RU2478646C1 - Method for producing high-affinity polyclonal antibodies - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses a method for producing high-affinity polyclonal antibodies. The method involves the stages as follows: a) application of an antibody solution on an affine sorbent with an immobilised antibody at an excess molar quantity of antigen antigen-specific antibodies; b) removal of proteins and low-affinity antibodies; c) removal of the bound medium-affinity antibodies by means of a moderately chaotropic agent, such as LiCl, MgCl₂, glycine, d) elution of the high-affinity antibodies by means of the chaotropic agent, e) removal of the latter and f) if needed, removal of antibody aggregates. The method provides producing the antibodies affinity and specificity of which substantially exceeds those of the polyclonal antibodies included in the state of the art.

EFFECT: use of the antibodies prepared by the method according to the invention enables prominently increasing sensitivity and specificity of immunoassay techniques.

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Description

Technical field

The invention relates to the field of immunology, biotechnology and medical diagnostics, in particular, to a method for producing high-affinity polyclonal antibodies. The use of antibodies obtained in accordance with the method of the present invention can significantly increase the sensitivity and specificity of immunoassay methods, as well as the effectiveness of the diagnosis, prognosis and postoperative monitoring of a number of diseases.

State of the art

Currently used methods of immunological analysis are based on the analysis of the reaction product of two immune agents: the antigen that determines the analyte, and recognizes its antibodies, which has significant specificity / selectivity for the antigen. An antibody is usually a tetramer consisting of two heavy chains (kappa, lambda, alpha, gamma, delta, epsilon or mu) and two light chains (kappa or lambda), each of which is represented in the serum by thousands of different variants that differ in the structure of the antigen-binding plot (variable region), resulting from the recombination of DNA in the cells of the immune system. Each antigen-binding region of an individual antibody recognizes a fragment of the primary or spatial structure of an individual antigen (epitope) or several antigens entering the body from the environment at the same time. Antigen immunization of laboratory animals (such as a mouse, rabbit, goat, camel, etc.) leads to the appearance of antigen-specific antibodies in their blood serum resulting from the proliferation of cells producing chains of antigen-binding immunoglobulins. The blood serum of such laboratory animals (the so-called antiserum) contains dozens of types of polyclonal (i.e., binding to different parts of the antigen) antigen-specific antibodies, mainly related to G immunoglobulins (A. Royt, J. Bryusstoff, D. Mail. Immunology - M .: Mir, 2000).

Purification of antigen-specific antibodies from antiserum allows a quantitative analysis of the presence of antigen in complex protein mixtures using immunoassay methods. In this case, after the addition of antibodies to the sample containing the antigen, non-specific complexes of the antigen and the antibody with the components of the sample are destroyed by washing with chaotropic or other buffers, and the amount of the remaining antigen-antibody complexes is determined using analytical tests based on: 1) conjugates of antibodies with the enzyme, the activity of which leads to a subsequent discoloration of the mixture as a result of the addition of an enzyme to the system of a chromogenic substrate (Ausubel FM et al (eds). Short protocols in molecular biology. 1995, p.10- 40. J. Willey & Sons Inc.); 2) conjugates of an antibody with a DNA reporter, the amplification of which allows to quantify the amount of antigen bound to the antibody by PCR (Sikes HD, Jenison R., Bowman CN Antigen detection using polymerization-based amplification. 2009. Lab Chip, 9, 653-656 )

Antisera obtained after immunization of laboratory animals with an antigen contain both high-affinity antibodies to the antigen (affinity constant, K _a > 10 ⁷ L / mol) and antibodies with medium and low affinity, comprising 90% of the produced antibodies (Notkins AL Polyreactivity of antibody antibodies 2004. Trends in Immunology, 25, 174-9). Since modern methods of purification of polyclonal antibodies do not allow complete removal of low- and mid-affinity antibodies from antiserum, their presence in antibody preparations leads to a sharp decrease in the sensitivity and specificity of the vast majority of immunological tests.

The only method described in the literature for the removal of low affinity antibodies from antiserum allows the preparation of polyclonal antibodies with an average affinity constant of 1.5 × 10 ⁷ L / mol (Shimizu F. et al. Separation of anti-dinitrophenyl antibodies according to affinity by fractionated elution from immunoadsorbent using thyocyanate. 1978. Res. Exp. Med. (Berl.) 173, 165-71). The method includes the following steps:

1) applying antiserum to a sorbent containing immobilized antigen used to immunize animals, the amount of which is in molar excess to the amount of high-affinity antibodies;

2) sequential washing of the column with solutions of 0.5 M, 2 M and 3 M potassium rhodanide (a chaotropic agent that partially destroys the intramolecular and intermolecular hydrogen and ionic bonds of the antigen and antibody);

3) removal of the chaotropic agent by dialysis.

The main disadvantage of this method is that when an antiserum is applied to a sorbent with an immobilized antibody under conditions of excess antigen, all antigen-specific antibodies formed as a result of immunization bind to the sorbent, which greatly complicates the subsequent removal of low- and medium-affinity antibodies.

The present invention aims to overcome this disadvantage of known methods for purifying polyclonal antibodies.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a method for producing high affinity polyclonal antibodies.

The proposed method includes the following stages:

a) applying an antibody solution to an affinity sorbent with an immobilized antigen with a molar excess of antigen-specific antibodies to the antigen;

b) removal of proteins and low-affinity antibodies by washing the sorbent with a buffer;

c) removal of medium affinity antibodies bound to the sorbent by a moderately chaotropic agent;

d) elution from the sorbent of high affinity antibodies with a strong chaotropic agent;

e) removal of a chaotropic agent;

e) if necessary, the removal of aggregates of antibodies.

According to the invention, antisera, immunoglobulins or affinity purified antibodies are used as antibodies. Immunoglobulins are prepared by precipitation, ultrafiltration or gel filtration, or commercially available immunoglobulins are used. Affinity purified antibodies are obtained by affinity chromatography or commercially available affinity purified antibodies are used.

According to the invention, an intact natural or recombinant polypeptide or fragment thereof or a chemically synthesized peptide conjugated to a carrier protein or a denatured natural or recombinant polypeptide or fragment thereof is used as an antigen.

According to the invention, the molar excess of antigen-specific antibodies to the antigen is more than 10: 1, preferably 50: 1, which on average corresponds to 50 ml of antiserum per 3-5 nmol of immobilized antigen.

According to the invention, LiCl, MgCl ₂ or glycine are used as a chaotropic agent; preferably, in step c), 1 M LiCl is used, and in step g), 5 M LiCl.

According to the invention, the chaotropic agent is removed by gel filtration, ultrafiltration or dialysis, preferably the removal of the chaotropic agent and the removal of antibody aggregates is carried out in one step by gel filtration.

A second aspect of the present invention provides high affinity antibodies prepared according to the proposed method, having an affinity constant of more than 10 ⁷ L / mol, preferably 10 ⁹ L / mol or more.

A third aspect of the present invention provides the use of high affinity antibodies obtained according to the proposed method for immunoassay, in particular for enzyme immunoassay, immunofluorescence analysis, immuno-PCR, Western blotting and dot blotting, immunohistochemical analysis and immunoprecipitation.

The invention will now be described in more detail with reference to certain illustrative examples and figures.

List of figures

Figure 1 shows the results of the purification of antibodies to the denatured antigen AKR1B10:

1A is the elution profile obtained in the process of affinity chromatography of antiserum using stepwise elution of antibodies with LiCl solution (OE - relative units);

1B is a gel filtration profile of the combined fractions obtained by elution of an affinity column of 5 M LiCl (OE - relative units).

Figure 2 shows the results of the separation of the combined peak fractions shown in Figure 1A by electrophoresis in 10% denaturing polyacrylamide gel. The numbers above the tracks correspond to: 1 - antibodies obtained after elution of the affinity sorbent 1 M LiCl; 2 - antibodies obtained after elution of the affinity carrier 5 M LiCl; 3 - antibodies obtained after gel filtration of the combined fractions of the first (minor) peak in figure 1; 4 - antibodies obtained after gel filtration of the combined fractions of the second (major) peak, in Fig.1B; 5 - antibodies applied to lane 4 with addition of 50 mM dithiothreitol to the application buffer (for separation of antibody heavy and light chains); 6 - a set of protein standards of molecular weight.

Figure 3 shows the results of Western blotting of total protein extracts of adenocarcinoma (T) and normal tissue (N) of the human colon using antibodies to the AKR1B10 protein obtained by the method of the present invention (A) and commercial antibodies to the same antigen (B). On the left is the position of molecular weight standards in kDa.

Figure 4 shows the results of Western blotting of total protein extracts of adenocarcinoma (T) and normal tissue (N) of the human colon using antibodies to the extracellular part of the EpCAM protein obtained by the method of the present invention (A) and commercial antibodies to the same antigen (B). On the left is the position of molecular weight standards in kDa.

Figure 5 shows the results of dot blotting of indicated amounts of purified recombinant EpCAM protein using antibodies to its extracellular part obtained by the method of the present invention (A) and commercial antibodies to the same antigen (B). On the left is the position of molecular weight standards in kDa.

Disclosure of invention

The objective of the invention is to increase the affinity of antigen-specific polyclonal antibodies.

The problem is solved using the method of producing high-affinity polyclonal antibodies.

The proposed method includes the following stages:

a) applying an antibody solution to an affinity sorbent with an immobilized antigen with a molar excess of antigen-specific antibodies to the antigen;

b) removal of serum proteins and low affinity antibodies by washing the sorbent with a buffer;

c) removal of medium affinity antibodies bound to the sorbent by washing it with a moderately chaotropic agent;

d) elution with a sorbent of high affinity antibodies by a chaotropic agent;

e) removal of a chaotropic agent;

e) if necessary, the removal of aggregates of antibodies.

Antibodies, immunoglobulins or affinity purified antibodies can be used as antibodies according to the invention.

An antiserum can be obtained by immunization with an antigen of laboratory animals such as mouse, rabbit, goat, camel, etc. (Cooper H.M., Paterson Y. Production of polyclonal antisera. 2001. Curr. Protoc. Immunol. Chapter 2: Unit 2.4.).

Alternatively, commercially available antisera can be used.

Immunoglobulins can be obtained by salting out with ammonium sulfate (sodium), precipitation (for example, using polyethylene glycol or caprylic acid), ultrafiltration or gel filtration, and affinity chromatography (in the latter case, sorbents with immobilized proteins A or G or thiophilic adsorption chromatography are used ) (BD Biosciences Clontech. Purification of immunoglobulins. 1998; Vijayalakalashmi MA. Antibody purification methods. 1998. Applied. Biochem. Biotechnol. 75, 93-102.)

Alternatively, commercially available immunoglobulins can be used.

Affinity purified antibodies can be obtained using affinity chromatography, which allows you to separate complex mixtures based on the specific interaction of the protein with its ligand (in this case, the antigen immobilized on the sorbent). In this case, the components of the mixture that were not strongly bound to the sorbent were removed by washing with a buffer, and the antibodies were eluted with acid (pH less than 2.5) or alkaline (pH more than 10) chaotropes (Fitzgerald J, Leonard P, Darcy E, O'Kennedy R. Immunoaffinity chromatography. 2011. Methods Mol Biol. 681, 35-59).

Alternatively, commercially available affinity purified antibodies can be used.

According to the invention, an intact natural or recombinant polypeptide or fragment thereof or a chemically synthesized peptide conjugated to a carrier protein or a denatured natural or recombinant polypeptide or fragment thereof can be used as an antigen.

According to the invention, antigen immobilization on a sorbent can be carried out as a result of the interaction of its amino, carboxyl or thiol groups with activated sorbent groups (for example, the interaction of antigen amino groups with bromine-activated hydroxyl groups of the sorbent), the interaction of periodate oxidized oligosaccharide residues of the natural antigen with hydrazide or hydroxylamine as well as antigen adhesion to microparticles (Kalia J., Raines RT Advances in Bioconjugation. 2010. Curr. Org. Chem. 14, 138-147).

According to the invention, the molar excess of antigen-specific antibodies to the antigen immobilized on the sorbent is more than 10: 1, preferably 50: 1, which corresponds to 50 ml of antiserum per 3-5 nmol of immobilized antigen (depending on the immune response of the laboratory animal used and the antigen immunogenicity). When using immunoglobulins or affinity purified antibodies as a solution of antibodies, the corresponding molar equivalent is used. When calculating the amounts of antiserum (or fractions of immunoglobulins or antibodies purified by other methods) necessary for applying to the sorbent, it should be assumed that the amount of antigen-specific antibodies is on average 10% of the total amount of immunoglobulins of the antiserum or their purified fraction, while high-affinity antibodies on average, 10-20% of the amount of antigen-specific antibodies (Reddy ST et al. Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. 2010. Nat. Biotechnol. 28, 965-71). In the case of low immunogenicity of the antigen used for immunization, the amount of antigen-specific antibodies in the antiserum preparation or fractions of immunoglobulins or antibodies purified by other methods can be preliminarily evaluated by enzyme immunoassay.

The use of a correctly calculated excess of antibodies to the immobilized antigen allows one to minimize the binding of low- and medium-affinity antibodies to the sorbent as a result of competition of these antibodies with high-affinity antibodies. A decrease in this ratio leads to binding of medium- and low-specific antibodies to the sorbent, which significantly complicates their subsequent removal, since some of these antibodies are not removed by moderately chaotropic agents (for example, as a result of the formation of strong hydrophobic bonds with the antigen epitope). The use of higher ratios of antigen-specific antibodies to the antigen reduces the yield of highly specific antibodies as they begin to compete with each other. In addition, the most affinity antibodies in this case cannot be eluted from the sorbent without avoiding their irreversible denaturation.

According to the invention, any chaotropic agent (mainly LiCl, MgCl ₂ or glycine), i.e. a substance that partially or completely destroys the three-dimensional structure of macromolecules as a result of the destabilization of intramolecular hydrogen, hydrophobic or ionic bonds. The composition of chaotropic agents in each case is determined empirically: to destabilize hydrogen bonds, solutions are used that increase the dipole moment of the solvent (for example, 2-8 M urea and 2-6 M guanidine chloride), the breaking of hydrophobic bonds is achieved as a result of the use of detergents (for example, 1% SDS or 1% sodium deoxycholate), while 0.1 M glycine-Na, pH 10.0 (screening of positive charges), as well as 0.1 M glycine with the addition of hydrochloric acid (pH 2, 3) are most often used to destabilize ionic bonds ) and 1 M acetic acid ota (screening of negative charges). For screening charges of both types and the simultaneous destabilization of hydrogen bonds, 3-5 M solutions of LiCl, MgCl ₂ , KCl, KI and potassium thiocyanate are used. The chaotropy of an agent can be changed as a result of a decrease or increase in its concentration (for solutions of salts and detergents) or pH (for alkaline and acid chaotropes), which allows one to obtain “moderate” and “strong” chaotropic agents (Dimitrov JD, Lacroix-Desmazes, Kaveri SV, Vassiliev TL Insight into the mechanism of the acquired antibody auto-reactivity. 2008. Autoimmun. Rev., 7, 410-414).

According to the invention, preferably 1 M LiCl is used at the stage of washing the medium affinity antibodies, and a solution of 5 M LiCl is preferably used at the stage of elution of high affinity antibodies. The use of these chaotropes allows the most softly (i.e., in the absence of irreversible denaturation) screening of ionic bonds and at the same time to destroy hydrogen bonds in antigen-antibody complexes, which allows one to obtain practically intact renatured antibodies with a native three-dimensional structure (according to the method of circular dichroism and constant estimation affinities).

According to the invention, a chaotropic agent after elution of antibodies can be removed by gel filtration, ultrafiltration or dialysis (Janson JC. Protein purification: principles, high resolution methods, and applications. 2011. Wiley series in methods of biochemical analysis, p.1-269) .

According to the invention, it is preferable to simultaneously remove the chaotropic agent and antibody aggregates (which are less active than individual antibodies due to the partial denaturation of their constituent antibodies) using gel filtration. When using ultrafiltration or dialysis, the simultaneous removal of the chaotrope and antibody aggregates becomes impossible, since these methods allow you to remove only substances with a low molecular weight.

The present invention also provides for high affinity antibodies produced by the method of the present invention having an affinity constant of more than 10 ⁷ L / mol, preferably 10 ⁹ L / mol or more. Assessment of the affinity of the antibodies to the AKR1B10 protein obtained by the method of the present invention showed that their average affinity constant (10 ⁹ L / mol) increases by almost a hundred times compared to the original antiserum (2 × 10 ⁷ L / mol) and commercial preparations (3 × 10 ⁷ l / mol) (Table 1). Thus, the method of the present invention can significantly increase the affinity of polyclonal antibodies as a result of their competition with low and medium affinity antigen-specific antibodies when applying antiserum to the sorbent. The number of antibodies obtained by the method of the present invention, on average, is 2-3% of the total number of antigen-specific antibodies contained in the antiserum (which corresponds to 1 mg of antibody per 50 ml of antiserum). However, the resulting preparations do not contain impurity proteins (Figure 2).

Evaluation of the specificity of antibodies (i.e., the absence of their cross-interaction with other antigens) by Western blotting showed that when using antibodies obtained by the method of the present invention to two different antigens (a denatured AKR1B10 protein and a denatured fragment of EpCAM protein) in total protein extracts of the colon were detected only polypeptides of the expected size (36 kDa and 35 kDa, respectively) in the complete absence of cross-reactivity of antibodies with other proteins, whereas commercial an titer to the same antigens under the same conditions revealed several bands, none of which corresponded to the expected size (Figs. 3-4). This result demonstrates a much higher specificity of the antibodies obtained in accordance with the method of the present invention, compared with commercial analogues.

The present invention also provides the use of high affinity antibodies obtained according to the proposed method for immunoassay (in particular, enzyme immunoassay, immunofluorescence analysis, immuno-PCR, Western blotting and dot blotting, immunohistochemical analysis and immunoprecipitation; Ausubel FM et al (eds). Short in molecular biology. 1995, p. 10-40. J. Willey & Sons Inc.).

A significant advantage of using antibodies obtained by the method of the present invention was demonstrated in experiments showing that such antibodies can increase the sensitivity of dot blotting by more than a hundred times compared with antibodies obtained by standard protocols (Figure 5). In this case, the use of antibodies obtained by the method of the present invention allows the detection of 10 pg of pure protein antigen by chemoluminescence of the spot, while the ultimate sensitivity of the test using commercial antibodies is only 1 ng.

Comparison of the results of antigen detection in complex protein mixtures by Western blotting allows us to conclude that the sensitivity of this test in the case of using antibodies obtained by the method of the present invention is more than a thousand times higher than the results obtained using commercial analogs (according to the results of scanning bands on Figure 3-4). The higher specificity of the antibodies obtained by the method of the present invention is a consequence of the removal of low- and medium-specific antibodies, which in many cases are polyreactive.

Finally, the calculations show that the use of antibodies obtained by the method of the present invention for enzyme-linked immunosorbent assay in a sandwich format (one of the most sensitive and popular immunochemical methods) will increase the sensitivity of the test ten thousand times or more. This is due to the fact that the sensitivity of the analysis is proportional to the product of the affinity constants of the capture and detecting antibodies used for specific immobilization of the antigen contained in the analyzed mixture and its quantitative detection, respectively.

Information confirming the possibility of carrying out the invention

The invention is illustrated in detail below with reference to specific examples representing its most preferred embodiments. For the described experiments, two recombinant human proteins were used: the full-sized protein AKR1B10 and the extracellular fragment of the EpCAM protein, including amino acid residues 24-265. Purification of antibodies to both antigens was carried out according to the same protocol, which can be used to purify antibodies to any protein antigen.

Example 1. Immobilization of the denatured protein AKR1B10 on a carrier

Antigen denaturation

To a solution containing 6 nanomoles (200 μg) of antigen in 150 μl of PBS, SDS (final concentration of 2%) and DTT (final concentration of 100 mM) were added, followed by denaturation of the antigen (5 min, 95 ° C). The antigen was transferred to immobilization buffer (0.1 M NaHCO ₃ , 0.5 M NaCl, 0.1% SDS, pH 8.3) by gel filtration on a HiTrap Desalting Column column (GE Healthcare, USA).

Antigen immobilization

0.2 g of CNBr-Sepharose (Sepharose 4 Fast Flow, GE Healthcare) was activated according to the manufacturer's recommendations. The resulting 0.7 ml of activated carrier was incubated with 200 μg of denatured antigen overnight at room temperature. Unbound antigen was removed by washing with immobilization buffer, and the remaining activated sorbent groups were blocked with 0.5 M Tris-HCl, pH 8 overnight at 4 ° C according to the manufacturer's procedure. The sorbent was washed with buffer (0.1 M Tris-HCl pH 8, 0.5 M NaCl), and then with a solution containing 0.1 M acetic acid and 0.5 M NaCl.

Example 2. Immunoaffinity purification of antibodies to the denatured protein AKR1B10

0.4 ml of a suspension of CNBr-sepharose containing 3.2 nanomoles (110 μg) of immobilized antigen was placed in a Tricorn 5/20 column (GE Healthcare, USA) and equilibrated with 5 ml of PBS. A 50 ml antiserum containing an average of 50 mg (300 nanomoles) of antigen antibodies (molar ratio of antibodies to antigen 1: 100) was applied to the column. The deposition rate of antiserum on the carrier was 50 μl / min. After applying the antiserum to the column, it was washed: 1) 12.5 ml of PBS; 2) 2 ml of 1 M LiCl in a buffer containing 10 mM Na ₂ HPO ₄ / NaH ₂ PO ₄ pH 7.2; 3) 6 ml of 5 M LiCl in the same buffer. Protein-containing pooled fractions obtained by washing the affinity column with 1 M LiCl were pooled and concentrated to a volume of 250 μl using an Amicon® Ultra-4 Centrifugal Filter Units ultrafiltration apparatus (10 kDa, Millipore, USA). The pooled fractions obtained by washing the affinity column with 5 M LiCl were treated similarly.

Example 3. Gel filtration of antibodies to the denatured protein AKR1B10

On a Superdex 200 column (10/30, GE Healthcare, USA) equilibrated with PBS, the pooled fractions obtained in Example 2 were sequentially applied after washing the column with 1 M LiCl and 5 M LiCl. Fractions corresponding to the peaks of intact, monomeric antibodies were pooled and concentrated using an Amicon® Ultra-4 Centrifugal Filter Units ultrafiltration apparatus (10 kDa, Millipore, USA).

Example 4. Characterization of purified antibodies: evaluation of their yield, purity, affinity and specificity

The amount of antibodies in the combined fractions of the peak obtained after elution of antibodies from an affinity sorbent of 5 M LiCl was determined by the Bradford method using a commercial kit (Bio-Rad, USA). The analysis results showed that the number of antibodies obtained by the method of the present invention, on average, is 2-3% of the total number of antigen-specific antibodies contained in the antiserum (which corresponds to 1 mg of antibody per 50 ml of antiserum).

The homogeneity of the samples obtained in example 3 was evaluated using electrophoresis in a 10% denaturing polyacrylamide gel, which showed the complete absence of impurity proteins in the preparation (Figure 2).

The affinity of antibodies was assessed by measuring their dissociation constant according to a standard protocol (Friguet B. et al. Measurements of the true affinity constant in solution of antigen-antibody complex by enzyme-linked immunosorbent assay. Journal of Immunological Methods, 1985, 77, 305- 19). The results of the analysis are given in Table 1.

The results of measuring the affinity constant of antibodies to the protein AKR1B10.

Table 1 Source of antibodies Affinity constant According to the method of the present invention 10 ⁹ l / mol Antisera Antibodies 2 × 10 ⁷ l / mol Commercial antibodies 3 × 10 ⁷ l / mol

Antibody specificity was assessed by Western blotting using standard protocols (Ausubel FM et al (eds). Short protocols in molecular biology. 1995, p.10-40. J. Willey & Sons Inc.) using the denatured antibodies obtained by the method of the present invention. protein AKR1B10 and the denatured fragment of the EpCAM protein in total protein extracts of the colon. The analysis revealed the presence of only polypeptides (antigens) of the expected size (36 kDa and 35 kDa, respectively) in the complete absence of cross-reactivity of antibodies with other proteins. At the same time, preparations of commercial antibodies to the same antigens under the same conditions revealed several bands, none of which corresponded to the expected size of the antigen (Figure 3-4).

Comparison of the sensitivity of antigen immunoassay using antibodies obtained by the method of the present invention with commercial analogs was carried out by Western blotting and dot blotting according to standard protocols (Ausubel FM et al (eds). Short protocols in molecular biology. 1995, p.10-40. J . Willey & Sons Inc.). The experiments showed that antibodies obtained by the method of the present invention allow:

1) to increase the sensitivity of Western antigen blotting in complex protein mixtures by more than a thousand times in comparison with commercial antibodies (according to the results of scanning the bands shown in Figures 3-4);

2) increase the sensitivity of dot blotting of pure antigen by more than a hundred times in comparison with commercial antibodies (according to the results of analysis of the chemiluminescence intensity of the spots shown in Figure 5).

All patents, publications, scientific articles, and other documents and materials cited or referenced herein are included in the present description, as if each of these documents was individually included or provided here in its entirety. The above detailed description of the invention and its specific embodiments, given in the Examples with reference to the figures, is intended solely to more fully clarify the essence of the claimed invention, but not to limit it. It will be clear to a person skilled in the art that various changes can be made, which, however, will correspond to the nature and scope of the present invention, which are determined by the attached claims.

Claims (13)

1. The method of obtaining high-affinity polyclonal antibodies, comprising the following stages:
a) applying an antibody solution to an affinity sorbent with an immobilized antigen with a molar excess of antigen-specific antibodies to the antigen;
b) removal of proteins and low affinity antibodies by washing with buffer;
c) removal of bound mid-affinity antibodies by a moderately chaotropic agent;
g) elution of high affinity antibodies with a chaotropic agent;
e) removal of a chaotropic agent;
e) if necessary, the removal of aggregates of antibodies. 2. The method according to claim 1, where the antibodies used are antiserum, immunoglobulins or affinity-purified antibodies that are in molar excess to the antigen. 3. The method according to claim 2, where the immunoglobulins are obtained by precipitation, ultrafiltration or gel filtration, or commercially available immunoglobulins are used. 4. The method according to claim 2, where the affinity purified antibodies are obtained using affinity chromatography or use commercially available affinity purified antibodies. 5. The method according to claim 1, where the intact natural or recombinant polypeptide or a fragment thereof or a chemically synthesized peptide conjugated to a carrier protein is used as an antigen. 6. The method according to claim 1, where the denatured natural or recombinant polypeptide or its fragment is used as an antigen. 7. The method according to claim 1, where the molar excess of antigen-specific antibodies to the antigen is more than 10: 1, preferably 50: 1. 8. The method according to claim 7, where the molar excess of antigen-specific antibodies to the antigen is in the range of 50 ml of antiserum for 3-5 nmol of immobilized antigen. 9. The method according to claim 1, where Liota, MgCl ₂ or glycine are used as a chaotropic agent. 10. The method according to claim 9, where 1 M LiCl is used as the chaotropic agent in step c). 11. The method according to claim 9, where 5 M LiCl is used as the chaotropic agent in step g). 12. The method according to claim 1, where the chaotropic agent is removed using gel filtration, ultrafiltration or dialysis. 13. The method according to claim 1, where the removal of the chaotropic agent and the removal of aggregates of antibodies is carried out in one stage using gel filtration.

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