RU2201256C2 - Immune specific lactoserum against helicobacter pylori and method of its preparing - Google Patents

Abstract

FIELD: medicine, immunology, microbiology. SUBSTANCE: lactoserum comprises antibodies raised against H. pylori, among them, against toxin and urease that are factors of its pathogenicity. Method of preparing involves immunization of calving cows with mixture of thermoinactivated culture H. pylori and antigenic extract from H. pylori biomass. Active fraction is isolated from milk in early period of lactation by its defatting, casein removal, clearing, diafiltration and concentrating. Lactoserum comprises antibodies raised to total antigen of H. pylori as active component with titer 1: 12 800, not above; to toxin 1:6 400, not less, and to urease 1:6400, not less, in the following ratio of components (as wt.-% of total protein content): immunoglobulin G, 25.95 ± 1.15; immunoglobulin A, 11.85 ± 0.75; immunoglobulin M, 16.95 ± 0.05; immunoglobulins of other classes, 0.75 ± 0.3; alpha-lactoglobulin, 10.65 ± 0.55; beta-lactoglobulin, 18.35 ± 0.25; serum albumin, 5.65 ± 0.65; residual proteins, the balance. EFFECT: improved method of preparing, enhanced specific activity of preparation. 3 cl, 1 tbl, 4 ex

Description

 The invention relates to the field of medicine, microbiology and immunology and can be used in the development of agents for the treatment and prevention of diseases associated with Helicobacter pylori infection.

 Helicobacter (H.) pylori - a pathogenic microorganism - affects the mucous membranes of the stomach and duodenum, is a leading etipatogenetic factor in mass diseases of the gastroduodenal sphere - gastritis, duodenitis, gastric ulcer and duodenal ulcer, and malignant lymphoma of the stomach [1].

 Specific immune preparations are known - lactoserum isolated from colostrum or milk of immunized cows and directed against pathogens affecting the gastrointestinal tract: shigella, salmonella, enteroviruses, as well as methods for producing such lactoserum [2, 3, 4].

 A specific immune lactoserum against pathogenic bacteria of the genus Shigella and a method for its preparation are known [3]. The specified lactoserum is a fraction of the milk of pregnant cows immunized intramuscularly and diathetically (by introducing antigen into the milk passages of the udder) with a suspension of inactivated shigella, lactoserum contains the following proteins: immunoglobulins - (56.27 ± 1.5)%, beta-lactoglobulins - (21, 0 ± 2.07)%, alpha-lactoglobulin - (10.3 ± 0.87)%, serum albumin - (6.0 ± 1.04)%, has a high activity of antibodies to the Shigella Sonne 0-antigen. Lactoserum is obtained in the following way: pregnant cows are immunized intramuscularly and diathetically with Shigella Sonne antigens 6-8 weeks before calving. Lactoserum is obtained from the early milk of immunized cows by degreasing it, removing casein, clarifying filtration and concentrating ultrafiltration, the content of specific antibodies and Shigella Sonne is controlled using the indirect hemagglutination reaction with erythrocyte diagnosticum - 0 Shigella Sonne.

 Specific immune lactoserums against H. pylori and methods for their preparation are not known.

 The invention is directed to the development of lactoserum against Helicobacter pylori, including against factors of its pathogenicity - toxin and urease, and a method for its preparation, while the specific activity of the drug is increased.

 The invention is reduced to the following. Specific immune lactoserum against Helicobacter pylori is a fraction of milk from pregnant cows immunized with H. pylori antigens, contains protein at a concentration of (35 ± 9.2) mg / ml and protein fractions in the following percentage: immunoglobulin G - (26.3 + 9 , 2) mg / ml, immunoglobulin A - (12.3 ± 1.9)%, immunoglobulin M - (17.9 ± 2.2)%, beta-lactoglobulin - (20 ± 2.5)%, alpha globulin - (10.5 ± 1.1)%, serum albumin - (6.0 ± 1.5)%; the preparation contains, as an active principle, antibodies to H. pylori antigens in the following titers: to the total H. pylori antigen - not less than 1: 12800, to the vacuolating H. pylori toxin, not less than 1: 6400, to urease - not less than 1: 6400.

 A method of obtaining a specific lactic serum of an immune specific against H. pylori provides for the immunization of cows in the pregnant period, the isolation of lactoserum from milk in early lactation by degreasing, removing casein, clarification, diafiltration and concentration. The specific activity of the preparation obtained is achieved through immunization of cows with a mixture of thermally inactivated H. pylori culture and antigenic extract from H. pylori biomass.

 The specific activity of the drug increases even more if, to obtain a thermally inactivated culture and antigenic extract, toxigenic ureazopositive H. pylori strains are used that have previously passed no more than 1 passage on artificial nutrient media after isolation from patients with gastroduodenal diseases.

 The invention is implemented as follows.

Example 1
Obtaining the total antigen of Helicobacter pylori.

 Prepare a culture medium for cultivation of Helicobacter pylori (H. pylori): horse normal serum is sterilely added to molten Colombian agar (Columbio Agar Base) to a final concentration of 7% and Iso Vitalex solution (Becton Dickinson Microbiology Systems) to a final concentration of 1%. Agar is poured into a petri dish and allowed to freeze.

Sowing the culture of 6 freshly isolated H. pylori strains on the agar surface is done with a bacterial loop (1 cup with inoculation for each H. pylori strain) and the applied material is distributed over the agar surface with a sterile spatula. The inoculated cups are placed in a GasPak 100 system anaerostat with a gas-generating package of the GasPak type and incubated at 37 ^{° C. for} 6 days. The grown H. pylori is checked microscopically for the purity of the culture, the biomass of the culture is removed with a sterile glass spindle and emulsified in 50 ml of a sterile 0.15 M NaCl solution containing 0.01 M phosphate-buffered solution pH 7.2 ± 0.2 (physiological buffered solution - PBS). For washing, the biomass is precipitated by centrifugation (15 min at 4 ^° C and a speed of 6 thousand rpm), the packed liquid is removed, and the biomass sediment is resuspended in 15.0 ml of PBS, a microbial suspension with a concentration of 15.5 billion microbial bodies is obtained in 1 ml according to the turbidity standard. 3.65 ml of PBS is added to it, as a result, a suspension is obtained with a concentration of 12 billion cells in 1 ml. Then, the resulting microbial suspension is heated in a water bath at 60 ^{° C.} for 60 minutes. To obtain an antigenic extract of H. pylori, inoculation from freshly isolated ureazopositive toxigenic cultures of this pathogen is performed as described above, however, emulsion of colonies removed with a spade is carried out in distilled water. The microbial suspension is centrifuged at 6 thousand revolutions (15 min, 4 ^° C), the supernatant is removed, and the sediment is resuspended in 15 ml of 0.1 M glycine buffer solution, pH 2.5, the suspended microbial suspension is kept on a rotary mixer for 10 minutes at ( 20 ± 5) ^o С and a rotation speed of 5 rpm, after which they are centrifuged, as described above, and the supernatant containing the antigenic extract is suctioned off (the precipitate is removed) and neutralized 1 N. NaOH solution, get antigenic extract with a protein concentration of 1.45 mg / ml

 12.5 ml of microbial suspension of H. pylori with a concentration of 12 billion cells in 1 ml is mixed with 12.5 ml of antigenic extract, 25 ml of antigen is obtained for immunization.

 5 cows 10 weeks before the expected calving are immunized intramuscularly, introducing to each animal 5 ml of antigenic mixture prepared in the manner described above.

 After 4 weeks and 8 weeks after intramuscular injection of antigen, animals are immunized diathetically, injecting 10 and 20 ml of antigen into the milk passages, respectively.

Milk to obtain lactoserum is taken on the 3rd day of lactation, degreased by centrifugation at 2000 rpm for 30 minutes, the fat layer is removed, 10 ml of skim milk is added to 10 l of 10% CaCl solution, stirred, the mixture is heated in a water bath to 37 ^o C and add to it 100 ml of a 10% solution of pepsin and mix for 10 minutes. After this, the mixture is left to curl at a temperature of (20 ± 3) ^o C for 18-20 hours. Clots of precipitated casein are separated by centrifugation at 2000 rpm for 30 minutes. The supernatant - lactoserum - is sucked off, the precipitate is removed. To lactoserum add 1 N. NaOH solution until a pH of 7.0 ± 0.5 is reached. Lactoserum is clarified by filtration through a plasma filter for plasmapheresis (Fresenins company) and dialysed by filtration through a fiber filter concentrating AP-01 (manufactured by the Kirishi TMB plant), using diafiltration 30 l of 0.001 M phosphate buffer solution pH 7.0. Diafiltration of the lactoserum is completed by the concentration operation — the diafiltrate is recirculated through the same AP-01 filter until the volume of the diafiltrate decreases to 2 liters.

 The concentrate is sterilized by filtration through a Millipor A. G. sterilizing membrane with a pore size of 0.45 μm.

The resulting preparation of lactic serum immune specific against H. pylori control:
1) for protein content by biuret method;
2) the content of protein fractions by polyacrylamide gel electrophoresis;
3) the content of immunoglobulins of class G, class A and class M in the reaction of radial immunodiffusion in agarose gel;
4) lactose concentration (by iodometry, by the difference between the quality of the taken and unreacted iodine by titration with Na ₂ S ₂ O ₂ );
5) the activity of specific antibodies against a) total H. pylori antigen; b) against the toxin; c) against urease - in the enzyme-linked immunosorbent assay (ELISA).

 ELISA is performed by known methods, as the antigen adsorbed on the solid phase (in the wells of a polystyrene tablet for immunological reactions), use: a) a neutralized antigenic extract from the microbial mass of H. pylori, obtained as described above, at a concentration of 5 μg / ml; b) recombinant Cag A-ipothein associated with vacuolating toxin at a concentration of 2 μg / ml; c) recombinant H. pylori urease at a concentration of 2 μg / ml. As a detector of immunoglobulins, peroxidase-labeled affinity-purified antibodies to cattle immunoglobulins in working dilution (Sigma) are used.

 Specific immune lactoserum obtained as described above is tested in ELISA in parallel with the study of lactoserum from a pool of milk from 20 non-immunized cows ("normal lactoserum"). The immune and “normal” lactoserum are pre-diluted with 1: 800 PBS and examined in ELISA in serial dilutions: 1: 1600, 1: 3200, 1: 6400, 1: 12800, 1: 25600. For the titer of antibodies in immune lactoserum, then its greatest dilution, in the study of which the ELISA index (optical density of the reaction product at a wavelength of 492 nm) is 2 times or more higher than the ELISA index of the “normal” lactoserum in the same dilution. For example: ELISA of immune lactoserum are equal: at a dilution of 1: 600 - 2.48; at a dilution of 1: 3200 - 2.01; at a dilution of 1: 6400 - 1.43; at a dilution of 1: 12800 - 0.742; at a dilution of 1: 25600 - 0.503. ELISA of "normal" lactoserum: 1: 1600 - 0.485; 1: 3200 - 0.438; 1: 6400 - 0.386; 1: 12800 0.308; 1: 25600 - 0.296. The largest dilution under which the condition is met is a 2-fold excess of the ELISA of immune lactoserum over the "normal" lactoserum - 1: 12800. This is the titer of antibodies of the immune lactoserum.

 As a result of the control prepared according to example 1 lactoserum receive the following data presented in the table. The percentage of protein fractions of the immune lactoserum is close to that of the known analogues and prototype. Immune lactoserum contains specific antibodies to H. pylori antigens in high titers, comparable to the titers of highly active blood sera of people infected with H. pylori [5]. According to the lactose content, the drug is at the level of areactogenic milk mixtures [2].

Example 2
Milk from immunized cows (see example 1) is collected on the 4th day of lactation and combined into a pool (from 5 animals). 10 l of milk pulp is used to obtain immune lactoserum in accordance with the conditions of example 1. Receive the following results, presented in the table, which are fundamentally close to the results of the control of immune lactoserum from unpopulated milk. An example confirms the possibility of obtaining a highly active specific drug in the isolation of lactoserum from a pool of milk.

Example 3
Individual milk samples from immunized cows taken on the 2nd, 6th, 8th, 10th day of lactation are fractionated and lactoserum obtained from them, as described in example 1.

 Get 20 samples of lactoserum, which determine the concentration of protein and the percentage of protein fractions, antibody titers and glucose concentration. The maximum levels of antibody titers are observed on the 2nd day of lactation: to the total H. pylori antigen 1: 25600, to the toxin 1: 25600, to urease 1: 12800. The minimum titer values are noted in lactoserums on the 10th day: 1: 3200, 1: 1600, 1: 800, respectively, which is significantly lower than the titers of lactoserum from the 8th day of lactation: 1: 12800, 1: 6400, 1: 6400, respectively. A significant drop in titers after the 8th day (4 or more times) indicates the fact that the use of milk after the 8th day of lactation to obtain an active drug is impractical.

 The study of the protein composition of the immune lactoserum isolated during the 2-8 days of lactation, reveals the following fluctuations in indicators: total protein content (35 ± 9) mg / ml; the content of immunoglobulins G - (26.3 ± 2.1)%; immunoglobulins A - (12.3 ± 1.9)%; immunoglobulin M - (17.9 ± 2.2)%; beta-lactoalbumin - (20 ± 2.5)%; alpha-lactoalbumin - (10.5 ± 1.1)%; serum albumin - (6.0 ± 1.5)%.

Example 4
Performed in accordance with the conditions of example 1 with the difference that the immunization is carried out with a thermally inactivated suspension of H. pylori, without adding antigenic extract. Lactic serum preparations with a low content of antibodies to toxin and urease are obtained. The maximum titers of antibodies to these antigens are noted in lactoserums from the 2nd day of lactation: 1: 1600 and 1: 1600, respectively, which is significantly lower than in the present invention.

 Thus, the presented examples of the invention demonstrate the possibility of obtaining a new drug - specific immune lactoserum against Helicobacter pylori, in contrast to the prototype and analogues, which has biological activity and high antibody titers, against the total H. pylori antigen, as well as against this toxin and urease microbe. Activity against the last two antigens is significant due to the fact that they are considered the main pathogenicity factors of H. pylori [6,7]. The proposed method for the production of immune lactoserum provides a preparation with the stated parameters. Moreover, the composition of the antigen for immunization of cows is essential for obtaining lactoserum with a high specific activity of antibodies. The mixture of thermally inactivated H. pylori culture and the antigenic extract from the biomass of this microbe proposed for immunization provides high titers of antibodies to toxin and urease, as well as to the total H. pylori antigen.

Sources of information:
1. Helicobacter pylori, Basio Mechanisnis to Clinical Cure. Edit Hunt RH, Tytgat GNJ Kluwer Acad, Publischers. Dordrecht / Boston / London, 1994.

 2. Gennadyeva T. Ya., Kolesnikova EN, Strelkova M.R. and others. The level of sensitization to cow's milk proteins in children with oral administration of immune lactoserum. In collection: "Acute intestinal infections", L. - 1986, the works of the NIIEM them. Pasteur, issue 10, p. 154-160.

 3. Nikolaeva T.A., Gennadyeva T.Ya., Kapelyush E.V. and other electrophoretic characteristics of anti-shigellosis lactoserum and the influence of the ultrafiltration process on the protein composition of the drug. In: “Acute intestinal infections”, L., 1987, Proceedings of the NIIEM them. Pasteur, issue 11, p. 67-70.

 4. Brussow Y. e.a. J. Clin. Microbiol., 1987, v. 25, 6, p. 982-986.

 5. Goodwin C.S. e.a. J. Infect. Dis., 1987, 3, 155: 488-494.

 6. Figura N. and Crabtree J.E. In: "Helicobacter pylori, Basic Mechanisnis to Clinical Cure". Kluwer Acad. Publaschcra Dordrecht / Boston / London, 1994, p. 222-231.

 7. Mobley H.T.L. and Foxall. Ibid., Pp. 41-58.

Claims (3)

 1. A method of obtaining an immune specific lactoserum, including immunization of cows during pregnancy, milk production in the early stages of lactation, skim milk, removal of casein, clarification, diafiltration and concentration, characterized in that the cows are immunized with a mixture of heat-inactivated culture of N. pylori and antigenic extract from the biomass of H. pylori.  2. The method according to p. 1, characterized in that to obtain a thermally inactivated culture and antigenic extract, toxigenic ureazopositive strains of H. pylori are used, having previously passed no more than 1 passage on artificial nutrient media after isolation from patients with gastroduodenal diseases. 3. Immune specific lactoserum obtained by the method of claim 1 or 2, characterized in that it contains antibodies to the total H. pylori antigen in a titer of not less than 1: 12800, to a toxin - not less than 1: 6400 and to urease - not less than 1 : 6400 in the following ratio,% of the total protein content:
Immunoglobulin G - 25.95 ± 1.15
Immunoglobulin A - 11.85 ± 0.75
Immunoglobulin M - 16.95 ± 0.05
Immunoglobulins of other classes - 0.75 ± 0.3
Alpha-Lactoglobulin - 10.65 ± 0.55
Beta-Lactoglobulin - 18.35 ± 0.25
Serum Albumin - 5.65 ± 0.65
Residual Proteins - Else

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