RU2102080C1 - Method of detection of high-affinity poly- and monoclonal antibodies raised to water-insoluble haptens - Google Patents

Abstract

FIELD: biotechnology, immunology. SUBSTANCE: method involves incubation of an antibody-containing examined sample with radiolabeled oligonucleotide which is condensed preliminary with the corresponding polycondensed aromatic or heterocyclic compound of the general formula O-S-R where R - the corresponding polycondensed aromatic or heterocyclic compound; S - diaminospacer, and O - radiolabeled oligonucleotide at mole ratio O : 1. EFFECT: simplified method of analysis. 6 dwg

Description

 The invention relates to biotechnology, and in particular to methods for the immunochemical determination of haptens (xenobiotics) in samples of biological origin and environmental objects, and can be used to detect high-affinity poly- and monoclonal antibodies to water-insoluble polycondensed aromatic and heterocyclic compounds.

 One of the most important characteristics of analytical systems is sensitivity. The sensitivity of immunoassay is limited by the binding constant of specific antibodies to the antigen, which determines the minimum concentration of immunoreagents at which an immune complex can still form. Thus, the identification of high-affinity antibodies is an important stage in the development of highly sensitive immunoassay systems.

 The analysis of antibodies to water-insoluble haptens is complicated by the fact that when water-insoluble haptens are transferred to aqueous solutions by known methods, the hapten molecules are physically connected to each other in the composition of water-soluble complexes. The obtained characteristics of the binding of antibodies to such water-soluble hapten complexes do not reflect real antibody binding constants, since both antigen-binding sites of the antibody and hapten molecules in the composition of water-soluble complexes can simultaneously participate in the binding of such hapten complexes have completely different physical characteristics.

A solid-phase enzyme-linked immunosorbent method for detecting antibodies is known, which includes the preparation of carrier proteins with covalently attached hapten, adsorption of a protein carrier with covalently attached hapten molecules on the solid phase (on the walls of test tubes or immunological tablets), incubation of the test sample of antibodies with adsorbed antigen, detection of antibodies, associated with an adsorbed antigen using reagents used to determine immunoglobulins (anti-specific antibodies, protein A, etc. .) [1]
Solid-phase immunoanalytic methods can detect antibodies to the analyzed compounds, however, since the reaction of the interaction of antibodies with the antigen does not take place in solution, but on the solid phase, these methods do not allow the determination of real antibody binding constants and are not suitable for the detection of high-affinity antibodies.

 The prototype closest to the proposed method is a method for detecting antibodies by competitive solid-phase immunoassay, which includes the following steps [2] conjugation of activated haptens derivatives with a protein carrier and isolation of modified proteins, adsorption of a protein carrier with covalently attached hapten molecules on the solid phase (on the walls of test tubes or immunological tablets), followed by washing the substance which is not bound to the solid phase, dissolving the hapten in an organic solution barely, drying the solution on the walls of the tube and transferring the hapten to a water-soluble complex by prolonged sounding in the detergent solution, incubating the test sample of antibodies with adsorbed antigen and various concentrations of the hapten in the form of a water-soluble complex with detergent, followed by washing from unreacted components, incubation with reagents, incubation with reagents, antibodies associated with an adsorbed antigen, determining the concentration of a specific immune complex by detecting an enzyme signal active reaction and calculation of the binding constant of antibodies to antigen.

 The disadvantage of the prototype is that the process of dissolving haptene in an aqueous solution occurs by embedding a water-insoluble compound in the micelles of the detergent with the formation of a water-soluble complex. After such dissolution, as a rule, several molecules of the analyte are located in one detergent micelle. Due to the large micelle size, both valencies of the antibody are able to interact with the micelle, and this interaction does not reflect the real binding constant. In addition, micelles are strongly heterogeneous in size, the molecule embedded in the detergent micelle already has other diffusion characteristics, which also does not allow us to estimate the real antibody binding constant.

 The described method for detecting antibodies is multi-stage and is characterized by high complexity.

 The technical task of the invention is to simplify the method for detecting antibodies and providing the ability to detect high affinity antibodies to water-insoluble haptens.

 The problem is solved by the proposed method for the detection of high-affinity poly- and monoclonal antibodies to water-insoluble haptens by incubating a test sample containing antibodies with a radioactively labeled oligonucleotide previously condensed with the corresponding water-insoluble polycondensed aromatic or heterocyclic compound.

 The proposed method is as follows.

 The radiolabeled oligonucleotide is condensed with a reactive derivative of a water-insoluble hapten and the reaction product is isolated - a water-soluble conjugate by known methods.

 A test sample containing antibodies is incubated with a conjugate solution.

 The formed immune complex is separated by fractionation with polyethylene glycol and the concentration of the bound fraction is determined by the radioactivity of the precipitate.

 Using the proposed method allows to determine the real constant binding of antibodies to water-insoluble hapten, since the reacting substances are in non-aggregated form, in contrast to the prototype, where water-insoluble haptens are embedded in micelles of detergents.

 The proposed method contains a smaller number of stages, each stage takes less time than in the prototype and contains fewer operations, so the method is faster and less time consuming.

 In addition, conjugation of a radiolabeled oligonucleotide with a hapten significantly facilitates the detection of the immune complex and makes the detection procedure more reproducible, since no additional reagents are used to detect the immune complex and there is no enzymatic reaction stage, but the physical quantity is measured directly - radioactivity independent of the chemical composition of the medium temperature, etc.

 The defining significant difference of the proposed method is the preliminary condensation of a water-insoluble hapten with an oligonucleotide, which results in a water-soluble conjugate with a hapten: oligonucleotide molar ratio of unity, capable of reacting with antibodies, which simplifies the known method and makes it possible to detect high affinity antibodies to water. When using the hapten: oligonucleotide ratio less than one, the sensitivity of the analytical system deteriorates, since only a part of the hapten binding to antibodies is radioactively labeled, and when the hapten: oligonucleotide ratio is more than one, the hapten molecules are physically bound, the binding of one antigen molecule by the antibody affects the ability of other molecules to be recognized by antibodies, in addition, difficulties arise with the dissolution of such conjugates and the determination of the degree of modification of the oligon cleotide.

 The previously described methods do not allow us to evaluate real constants, since the binding reaction takes place in the heterophase system and the presence of two binding centers in antibodies significantly increases the apparent antibody binding constant.

 Currently, oligonucleotide compounds with certain heterocyclic and aromatic compounds are described that are obtained by condensation of activated derivatives of these compounds with 5 terminal phosphate via spacers containing amino groups [3, 4] However, such modified oligonucleotides have not been used to detect high affinity antibodies. Only their use is described to increase the strength of the complementary complex of the oligonucleotide with the matrix.

 The use of oligonucleotide compounds with water-insoluble polycondensed aromatic and heterocyclic compounds to increase the solubility of the compounds and the presentation of water-insoluble compounds to antibodies is not obvious, since it follows from the above studies that the hydrophobic part of the conjugate is in complex with the nitrogenous bases of the oligonucleotides, it is not exposed to the solution and not able to interact with antibodies.

 Thus, due to the fact that in the known scientific, technical and patent sources, information about the claimed method is not found, we can conclude that the proposed technical solution meets the criteria of "novelty" and "inventive step".

 The invention is illustrated by the following examples.

 Obtaining conjugates of oligonucleotides with water-insoluble polycondensed aromatic and heterocyclic compounds.

 Example 1. For the synthesis of conjugate take 1,6 O.E. (50 μg) of sixteen-link oligonucleotide with radioactive 5 terminal phosphate and specific radioactivity 0.5-1 mKu / O.E, purified by polyacrylamide gel electrophoresis.

The oligonucleotide is converted to cetavlon salt. For this, 2 μl of an 8% solution of cetavlon is added to 10 μl of a solution of oligonucleotide in distilled water, the cetavlon salt of the oligonucleotide is precipitated by centrifugation, the supernatant is removed, the precipitate is dissolved in 50 μl of methanol and dried under vacuum. The cetavlon salt of the oligonucleotide is dissolved in 50 μl of dimethyl sulfoxide (DMSO), solutions of triphenylphosphine (7.8 mg in 25 μl of DMSO), dipiperidyl disulfide (6.6 mg in 25 μl of DMSO) and 4.8 μl of N-methylimidazole are added. After incubation for 10 minutes at room temperature, 4 μl of triethylamine, 5 μl of diaminopentane are added to the reaction mixture and incubated for another 15 minutes. An oligonucleotide with a covalently attached diamine is precipitated with 10 volumes of acetone with 2% LiCLO ₄ , washed with acetone and dried. The dry precipitate is dissolved in 2.5 μl of water, 47.5 μl of dimethylacetamide (DMA), 30 μl of a solution of 5-fluoro-7- (N-hydroxysuccinimidyl) carboxymethyl-12-methyl-benz [a] anthracene (2 mg / ml) are added DMA), 5 μl of triethylamine and incubated for 1 h at room temperature. The conjugate was reprecipitated with acetone with 2% LiCLO ₄ , washed with acetone, dried and re-dissolved in 20 μl of water. The conjugate was purified by electrophoresis on a 20% polyacrylamide gel with 7 M urea in 50 mM Tris-borate buffer, pH 8.3. The conjugate in the gel was detected by exposure to an X-ray film, the gel with the conjugate was cut out and the conjugate was eluted on DEAE cellulose in 5 mM Tris-borate buffer, pH 8.3. The conjugate was washed with 2 M LiCLO ₄ , reprecipitated with acetone, dried and dissolved in 50 μl of water. Based on the specific radioactivity of the oligonucleotide and the radioactivity of the conjugate solution, the concentration of the conjugate is calculated. The structure of the obtained conjugates of 5-fluoro-7-carboxymethyl-12-methyl-benz [a] anthracene (FMBA) with an oligonucleotide was confirmed by electrophoresis in a 20% polyacrylamide gel in TBE buffer with 8 M urea (Fig. 1) and using antibodies to FMBA (Fig. 2). In FIG. 1. The change in the electrophoretic mobility of the oligonucleotide after the addition of diamine and FMBA was demonstrated. On the 1st track of the radiograph, the initial oligonucleotide and reaction products of the original p (T) ₁₆ with diamine NH ₂ (CH ₂ ) ₅ NH _{2 are} marked, on the 2nd track, the reaction products of the amino derivative oligonucleotide and FMBA N-oxysuccinimide ester.

Example 2. For the synthesis of conjugate take 1,6 O.E. (50 μg), purified by electrophoresis, in a polyacrylamide gel of a sixteen-link oligonucleotide with radioactive 5 terminal phosphate and specific radioactivity of 0.5-1 mKu / O. E. A conjugate of N- (3,7,8-trichlorodibenzo dioxi nyl-1) adipamic acid N-hydroxysuccinimide ester with an oligonucleotide was synthesized and isolated as in Example 1. The formation of a conjugate was detected by the appearance of a product with lower electrophoretic mobility ( Fig. 3) and according to the ability of the conjugate to interact with antibodies to 1-N- (adipo mino) -3,7,8-trichlorodibenzodioxin (TCDD) (Fig. 4). In FIG. Figure 3 shows a gel radioattractograph (20% acrylamide, 8 M urea) after electrophoresis of products obtained at different stages of synthesis (TCDD) of NH (CH ₂ ) ₅ NHp (T) ₁₆ . On the 1st track, the initial p (T) ₁₆ oligonucleotide is plotted; on the 2nd track, the reaction products of p (T) ₁₆ with diamine NH ₂ (CH ₂ ) ₅ NH ₂ ; on the 3rd track, the reaction products of the amino derivative oligonucleotide and N- oxysuccinimide ether TCDD. From FIG. 3 it follows that the addition of diamine occurs almost quantitatively, and TCDD N-oxysuccinimide ester is included in the conjugate by 5-10%. The concentration of the conjugate was determined based on the specific radioactivity of the starting oligonucleotide and the conjugate radioactivity.

 Example 3. Detection of antibodies to FMBA.

 BALB / c mice were immunized with bovine serum albumin pre-modified with 5-fluoro-7- (N-hydroxysuccinimidyl) carboxymethyl-12-methyl-benz [a] anthracene. For analysis, dilutions of immune and non-immune sera are prepared 100, 200, 400, 800, 1600 and 3200 times with a buffer solution of 0.01 M Tris / HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20 (TBS ) To 100 μl of antiserum dilution, 100 μl of conjugate solution (500-fold dilution per TBS) is added, and as a control of non-specific sorption, 100 μl of a solution of the initial radiolabeled oligonucleotide with the same final concentration as the conjugate is added to 100 μl of antiserum. The same plantings are placed on non-immune serum. After incubation for 12 hours at room temperature, 50 μl of a human IgG solution (5 mg / ml) and 800 μl of a 15% solution of polyethylene glycol mv are added to each tube. 6000 on the water. After centrifugation, the supernatant is drained and the radioactivity of the precipitates is determined on a radioactivity counter (RacBetta, LKB). In FIG. 2. presents the dependence of conjugate binding on antiserum concentration. The figure shows that the antibodies of non-immune mouse serum do not bind the conjugate, and for the mouse antiserum immunized with albumin modified with FMBA, a typical growth curve is observed, which indicates that the conjugate contains the FMBA molecule. From the plant curve, it is determined that an immune antiserum at a dilution of 1: 400 binds half of the maximum conjugate binding. In tubes containing 100 μl of antiserum diluted 400 times, 100 μl of conjugate solutions with a final concentration of 6, 3, 1.5, 0.75, 0.375 and 0.187 nmol are added. For each concentration of the conjugate, non-specific sorption is determined by incubating the conjugate with buffer without antiserum. After incubation, precipitation and counting the radioactivity of the precipitates, the antibody binding constant is determined to be 1.12 nmol (Fig. 5).

 Example 4. Identification and determination of the binding constants of antibodies to 1-N- (adipo mino) -3,7,3-trichlorodibenzodioxin (TCDD).

 Immunoanalytic procedures were performed as described in Example 3. To produce antisera, BALB / c mice were immunized with a conjugate of N- (3,7,3-trichlorodibenzodioxi nyl-1) adipamic acid N-hydroxysuccinimide ester with bovine serum albumin. In FIG. Figure 4 shows the growths of the immune and non-immune antisera on a conjugate of dioxin with an oligonucleotide. From the plant curves it follows that the antibodies of the non-immune mouse serum do not interact with the conjugate, and the immune antiserum binds the dioxin conjugate to the oligonucleotide. From the plant curve, it is determined that an immune antiserum at a dilution of 1: 800 binds half of the maximum conjugate binding. Using the conjugate screening on an antiserum diluted 800-fold, the binding constant of antioxidant antibodies is determined to be 0.375 nmol (Fig. 6).

 Sources of information.

 1. Toxicol. Appl. Pharmacol, 50 (1979) 147.

 2. Experimental Toxicology and Chemistry, 7, 859-870, 1988.

 3. Journal of Molecular Recognition, vol. 7, 177-188 (1994).

 4. Bioconjugate Chem. 3, 554-558 (1992).

Claims (1)

 A method for detecting high-affinity poly- and monoclonal antibodies to water-insoluble haptens by incubating the test sample with an antigen, isolating the resulting immune complex, followed by its detection, characterized in that the corresponding water-insoluble polycondensed aromatic or heterocyclic compound precondensed with a radio-nucleotide formula is preliminarily condensed with OSR, where R is the corresponding polycondensed aromatic or hetero an acyclic compound, S is a diaminospacer, O is a radiolabeled oligonucleotide, wherein the molar ratio of O R is unity.

Images