RU2795322C1 - Method for obtaining monospecific antibodies to human lactoferricin - Google Patents

Abstract

FIELD: biochemistry; biotechnology.

SUBSTANCE: method for obtaining monospecific antibodies to human lactoferricin is proposed. Acid hydrolysis of skimmed human milk proteins is carried out using porcine pepsin. The resulting peptides are separated by precipitation with trichloroacetic acid and centrifuged. The deproteinized filtrate is neutralized and conjugated with bovine serum albumin. After incubation, sodium borohydride is added to the reaction mixture. The reaction is stopped with glacial acetic acid. Rabbit immunization cycles are carried out by introducing the BSA-lactoferricin conjugate with complete Freund's adjuvant until a hyperimmune response is formed. In the obtained rabbit sera, ballast impurities are eliminated to obtain monospecific antibodies to human lactoferricin.

EFFECT: invention can be used to obtain antibodies for the development of a test system for human lactoferricin and is aimed at simplifying and improving the accuracy of determining human lactoferricin.

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Description

The invention relates to medicine, namely to biochemistry and biotechnology, and can be used to obtain antibodies and develop a test system for the human lactoferricin peptide.

The structural domain of lactoferrin responsible for bactericidal properties is a pepsin-cleavable fragment called lactoferricin (Bellamy W., Takase M., Yamauchi K. [et al.] Identification of the bactericidal domain of lactoferrin // Biochim. Biophys. Acta. - 1992 - Vol. 1121, No. 1-2 - P. 130-136).

The peptide lactoferricin (LFC) is active against all tested bacteria (E. coli, Klebsiella spp., Pseudomonas spp., Listeria spp., Staphylococcus spp.) and is effective even against infections caused by antibiotic-resistant Staphylococcus aureus and Klebsiella pneumoniae (Oda H ., Wakabayashi H., Yamauchi K. [et al.] Isolation of a bifidogenic peptide from the pepsin hydrolysate of bovine lactoferrin Appl Environ Microbiol 2013 Vol 79(6): P 1843 -1849). Human LFC, in addition to antimicrobial, antiviral and antifungal activity, has anticarcinogenic activity (Perroden J.P. Method for the production of lactoferrin, a fraction containing lactoferrin, and its applications / Patent for invention RU 2579661, 10.04.2016).

The peptides formed during pepsin hydrolysis of human lactoferrin, after removing unreacted proteins with trichloroacetic acid (TCA), have a molecular weight in the range of 2-5 kDa and do not have immunogenic properties. According to the information contained in the UniProt database, human LPCs consist of 48 amino acid residues and have a molecular weight of 5345 Da (Serebryakov A.A., Kokhanova A.V. Dynamics of lactoferricin and alpha-fetoprotein in the blood of rabbits after various types of liver damage // Crimean Journal of Experimental and Clinical Medicine - 2022. - V. 12, No. 2. - P. 41-48).

Typically, low molecular weight proteins and peptides are non-immunogenic and poorly adsorbed to plastics, making them difficult to screen and monitor in biological fluids.

However, despite complete information about the structure of this important antibacterial peptide, there is still no immunochemical method for determining lactoferricin.

The immunochemical method of registration of antimicrobial peptides, based on the antigen-antibody reaction, has a great advantage over spectrophotometric and other methods of registration of the measurement result, which include universality, selectivity, specificity, high sensitivity (Frimel X. Immunological methods. M .: Medicine, 1987 - 472 pp.; Chekhonin VP, Dmitrieva TE, Zhirkov Yu.A. Immunochemical analysis of neurospecific antigens. - 2000. - M.: Medicine - 416 pp.).

At the same time, technologies for conjugation of low molecular weight ligands with macromolecular carriers have long been developed to obtain immunogenic combinations. To convert non-immunogenic peptide molecules into an immunogenic state, they can be aggregated into larger particles or attached to an appropriate carrier. It is preferable to use the second method (Frimel X. Immunological methods. M .: Medicine, 1987. - 472 p.).

For example, a method is known for conjugating antibacterial peptides by immobilizing them on insoluble carriers made from polystyrene, polypropylene, polyamide, cellulose, agarose, polyacrylamide, dextran or vinyl polymeric materials (Blondelle Sylvie E. (US) et al. Antibacterial peptides // RF Patent No. 2468033 dated 07/10/2007).

However, as it turned out, methods for preparing conjugates based on insoluble polymers do not allow obtaining antibodies to the non-immunogenic peptide lactoferricin in rabbits.

To obtain soluble complexes of protein carriers with non-immunogenic peptides, heterogeneous proteins are most often used: bovine serum albumin, keyhole limpet hemocyanin (KLH), thyroglobulin, or ovalbumin. For example, known methods of immobilization by covalent conjugation of the peptide with cochlear hemocyanin (Shirakawa Kamon. Specifically purified antibodies against presepsin // RF Patent No. 2739607 dated 25.08.2015).

However, the most reliable and most commonly used carrier for peptides is bovine serum albumin (BSA) (Heisch R.D., Hufner M. Highly specific antibodies to triiodthyronine. - Acta biol. Med. Germ., 1972. - Vol. 28. - P. 861 -864.).

To obtain conjugates of various haptens with BSA, methods of diazotization or the reaction of the interaction of the aldehyde group with the amino group of BSA are often used (Kovalev I.E., Polevaya O.Yu. Biochemical bases of immunity to low molecular weight chemical compounds. M., 1985, p. 14-25; Kostyanko M.V., Glushkov A.N. Method for obtaining a hapten-protein conjugate / Patent for invention RU 2141114, 16.02.1998). However, these conjugation methods are not suitable for a lactoferricin-type peptide that carries a significant positive charge.

Due to the lack of an enzyme immunoassay method for detecting lactoferricin in the literature, our article on the determination of lactoferricin in blood serum and other biological fluids by the spectrophotometric method was used as a prototype (Serebryakov A.A., Kokhanov A.V., Lutseva O.A., Taspenova G. K., Muldasheva N.R. Lactoferrin and lactoferricin in urine and faeces in patients with urgent urological and surgical pathology // Modern problems of science and education. - 2021. - No. 4. (Electronic journal); URL: http://www .science-education.ru/article/view?id=27717).

The invention is aimed at simplifying and improving the accuracy of determining human lactoferricin.

The goal set in the invention is achieved by the fact that with the help of the pig pepsin enzyme with an activity of 90 IU/mg, acid hydrolysis of skimmed human milk proteins is carried out for 4-6 hours at 37 ° C, the peptides formed during hydrolysis are separated from unreacted milk proteins by precipitation of 10% trichloroacetic acid, followed by centrifugation at 5000 g for 10 min, after neutralization of the deproteinized filtrate, it is conjugated with bovine serum albumin at the rate of 2-3 mg of filtrate protein per 100 mg of bovine serum albumin in 0.1 N NaOH, after a three-hour incubation at room temperature 20 mg of sodium borohydride reducing agent is added to the reaction mixture and after 30 minutes the reaction is stopped with glacial acetic acid, bringing the pH to 7.2-7.4, rabbits are immunized with three weekly intramuscular injections of the BSA-lactoferricin conjugate with complete Freund's adjuvant for 28 -35 days before the formation of a hyperimmune response in rabbits, in the obtained rabbit sera, ballast impurities are eliminated by adding 200 mg of lyophilized human plasma and 50 mg of commercial bovine serum albumin to 10 ml of serum to obtain monospecific antibodies to human lactoferricin.

Examples of the invention.

Example 1

The source for obtaining monospecific antibodies to human lactoferricin is skimmed human milk. To obtain a peptide fraction from milk protein, the solution is acidified with hydrochloric acid to pH 2.0, a solution of porcine pepsin with an activity of 90 IU/mg (Sigma, USA) is added, and the resulting mixture is kept in a thermostat for 4 hours at 37°C. During this time, peptides with a molecular weight below 4 kDa accumulated in the incubation medium, which are separated from unreacted protein components by adding 1 ml of a 20% trichloroacetic acid solution, followed by centrifugation at 5000 g for 10 min. The deproteinized filtrate containing lactoferricin is neutralized with sodium bicarbonate to pH 7.6-7.8 and after centrifugation at 5000 g for 20 minutes, stored until the next stage of obtaining the BSA-LFC conjugate in a freezer at -20°C. The concentration of the lactoferricin peptide in the obtained preparations of skimmed human milk, calculated spectrophotometrically by the Scopes method (Scopes R. Methods for purification of proteins: Lane from English - M .: Mir, 1985. - 358 p.) at wavelengths of 260 and 280 nm, is 2-3 mg/ml.

The presence of positively charged groups in the lactoferricin peptide makes it possible to obtain immune complexes with bovine serum albumin only by adsorption without the use of additional spacer functional groups. To obtain BSA-lactoferricin conjugates, 1 ml of a lactoferricin preparation containing 2-3 mg of the peptide is added to 0.1 gram of BSA dissolved in 1 ml of 0.1 N sodium hydroxide solution. After three hours of stirring at room temperature, 20 mg of sodium borohydride reducing agent is added to the solution, and after 30 minutes the reaction is stopped with glacial acetic acid, bringing the pH to values of 7.2-7.4. After centrifugation at 5000 g for 20 min, the BSA-lactoferricin conjugates are stored until the next stage of immunization in a freezer at -20°C. According to spectrophotometry data, 40-60 mol of lactoferricin is accounted for in the resulting conjugate per 1 mol of BSA.

To obtain antisera to the human lactoferricin peptide, chinchilla rabbits weighing 3-4 kg are immunized according to the scheme developed by Kalashnikov [Kalashnikov V.V. Cancer-embryonic human proteins: Dis. … dr. honey. Sciences. - M., 1986]. The BSA-lactoferricin conjugates obtained at the previous stages were used as antigens.

Rabbits were immunized with the resulting BSA-lactoferricin conjugate according to the following scheme:

a) 10 mg of the conjugate dissolved in 5 ml of saline was carefully suspended with 1 ml of complete Freund's adjuvant and injected subcutaneously into rabbits at 6-8 sites;

b) a week later, 10 mg of the drug dissolved in 5 ml of saline was suspended with 1 ml of incomplete Freund's adjuvant and injected subcutaneously into rabbits in 6-8 places;

c) after a week break, the procedure for immunizing rabbits with the same preparation was repeated, repeating the conditions described in paragraph b);

c) a week after the last injection of rabbits, a test blood sampling was carried out from the marginal vein of the auricle.

With a low titer of antibodies to the BSA-lactoferricin conjugate, after 2.5-3 months, the animals were immunized again by a single subcutaneous injection of 2-3 ml of the conjugate containing 8-10 ng of protein.

With a high quality of serum, blood sampling from the marginal vein of the rabbit's ear was carried out strictly 7 days after the last injection, while 50-100 ml of blood was taken from the rabbit once. Blood sampling was repeated after 2-3 days and continued in this mode for 15-20 days.

All obtained rabbit sera, after testing for the presence of antibodies to lactoferricin, were additionally subjected to the procedure for eliminating ballast impurities by adding 200 mg of lyophilized human plasma and 50 mg of commercial bovine serum albumin to 10 ml of rabbit serum.

Two hours later, the obtained rabbit sera were centrifuged and the completeness of depletion was checked by the immunodiffusion method with the serum and blood plasma of donors and the blood serum of cows.

After that, the precipitating ability of the obtained rabbit sera with lactoferricin was determined in specific reactions of immunodiffusion analysis. At the same time, if the obtained rabbit serum gave a single line of precipitation with the peptide lactoferricin (LFC), it was considered monospecific.

Example 2

The source of lactoferricin for obtaining the complex of this peptide with BSA was preparations of pure human lactoferrin protein, provided for research by Professor A.A. Nikolaev. (Department of Chemistry, Astrakhan State Medical University). To obtain a peptide fraction from lactoferrin, 200 mg of lyophilized LF protein was dissolved in 1 ml of water, the resulting solution was acidified with hydrochloric acid to pH 2.0, a solution of porcine pepsin with an activity of 90 IU/mg (Sigma, USA) was added, and the resulting mixture was kept in a thermostat at for 4 hours at 37°C.

All further steps for obtaining a lactoferricin preparation from purified human lactoferrin, obtaining a BSA-lactoferricin conjugate, immunizing rabbits, obtaining hyperimmune rabbit sera, procedures for eliminating ballast impurities from sera to obtain monospecific rabbit antibodies to human lactoferricin are similar to example 1.

At the same time, if the obtained rabbit serum gave a single line of precipitation with the peptide lactoferricin (LFC) and did not react with antibodies to lactoferrin (LF) and any other substances, it was considered monospecific and the obtained antibodies were used to construct a test system for lactoferricin.

Checking the quality of the test system for human lactoferricin using monospecific antibodies to human lactoferricin by immunodiffusion analysis. This variant of immunochemical analysis makes it possible to study various antigens in complex mixtures without their purification and isolation from blood serum or other biological fluid. The method has a sufficiently high sensitivity and eliminates errors associated with the use of insufficiently depleted antisera.

The results of a comparison of two monospecific test systems for human lactoferrin and lactoferricin by double radial diffusion in Ouchterloni agar [Frimel X. Immunological methods. M.: Medicine, 1987. - 472 p.] in the format of the stamp "seven" are presented in Fig. 1.

At the same time, monospecific antibodies to human lactoferricin are introduced into the central well of the seven-stamp in agar gel, and samples of the purified lactoferricin preparation are introduced into the central upper and lower wells. For comparison, test antigen and test serum for lactoferrin from the set of the Department of Chemistry of the Astrakhan State Medical University are introduced into the side holes of the seven stamp.

1 - preparation of purified lactoferricin peptide;

2 - antiserum against lactoferricin peptide;

3 - preparation of purified lactoferrin, provided by Professor Nikolaev A.A. (Department of Chemistry, Astrakhan State Medical University);

4 - antiserum against human lactoferrin from the set of the Department of Chemistry of the Astrakhan State Medical University.

The result obtained indicates the absence of a cross-reaction between antibodies to human lactoferrin and antibodies to human lactoferricin.

Example 3

The steps for obtaining a lactoferricin preparation from purified human lactoferrin, obtaining a BSA-lactoferricin conjugate, immunizing rabbits, obtaining hyperimmune rabbit antisera, procedures for their depletion and formation of monospecific antisera to human lactoferricin are similar to example 2.

If the concentration of specific antibodies to LFC in rabbit antisera does not exceed 10% of the total amount of serum gamma globulins, then such rabbit sera cannot be used to activate polystyrene plates and to prepare conjugates with enzymes. In this case, the step of isolating a fraction of monospecific immunoglobulin IgG to lactoferricin from rabbit serum using a combination of conventional biochemical methods of salting out and chromatography on Sephadex G-150 is included.

However, the best results for obtaining monospecific antibodies to human lactoferricin are obtained by using immunosorption methods.

To isolate specific antibodies from polyclonal rabbit sera, we used immunosorbents prepared on the basis of Sepharose 4B activated with cyanogen bromide (Sigma, United States). The purified lactoferricin is chemically bound to this carrier.

Before binding, 10 g of Sepharose are soaked for 20 min in 500 ml of 1 mM HCl solution, then washed with 2 L of the same solution on a glass filter. To the thus activated gel is added a solution of lactoferricin in 0.1 M carbonate buffer pH 8.3 containing 0.5 M NaCl (at the rate of 5-10 mg of lactoferricin peptide per 1 ml of gel). The resulting mixture is incubated for 2 hours at room temperature, with stirring on a rotary stirrer (a magnetic stirrer can destroy the gel structure). Then the immunosorbent is washed with 4 l of the same buffer, the remaining unbound active groups of the carrier are blocked by treatment with 1M glycine solution (2 hours). Excess glycine is washed off by washing alternately with 0.1 M carbonate buffer pH 8.3 and 0.1 M Na-phosphate buffer pH 4 (both buffers contain 0.5 M NaCl). The last wash is with carbonate buffer.

The immunosorbent prepared in this way is placed in a small column (usually a layer of sorbent with a volume of 3-5 ml is enough), through which 10-20 ml of monospecific antiserum is passed. The extraction of antibodies is carried out in slow recirculation mode at a flow rate of 10 ml/h. As the results of immunodiffusion analysis show, for complete extraction of antibodies, the indicated volume of serum must be passed at least 3-5 times through a column with an immunosorbent. After that, the column is washed with the same carbonate buffer until the optical density of the eluate <0.02 is reached (the optical density is recorded using a flow photometer, for example, Uvicord-S from LKB, Sweden). The adsorbed antibodies are eluted with 0.2 M glycine buffer pH 2.2 and the eluate is immediately neutralized with saturated TRIS solution. After dialysis against distilled water, the antibodies are concentrated by ultrafiltration through an XM-100A membrane (Amicon) and lyophilized. Dry preparation of antibodies is stored at 4°C.

Example 4

The steps for obtaining a lactoferricin preparation from purified human lactoferrin, obtaining a BSA-lactoferricin conjugate, immunizing rabbits, obtaining hyperimmune rabbit antisera, procedures for their depletion and formation of monospecific antisera to human lactoferricin are similar to example 2.

To characterize the preparation of antibodies to human lactoferricin, they are evaluated according to two parameters:

• as antigens using antiserum to rabbit immunoglobulins;

• as antibodies using a standard test system for lactoferricin.

This approach allows, on the one hand, to determine the concentration of immunoglobulins, and on the other hand, to determine what proportion of the total number of immunoglobulins are specific precipitating antibodies. In this case, the calculations are carried out using the following constants:

• the minimum concentration of immunoglobulins detected using antiserum to rabbit immunoglobulins ("antibodies as antigens") by Ouchterlony double immunodiffusion method, which is 10 µg/ml;

• the minimum concentration of antibodies detected using a standard test system for the studied antigen ("antibodies as antibodies"), which is 80 µg/ml.

Multiplying by 10 the maximum dilution of antibodies, which still gives a precipitation reaction with antiserum to rabbit immunoglobulins, you can get the total concentration of immunoglobulins in the analyzed solution. This value should be compared with the calculated concentration of precipitating specific antibodies, which is obtained by multiplying by 80 the maximum dilution of antibodies that react as antibodies in the standard test system for lactoferricin. Close values of these values indicate the high quality of the resulting antibody preparation.

The introduction of this method for the determination of lactoferricin is superior to the spectrophotometric method for determining this peptide in terms of sensitivity, it makes it possible to increase the accuracy and simplify the procedure for determining the concentration of the non-immunogenic antibacterial peptide lactoferricin.

The proposed method for obtaining monospecific rabbit antibodies to human lactoferricin can be used as a tool for quantitative determination of the concentration of lactoferricin peptide in biological fluids by any immunochemical method of analysis (IDA, ELISA, ICA, etc.).

Claims (1)

A method for producing monospecific antibodies to human lactoferricin, which consists in the fact that using the pig pepsin enzyme with an activity of 90 IU/mg, acid hydrolysis of skimmed human milk proteins is performed for 4-6 hours at 37 ° C, the peptides formed during hydrolysis are separated from unreacted proteins milk by precipitation with 10% trichloroacetic acid, followed by centrifugation at 5000 g for 10 minutes, after neutralization of the deproteinized filtrate, it is conjugated with bovine serum albumin at the rate of 2-3 mg of filtrate protein per 100 mg of bovine serum albumin in 0.1 n NaOH, after a three-hour after incubation at room temperature, 20 mg of sodium borohydride reducing agent is added to the reaction mixture and after 30 minutes the reaction is stopped with glacial acetic acid, bringing the pH to 7.2-7.4, rabbits are immunized with three weekly intramuscular injections of the BSA-lactoferricin conjugate with a complete adjuvant Freund for 28-35 days before the formation of a hyperimmune response in rabbits, in the obtained rabbit sera, ballast impurities are eliminated by adding 200 mg of lyophilized human plasma and 50 mg of commercial bovine serum albumin to 10 ml of serum to obtain monospecific antibodies to human lactoferricin.

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