions for performing one or more of the disclosed methods. Preferably, the kit components are optimized to operate in conjunction with one another.

In some embodiments, the present teachings provide a kit for quantitating one or more target antibodies in solution comprising; a pair of proximity probes comprising binding moieties with affinity for the target antibody and each provided with an oligonucleotide capable of interacting with each other, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody. In some embodiments, the kit further comprises a ligase and a splint template for joining the oligonucleotides. In some embodiments, the kit further comprises PCR primers which correspond to each of the oligonucleotides. In some embodiments, the kit comprises a first pair of binding moieties being a first pair of antibodies with affinity for the target antibody; and a second pair of binding moieties being a second pair of antibodies directed against the Fc portion of the first pair of antibodies.

Although the disclosed teachings have been described with reference to various applications, methods, and kits, it will be appreciated that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are provided to better illustrate the present teachings and are not intended to limit the scope of the teachings herein. Certain aspects of the present teachings may be further understood in light of the following claims.

Claims

1. A method for quantitating a target antibody in solution, the method comprising;

binding two proximity probes to two binding sites on the target antibody, wherein the proximity probes are comprised of a binding moiety and a thereto coupled oligonucleotide, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody;

allowing the binding moieties to bind to the target antibody and allowing the oligonucleotide to interact with each other if they are in close proximity to each other; and,

quantitating the degree of interaction between the oligonucleotides.

2. A method according to claim 1 further comprising amplifying the interacted oligonucleotides to form an amplification product, and quantitating of amplification product.

3. A method according to claim 1, wherein the binding moieties of the proximity probes are selected from a protein, such as a monoclonal or polyclonal antibody, lectin, soluble cell surface receptor, combinatorially derived protein from phage display or ribosome display, peptide, carbohydrate, nucleic acid, such as an aptamer, or combinations thereof.

4. A method according to claim 1, wherein the interaction of the oligonucleotides coupled to the binding moieties is through hybridization to a common splint template and ligation of the oligonucleotides.

5. A kit for quantitating one or more target antibodies in solution comprising;

a pair of proximity probes comprising binding moieties with affinity for the target antibody and each provided with an oligonucleotide capable of interacting with each other, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody.

6. The kit according to claim 5 further comprising a ligase and a splint template for joining the oligonucleotides.

7. The kit according to claim 5 further comprising PCR primers which correspond to each of the oligonucleotides.

8. A reaction composition comprising;

two proximity probes bound to two binding sites on a target antibody, wherein the proximity probes are comprised of a binding moiety and a thereto coupled oligonucleotide, wherein the binding moities of each proximity probe are the same, and wherein the binding sites for the binding moieties of the proximity probes are on one and the same target antibody.

9. The reaction composition according to claim 8 further comprising a splint oligonucleotide, wherein the splint oligonucleotide is hybridized to the oligonucleotides of the proximity probes.