Synthetic antigen based on copolymers of n-vinylpyrrolidone, crotonic acid and para-crotonoyl aminophenol comprising covalently joined 2-naphthylamine as hapten

FIELD: organic chemistry, polymers, immunochemistry of high-molecular compounds.

SUBSTANCE: invention describes a synthetic hapten based on copolymers of N-vinylpyrrolidone, crotonic acid and p-crotonoyl aminophenol comprising covalently joined 2-naphthyl amine as a hapten of the following general formula:

wherein m = 88.5 mole%; n = 6.8-10.3 mole%, and l = 1.2-4.7 mole%. Invention provides specificity of antibodies raised against 2-naphthylamin as a hapten and possibility for preparing highly specific antibodies raised against carcinogen 2-naphthylamine as a hapten by immunization of rabbits with synthetic antigen comprising a copolymer of N-vinylpyrrolidone, crotonic acid and p-crotonoyl aminophenol as a macromolecular carrier. Also, invention provides possibility for practice using these antibodies for immunological recording 2-naphthylamine as a hapten in blood of industrial workers subjected for exposition with 2-naphthylamine. The proposed compound elicits antigenic features and can be used for detection of oncological risk among workers subjecting with contact with 2-naphthylamine.

EFFECT: valuable properties of antigen.

1 tbl, 1 dwg, 6 ex

 

The invention relates to the immunochemistry of macromolecular compounds possessing antigenic properties.

The invention may find wide use in immunological determination in biological fluids 2-naphtylamine-protein adducts, which are specific markers of occupational cancer risk, resulting in individuals having contact with 2-naphthylamine or substances containing it in the structure of the molecule, for example, aniline dyes.

It is known that 2-naphtylamine, widely used in various industries, is cilnm organotropic carcinogenic, causing people and dogs bladder cancer, and mice - hepatoma (Temkin I.S. bladder Tumors induced carcinogenic amino compounds. - M.: Medicine, 1957 - p.36-47). The most typical for the early stages of chemical carcinogenesis immunometabolic violations include education in the serum and in the target tissue of animals carcinogen-protein antigens (KBA) (Korosteleva T.A. About changes to tissue antigens during experimental carcinogenesis. - L.: Medicine, 1966, s). KBA are detected at early stages of carcinogenesis by the action of the animal carcinogens-ortho-amino-azotoluene, benzidine, 2-naphtylamine, 3-methyldiethanolamine, 3-Oxandrolone acid. KB is detected when the cancer risk in persons, contact with carcinogens (Korosteleva T.A., Belohvostova A.T. the Possibility of using immunological markers for targeting cancer risk in the production contingents (Collection of scientific papers. - Practical and scientific basis for the prevention of carcinogenic effects. - Leningrad, 1984. - c.159).

The original copolymer of N-vinylpyrrolidone (VP) with crotonic acid (QC), used to obtain a ternary copolymer of VP-KK-n-crotonylene and the claimed compounds on the basis described in the literature: Snesarev, Wasreached, Lbottomnav, Rigorous, Timerclose, "copolymerization of crotonic acid and vinyl pyrrolidone"//high-Molecular compounds, I. IX (A), No. 8, s-1813, 1967.

These factors formed the basis for the development of immunological methods for registering 2-naphtylamine a hapten in biological materials as a marker of cancer risk.

Described antigen containing covalently attached to a protein carrier 2-naphtylamine as hapten (Jumps A.P. Study of tissue antigens and antibodies in the early stages of carcinogenesis under the action of 2-naphtylamine. Abstract of Cand. Diss., L., 1970). This antigen is the prototype of the claimed invention. The author used antibodies against 2-naphtylamine-antigen as reagents for the detection of 2-naphtylamine, vhodyashih is in antigenic complexes, formed in the body of animals with the introduction of the carcinogen. Search KBA was given when using the precipitation reaction in the gel.

A disadvantage of the known comprehensive solutions aimed at obtaining synthetic antigen containing covalently attached 2-naphtylamine as hapten, is the need to use as macromolecular carriers serum proteins of various animals that have obselidia antigens. Immunization with antigens containing the remainder of the 2-naphtylamine, attached to a protein, inevitably leads to the formation side of antibodies to observedby antigens, which complicates the qualitative and quantitative interpretation of the final results of the determination of 2-naphtylamine-protein antigens, formed in the body. In this regard, the author of the prototype was forced to put additional reactions depletion observedby antigens.

The technical result of the invention is to improve the specificity of antibodies against 2-naphtylamine as the hapten.

This is achieved by using for the production of antibodies against 2-naphtylamine synthetic antigen, representing isoprostane 2-naphtylamine-based copolymer of N-vinylpyrrolidone with crotonic acid and crotononitrile General formula I:

GDEM=88,5 mol.%; n=6,8÷10,3 mol.%; l=1,2÷4,7 mol.%.

The method of obtaining this antigen is as follows:

1. The amino group of 2-naphtylamine diasterous with getting chloride 2-naphthalenedione. Spend the azo coupling copolymers of N-vinylpyrrolidone with crotonic acid and n-crotononitrile II chloride with 2-naphthalenethiol III in aqueous solution at 0÷5°first in acidic (pH 4.2÷4,6), and then in alkaline (pH 9,0÷9,4) environment.

2. When this molar number of terpolymer (one per link n-crotononitrile) chloride 2-naphthalenedione be 1:2÷2,2. The scheme of synthesis of antigen 1 can be represented as follows:

Analysis of the known level of science and technology has shown a lack of information about the ternary copolymers of General formula 1.

The structure of terpolymer 1 confirmed method PMR spectroscopy. In the PMR spectrum of terpolymer 1, recorded in deuterated dimethylformamide, in the field of 0.5÷5,0 ppm stored signals of the original ternary copolymer of vinylpyrrolidone with crotonic acid and n-crotononitrile (n-KAF). Unlike the PMR spectrum of terpolymer II in the PMR spectrum of the product result, I found intense signal in the area of 7.9 to 8.5 ppm, related to the protons of the benzene nucleus of the link p-KAF, odawise in ortho - and meta-positions to sogrape. The absence of a signal of 6.8 to 7.3 ppm allows to assert that the substitution of the protons on the balance of azo-2-naphtylamine occurred in two places benzene ring link n-KAF. Comparison of the peak areas in the area between 7.4 and 7.8 ppm and in the field of 7.9 to 8.5 ppm in the PMR spectrum of conjugate I allowed to determine the location of the substitution of the protons of the benzene nucleus of link n-KAF for the remainder of the azo-2-naphtylamine. The observed peaks in the spectrum is equal to the area corresponds to only the substitution in positions 2 and 6 of the benzene ring link n-KAF.

To determine the residues of 2-naphtylamine in the product result was shooting its UV spectrum in ethanol in reference to the solution of the original copolymer II. Used the calibration of the D280- C (g/ml), previously set for 2-naphtylamine, in the UV spectrum which has an intense absorption band (lg∑=4,2) with a maximum at 280 nm.

For a better understanding of the essence of the claimed invention, examples of its specific implementation.

A. Preparation of an aqueous solution of a chloride 2-naphthalenedione.

To a suspension of 0.001 kg 2-naphtylamine in a mixture of 0.01 l of distilled water and 0.003 l of concentrated hydrochloric acid, cooled to 0°C, dropwise with stirring, add the cooled solution of 0.58·10-3kg sodium nitrite 0.01 l of water. The obtained clear solution is dark yellow stirred at 0° C for 30 minutes

B. Examples 1-6 on the synthesis of antigen I.

Example 1.

To a cooled to 5°With the solution terpolymer II (m=88,5 mol.%, n=10,3 mol.%, 1=1,2 mol.% with the molecular weight of 11,000) 0.01 liters of water poured dropwise with stirring to 1.5·10-3l the obtained chloride 2-naphthalenedione containing 0,06·10-6kg diazonium salts, and the addition of dry sodium bicarbonate bring the solution pH to 4.3. The mixture is stirred for 30 minutes and bring the pH of the reaction solution to 9.2 using 5% NaOH solution. The azo coupling is carried out at the pH for 1 hour at 5°and for another 3 hours at room temperature. Then dialysis of the resulting solution against water for 3 days. The reaction product produce by freeze drying. Get 1,23·10-3kg (79.3 percent) antigen I (m=88,5 mol.%, n=10,3 mol.%, l=1,2 mol.%), containing 3.3 wt.% linked 2-naphtylamine.

Conducting the immunization of outbred rabbits wiring Repolovskoye kennel weighing 1.5-2 kg of a 5% aqueous solution of the obtained antigen according to the following scheme:

1. Once a week intradermally at 10 points along the spine at a distance of 1.5-2 cm from it in both directions (5 points on each side) to impose 1,0·10-3l aqueous solution of antigen I.

2. After a week repeated intradermal injection of the antigen I of the same dose in 10 points of the pins.

3. After 2 weeks, introduce a solution of the antigen I, under both blades subcutaneously and intraperitoneally in the dose of 1.5·10-3L.

4. After 1.5 months, introduce a solution of the antigen I of 0.5·10-3l intravenously every other day three times a week. Blood will be drawn on the 9th day after the last injection. In the future, rabbits reimmunized 3-4 months, using only intravenous antigen.

The next day after taking the blood serum separated from the clot by centrifugation for 10 min at 400g. Whey concentrate 2 times in cellophane tubes under the fans, can boric acid and used for the given reaction counter immunodiffusion in agar and reaction of complement fixation (RAC).

For the given reaction precipitation in agar 1% aqueous solution of agar, Difco. For the production RAC use ready hemolytic serum against sheep red blood cells and dry the complement. As the antigens used aqueous solutions of antigen I and the heterologous isoproterenol containing 2-naphtylamine, attached to proteins in human serum. Titration of antigens carried out before the production of each experience. Binding of complement is carried out at a volume of 0.5·10-5l motherboard. RAC put on cold at +4° (18 hours).

The results of the tests immersivity lead is received in the table. In column a indicates the contents in mol.% antigen I of the last dilution giving visible reaction precipitation in agar with serum intact (control) and immunized with the antigen I of rabbits. Column B lists the titers of antibodies against 2-naphthylamine in RAC.

Example 2.

In the conditions of example I of 0.001 kg of terpolymer II (m=88,5 mol.%, n=9,0 mol.%, 1=to 2.5 mol.%) and of 0.081·10-3kg chloride 2-naphthalenedione get 0,74·10-3kg (69,2%) antigen I (m=88,5 mol.%, n=9,0 mol.%, l=2.5 mol.%), containing 6.6 wt.% 2-naphtylamine.

According to the scheme described in example 1, conducting the immunization of rabbits received the antigen. The test results obtained immersivity on the presence and level of antibodies against 2-naphtylamine shown in the table.

Example 3.

In the conditions of example 1 from 0,7·10-3kg terpolymer II (m=88,5 mol.%, n=7,6 mol.%, l=3,9 mol.%) and 0,093·10-3kg chloride 2-naphthalenedione get 0,58·10-3kg (75,3%) antigen I (m=88,5 mol.%, n=7,6 mol.%, l=3,9 mol.%), containing 9.8 wt.% 2-naphtylamine.

According to the scheme described in example 2, conducting the immunization of rabbits received the antigen. The test results obtained immersivity shown in the table.

Example 4.

In the conditions of example 1 from 0,8·10-3kg terpolymer II (m=88,5 mol.%, n=6,8 mol.%, 1=4,7 mol.%) and to 0.127·10-3kg chloride 2-naphthalenedione get 0,67·10- kg (74,2%) antigen I (m=88,5 mol.%, n=6,8 mol.%, l=4,7 mol.%), containing 11.5 wt.% 2-naphtylamine.

According to the scheme described in example 1, conducting the immunization of rabbits received the antigen. The test results obtained immersivity shown in the table.

Example 5 (control).

In the conditions of example 1 of 0.001 kg of terpolymer II (m=88,5 mol.%, n=5,7 mol.%, l=5,8 mol.%) and 0,19·10-3kg chloride 2-naphthalenedione get 0,82·10-3kg (70,7%) antigen I (m=88,5 mol.%, n=5,7 mol.%, 1=5,8 mol.%), containing 13.7 wt.% 2-naphtylamine.

The resulting antigen is not dissolved in water, therefore, immunization of rabbits with this antigen did not.

Example 6 (control).

In the conditions of example 1 of 0.001 kg of terpolymer II (m=88,5 mol.%, n=10,6 mol.%, l=0.5 mol.%) and 0,016·10-3kg chloride 2-naphthalenedione get 0,65·10-3kg (64,3%) antigen I (m=88,5 mol.%, n=10,6 mol.%, l=0.5 mol.%), containing 1.4 wt.% 2-naphtylamine.

According to the scheme described in example 1, conducting the immunization of rabbits received the antigen. The test results obtained immersivity showed that there are no antibodies against 2-naphtylamine.

As can be seen from the table, the test results of immunoseparation as in the reaction counter immunodiffusion in agar, and the RAC suggests that the immunization of animals with antigen I, obtained according to examples 1-4, can be poluchenierazreshenija, containing antibodies against 2-naphtylamine as the hapten. The highest antibody titer detected in immunbiologie, obtained by immunization of rabbits with antigen I according to example 3. To confirm the specificity present in her antibodies against 2-naphtylamine as the hapten used the reaction of partial identity by exploring immersivity with antigen I and heterologous antigen containing the same hapten-2-naphtylamine, but other media - albumin horse serum.

Table
The test results of immunoseparation, obtained by immunization of rabbits with antigen I.
# example applicationAntigen I
mI, mol. %nI, mol. %1I mol.%Contents 2-naphtylamine wt.%The test results of immunoseparation
Using the immunodiffusion in agarUsing RAC
188,510,31,23,3--
288,59,02,56,693· 10-51:256
388,57,6a 3.99,81,2·10-81:8192
488,56,8the 4.711,511,2·10-51:128
5 control88,5the 5.75,813,7Not defined because the antigen is not soluble in water
6 test88,510,60,51,4No antibodies against 2-naphtylamineNo antibodies against 2-naphtylamine

The photo shows the results of the reaction of partial identity with the interaction of immersivity rabbit immunized with the antigen I of the example 3, containing 2-naphtylamine (1), with this antigen (2) and heterologous antigen containing the same hapten-2-naphtylamine, but other media - albumin horse serum (3). Education "spurs", visible in the photo indicates the presence of antibodies against 2-naphtylamine in immuneworks rabbit, giving a reaction of partial identity with their interaction with antigens containing the same hapten-2-naphtylamine, but different macromolecular carriers. As seen in the photo, came the actions of this immersivity with protein carrier - albumins horse serum (4), was not observed.

Immersivity containing antibodies against 2-naphtylamine a hapten, obtained by immunization of a rabbit 2-naphtylamine-polymeric antigen according to example 3, was used as the test system for the determination of 2-naphtylamine in the serum of workers LPO "Red triangle", in which the possible exposure to 2-naphthylamine. With this immersivity 2-naphtylamine-antigen was detected in 5 of 43 surveyed workers, which is 11.6%. Using immersivity rabbit, obtained by immunization of 2-naphtylamine-antigen containing protein carrier (albumin horse serum), 2-naphtylamine-antigen was detected in 3 out of 50 workers, which is 6.0%. These data indicate a positive effect by improving the accuracy of determination of 2-naphtylamine as the hapten in the blood of workers in comparison with the prototype when used as a test system immersivity obtained by immunization of rabbits with 2-naphtylamine-polymeric antigen.

The analysis of the table allows you to confirm the criteria values of the inventive compositions antigens I:

A. the Declared content of groups of n-crotononitrile in the original terpolymer II, equal to 1.2÷4,7 mol.%. The decrease in the content of this link to 0.5 mol.% (example 6) leads to the loss of positive EF is known.

B. the Molar ratio of n-crotononitrile links chloride and 2-naphthalenedione is 1:2÷2.2, which provides the introduction of two molecules of 2-naphtylamine in the benzene ring of the specified element.

Synthetic antigen based on copolymers of N-vinylpyrrolidone, cretonne easy acid and n-crotononitrile containing covalently attached 2-naphtylamine as hapten, the General formula

where m=88,5 mol.%, n=6,8÷10,3 mol.%, l=1,2÷4,7 mol.%.



 

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