RU2287342C1 - METHOD FOR OBTAINING IMMUNOGENIC Trichinella spiralis ANTIGEN - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: the present innovation deals with infecting laboratory animals, separating muscular tissue to carry out its proteolysis in artificial gastric juice, isolating invasion larvae and their cultivation in artificial nutritive medium, separating protein product followed by its purification and immunochemical screening. Moreover, proteolysis of muscular tissue of a laboratory animal in artificial gastric juice should be carried out at 38.5-39.0° C. Isolated invasion larvae of Trichinella should be washed out 5-7-fold with a sterile physiological solution which should be supplemented with antibiotics of different spectrum of action. Cultivation of invasion larvae should be fulfilled for 3-5 d at 38.5° C. After isolating protein product due to carrying out its dialysis and concentration it is necessary to isolate an excretory-secretory antigen. Trichinella larvae remained after separation of protein product should be reduced, the obtained homogenate should be extracted and centrifuged. Out of somatic extract due to its fractionating upon a column with sephadex G-100 one should additionally obtain fractionated somatic antigen being of high specificity.

EFFECT: higher efficiency.

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Description

The invention relates to veterinary medicine, in particular to immunodiagnosis and immunoprophylaxis of dangerous anthropozoonosis (trichinosis). It can be used for in vivo serodiagnosis of this disease in humans and animals and as a main component for the manufacture of a vaccine preparation.

Annually in the Russian Federation up to 700 cases of the disease with this dangerous helminthiasis of people are recorded, and in 40% they are of a group nature. Currently, regions of the North Caucasus, Eastern Siberia, the Far East, and the Kirov Region remain permanently dysfunctional for trichinosis. Given the wide range of susceptible animals and the diversity of their trophic connections, the transmission of the pathogen is carried out at several levels with the involvement of circulatory insects and small mammals, carnivores, omnivores and large predators. There is a potential epizootic relationship between natural and synanthropic biocenoses, which, with an increased interest in hunting with dogs of the corresponding breeds, can lead to the spread of trichinosis, since in the process of hunting dogs are constantly in contact with wild animals. Therefore, it is necessary to regularly examine domestic carnivores after staying in the natural foci of trichinosis. One of the most reliable methods for intravital detection of this tissue helminthiasis is serodiagnosis, based on the determination of the presence of specific antibodies in the blood during their interaction with Trichinella antigens in IGF.

The problem of prevention and immunodiagnostics of this socially dangerous tissue helminthiasis is a priority task of helminthological science and practice, and therefore the methods for producing and researching Trichinella antigens are of great scientific and practical value. Foreign researchers are involved in these issues. H.R. Gamble (3, 4) received the excretory-secretory products of Trichinella larvae by cultivation in a DMEM nutrient medium supplemented with glutamine and antibiotics (penicillin, streptomycin, gentamicin). Tyrcekova L. et al. (9) used RPMI-1640 medium for similar purposes. All authors carried out work under strictly sterile conditions using a CO ₂ incubator with a 5-10% carbon dioxide content, at a temperature of 37 ° C, the larval cultivation time varied from 18 to 48 hours. In the excretory-secretory products, the researchers revealed 5 molecular proteins weighing from 43 - to 63 kDa.

The most informative method of immunochemical study of antigens is currently recognized Western Blot analysis. Tyrcekova L. et al. (9) carried out immunoblotting of Trichinella antigens with hyperimmune rabbit sera and established the presence of 6 immunogenic proteins with a molecular weight of 43 to 70 kDa. These data are comparable with the results of Ko and Fan, 1996 (6).

Western Blot analysis of Trichinella antigens was performed by Ariaga et al (2), he identified five major proteins with a molecular weight of 43-63 kDa, his data coincide with Tyrcekova L. (9), and according to the author, they contain the main antigenic epitopes of antibody binding and are produced helminth throughout the invasion.

A specific antigen is the main component of an immunopreparation that promotes the development of humoral-cellular mechanisms of directed action, which block the vital functions of the parasite and ensure the development of immunity to subsequent infection.

The value of the invention is to optimize the production of preparative amounts of the main, so-called "major" Trichinella antigens, modern methods of monitoring the quality of the produced product are proposed.

The purpose of the invention is to obtain, in a biotechnological way, the main component - immunogenic T. Spiralis antigens for intravital serodiagnosis and vaccine prophylaxis of this dangerous anthropozoonosis.

The essence of the invention consists in the development of a method for producing immunogenic Trichinella proteins, which are the main component of an enzyme-linked immunosorbent assay and vaccine.

The closest prototype of the invention are the work of Gamble et all, 1984, 1985 (3, 4), Klimenko V.V. 1987 (1).

The disadvantages of the prototypes can be considered a complex, lengthy and more expensive process of fractionation and purification of antigens, a different combination of antibiotics used, a short period of cultivation of larvae in artificial nutrient media, even in the presence of expensive equipment (CO ₂ incubator, sterile box). Previous authors have no indication that it is advisable to use cultured invasive larvae that have significantly increased in size due to the consumption of nutrients from the DMEM medium to obtain a complete somatic extract and fractionated antigen. We also consider it not rational to carry out the process of proteolysis in IHL at the temperature indicated by previous authors, more physiological for this parasite is proposed by us.

Our work is characterized in that:

1. A more optimal mode of digestion of muscle tissue of animals invaded by Trichinella in artificial gastric juice is proposed.

2. Used a more rational use of antibiotics to destroy concomitant microflora.

3. Simplified and cheapened the process of biological cultivation of invasive larvae, because does not require the presence of a CO ₂ incubator, sterile box.

4. Due to an increase in the concentration of glutamine in the DMEM medium, the period of production of excretory-secretory proteins by living larvae was increased to 5 days.

5. The methods of immunoblot and IGF have proved the possibility of using protein antigens from the biomass of larvae remaining after cultivation in DMEM medium to obtain somatic extracts, fractionation, and hyperimmunization of animals in order to obtain high titer sera.

6. Optimal inexpensive biochemical methods have been developed (and first applied) for the fractionation of Trichinella proteins using the Sephadex-G100 sorbent.

7. A complex of economical methods of purification, concentration, immunochemical analysis of antigenic material is proposed.

8. The experiments showed a high immunogenicity of the obtained antigens, their high sensitivity and specificity in IGF.

The method includes the following steps:

1. Obtaining invasive material of Trichinella larvae.

2. Biological cultivation of larvae, obtaining excretory-secretory products.

3. Obtaining a complete somatic extract, fractionated antigen.

4. Analysis of the diagnostic properties of somatic fractionated and excretory-secretory antigens of T. spiralis larvae.

5. Immunochemical analysis of the obtained protein antigens.

6. The study of the protective properties of excretory-secretory products of Trichinella.

Preliminary preparation of laboratory glassware, instruments and their sterilization was carried out according to generally accepted methods from the guidelines "Animal Cell Biotechnology", ed. Spier R.E. (1989). Directly for the cultivation of Trichinella larvae, sterile disposable Petri dishes were used (produced by Lenpolymer), and immunological plates of domestic manufacture or Costar (USA) were used for IGF. Biological cultivation was carried out in an artificial nutrient medium DMEM with the mandatory addition of glutamine.

1. Obtaining invasive larvae of Trichinella

Outbred white rats are orally infected with T. spiralis larvae at a dose of 10 l / g live weight. After 1.5-2 months, the animals are killed by the method of cervical dislocation, skinned, internal organs are removed and the carcasses are digested in artificial gastric juice, changing the temperature regime in comparison with the conventional method. Digestion of muscle tissue to release Trichinella larvae is more appropriate not at 37 ° C, but at 38.5 ° -39.0 ° C, since this is the normal body temperature of carnivores, the main hosts of this helminth. The obtained Trichinella larvae are repeatedly washed with sterile saline with the addition of antibiotics, the composition of which is determined in such a way that several inhibitory agents act on concomitant microorganisms and mold fungi: β-lactam antibiotics have a bactericidal effect on microorganisms in the growth phase, inhibiting their cellular biosynthesis walls; tetracycline series - inhibit protein biosynthesis at the level of ribosomes; on fungi, aspergillus, fungi of the genus Candida, the dispersion of levorin is depressing. In this regard, 5 μg / ml of ampicillin, 6 μg / ml of gentamicin, 50 IU / ml of levorin, 2 μg / ml of doxycycline are added to the washing physiological solution and the invasive trichinella larvae are washed with it 5-7 times.

2. Biological cultivation of invasive larvae

To obtain excretory-secretory products of Trichinella larvae, further work is carried out in boxing conditions. Trichinella larvae are cultured in Petri dishes in DMEM nutrient medium supplemented with L-glutamine (40 μl / ml) and antibiotics (gentamicin 2 μl / ml and ampicillin 5 μl / ml). Sowing density 5 thousand liters / ml.

The protein products of Trichinella larvae are obtained by significantly simplifying the Gamble HR method (1): without using a CO ₂ incubator, a fixed level (10%) of carbon dioxide and (70%) moisture; in a conventional thermostat at 38.5 ° C. Protein products are taken once every 24 hours for 3-5 days, after each selection of excretory-secretory products, the initial volume is replenished by adding a new portion of the nutrient medium with L-glutamine. The viability of Trichinella larvae is daily evaluated under a microscope, if more than 30% of dead are detected, cultivation is stopped, and Trichinella biomass is used to obtain a somatic extract and fractionated antigen.

The obtained excretory-secretory products for different cultivation periods are dialyzed for 48 hours against distilled water with the addition of physiological saline (NaCl 1:10) at a temperature of 4 ° C with a buffer change every 6-8 hours. After dialysis, the antigen is concentrated against PEG-6000, the protein concentration is determined on a spectrophotometer at a wavelength of 280 nm, after which it is passed through a bacterial membrane with a pore diameter of 0.22-0.24 microns. The excretory-secretory antigen is stored at -18 ° C or in a kelvinator at -70 ° C.

3. Obtaining fractionated Trichinella antigen

To obtain fractionated somatic antigen, trichinella remaining after biotechnological cultivation is washed repeatedly with physiological saline, and then the larvae are crushed mechanically with periodic freezing and thawing. The resulting homogenate was extracted for 18 h at 4 ° C with the addition of physiological saline on a magnetic stirrer. After centrifugation at 12000 g for 20 minutes, the supernatant was collected and the resulting somatic extract was dialyzed against buffered saline (1:10) for 48 hours at 4 ° C. After dialysis, the proteins are concentrated against PEG-6000, the protein concentration is determined at a wavelength of 280 nm.

A complete somatic extract in a volume of 5 ml was fractionated on a Sephadex G-100 column, 2.5 cm in diameter, 80 cm in length, 2 protein fractions were selected. The protein concentration is determined by the method of Bradford (see drawing).

4. Analysis of the sensitivity and specificity of the obtained IFM antigens

The activity and specificity of excretory-secretory proteins and antigens of the second protein fraction of T. spiralis is determined in the ELISA reaction according to the generally accepted method (5).

Hyperimmune polyvalent trichinosis serums are obtained by immunization of rabbits according to a previously developed technique with a complete somatic antigen in a total dose of 4.5 mg. The maximum titer of antibodies is set in the preliminary ELISA, the resulting antigens are used at a concentration of 1 μg per well.

Examples of specific performance:

Example No. 1

In this experiment, diagnostic efficacy in the IGF of somatic fractionated and excretory-secretory T.spiralis antigens with blood serum of laboratory animals was studied. In the experiment, serum from rabbits was used; experimentally infected with trichinosis at a dose of 10 lich / g live weight; tissue helminths vaccinated with antigens, as well as spontaneously infected with parasitic diseases. The results presented in table No. 1 indicate that the sensitivity and specificity of both fractionated somatic and excretory-secretory antigens with blood serum of laboratory animals amounted to 100%.

Example No. 2.

In this experiment, the diagnostic efficacy of IGF was determined with somatic fractionated and excretory-secretory antigens of T.spiralis with blood serum of spontaneously infected pigs from their trichinosis-poor households and experimentally infected pig trichinosis (VIGIS serum bank). The results are presented in table No. 2. According to the results of IGF, the sensitivity of the test with the blood serum of pigs using fractionated antigen was 96.4%, and excretory-secretory - 93.5%; the specificity of both antigens was 84%.

Example No. 3.

In this experiment, a comparative analysis of the diagnostic effectiveness of antigens 2 of the Trichinella protein fraction in ELISA and Western blot was performed. Serums of humans and pigs with a confirmed diagnosis of trichinosis in both reactions were positive, and out of 42 sera from a trichinosis-poor farm, 10 positive results were recorded in an ELISA; Western blot analysis revealed 2 out of 10 positive sera that reacted in ELISA. Based on the data obtained, it can be concluded that Western blot analysis can be used not only to clarify the diagnosis, but also for the direct diagnosis of this disease. The data are presented in table No. 3.

5. Immunochemical analysis of Trichinella antigens.

Conducted gradient electrophoresis (polyacrylamide concentration from 6 to 18%) followed by computer data processing showed that all Trichinella protein products contain peptides with a molecular weight of 30, 43, 52-57, 64-69 kDa, with major proteins as in fractionated somatic , and in secretory-excretory at all periods of incubation, are antigens with a molecular weight of 43 and 53-57 kDa. The data are comparable with the results of studies by foreign authors (10).

Protein products were separated by denaturing electrophoresis in 12% separation and 4% concentration PAGE using a Bio-RAD mini protean Slab Cell. 20 μl of sample is applied per well and separation is carried out at 240 V for 50 minutes. Proteins are transferred from the gel to the filter electrophoretically using nitrocellulose filters (Schleicher-Schuell, Germany) with a pore diameter of 0.24 μm. The transfer is carried out in a makeshift semi-dry transfer chamber for 2 hours at room temperature at a current of 80 mA. In this case, the contact of the "sandwich" of the gel and nitrocellulose with lamellar graphite electrodes is ensured by gaskets of filter paper soaked in buffers.

At the end of the transfer, part of the gel is cut off and stained to determine the quantitative transfer of proteins, the filter is used for immune staining using polyvalent serums. Before this, the nitrocellulose membrane was incubated for 1 h at 37 ° C with 2% skimmed milk powder (Sigma USA), in 0.05% Tween-20. After washing with phosphate-buffered saline (PBS), the membrane is incubated with hyperimmune serum at a dilution of 1: 100 for 1 h, at the end of the incubation, the filter is washed with PBS from excess antibodies and then incubated with secondary antibodies (anti-rabbit IgG at a dilution of 1: 12000) and again wash off excess PBS secondary antibodies. The washed filter is immersed for 30 min in a 0.05% solution of 4-chloro-1-naphthol in PBS containing 0.06% hydrogen peroxide. After color development, the reaction is stopped by washing the filter with water. The filter is dried and stored in the dark to prevent fading. According to the results of Western blot analysis, excretory-secretory and fractionated somatic antigens contain major bands with a molecular weight in the range of 43-, 50-55 kDa. The results obtained are comparable with the data of foreign authors (5).

6. Protective properties of an immunopreparation based on Trichinella excretory-secretory antigens.

Example No. 4.

Studies were performed on 44 white mongrel mice weighing 18-20 g, divided into 3 groups:

the first group was immunized subcutaneously twice with an interval of 14 days with a preparation consisting of an excretory-secretory antigen (30 μg of protein in 0.125 ml by volume) and an equal amount of Freund's complete adjuvant (0.125 ml);

the second group of mice was immunized subcutaneously with Freund's adjuvant alone in the same dose twice with an interval of 14 days;

the third group was injected twice subcutaneously with sterile saline according to the same scheme.

After 14 days after the last immunization, all three groups of mice were infected with Trichinella invasive larvae at a dose of 100 larvae per animal. Accounting for the results of the experiment 60 days after infection is presented in table No. 4. In the first group of animals immunized with E / C AG + PAF, the number of larvae in 1 g of muscle tissue after digestion in IHL was on average 75.2 larvae in the group; in animals of the 2nd group (PAF + physical solution), the same indicator was 1622 l / g. In the control group (physical solution), the invasion intensity was 2109.3 l / g. The above calculations, as can be seen from the table, show that the level of protection during experimental infection of T. spiralis in mice vaccinated with this drug was 96.4% (((2109.3-75.2): 2109.3) × 100% = 96.4%), which significantly exceeds the protective effect obtained by foreign authors (8).

7. The study of the protective properties of the drug based on excretory-secretory antigens of Trichinella in rabbits. 8.

Example No. 5.

The studies were carried out on 16 young rabbits of the breed "Soviet Chinchilla", divided into two equivalent groups - experimental and control. The experimental group of animals was twice (with an interval of 10 days) subcutaneously immunized with a preparation consisting of equal volumes (0.25 ml) of Freund's complete adjuvant and excretory-secretory antigen (protein dose of 250 μg).

The control group of rabbits was injected at the same time with sterile saline. After 14 days after the last immunization, both groups were infected with invasive larvae of Trichinella at a dose of 4 thousand per animal.

Slaughter of experimental animals was carried out 60 days after infection with Trichinella. All muscle tissue was separated from the carcasses of rabbits and ground in an electric meat grinder (each separately), thoroughly mixed and proteolysis of three aliquots (50 g) of minced meat from each carcass in artificial gastric juice was performed. The results of the experiment are presented in table No. 5. The indicator of the average number of larvae in 1 g of muscle tissue over the whole group of rabbit Trichinella vaccinated with E / C AG was 38.8; while in the control group this indicator was 1390.6 l / g. The above calculations show that the level of protection during experimental infection of T.spiralis in rabbits twice vaccinated with this drug was 97.2% (((1390.6-38.8): 1390.6) × 100% = 97.2%) .

The experiments showed a high immunogenicity of the drug based on the Trichinella excretory-secretory antigen with PAF when administered twice subcutaneously to laboratory animals, the protection effect was 96-97%.

The immunochemical analysis fully characterizes the main immunogenic Trichinella antigens at the molecular level, and the IFM shows their high specificity and sensitivity in serological reactions with blood serum of both several animal species and people infected with trichinosis.

Information sources.

1. Klimenko V.V., Belozerov S.N. Bull. All Institute of helminthol. - 1986. - issue 44 - S.31-38.

2. Arriaga C., Muniz E., Morilla A., Ortega-Pierres G. Trichinella spiralis: Recognition of muscle larva antigens during experimental infection of swains and its potential use in diagnosis. Exp. Parasit. 1989. 69: 363-372.

3. Gamble H.R. Trichinella spiralis: Immunization of Mice Using Monoclonal Antibody Affinity-Isolated Antigens. Exp. Parasitology 59, 398-404, 1985.

4. Gamble H.R., Graham C.E. Monoclonal antibody-purified antigen for the immunodiagnosis of swine trichinosis. Vet. Res., 45, 67-74, 1984.

5. Helene Y., Shakir A. Development and Evaluation of a Western Blot Kit for Diagnosis of Human Trichinellosis (PubMed), 2003.

6. Co., O.S., Fan L. Heat shock respons to Trichinella spiralis and T. pseudospiralis. Parasitology 1996, 112: 89-95.

7. Parkhouse R.M., Philip M., Ogilvie V.M. Characterization of surface antigens of Trichinella spiralis infective larvae. Parasite Immunol., 1981, 3: 339-352.

9. Silberstein D.S., Despommier D.D. Antigens from Trichinella spiralis that induce a protective response in the mouse. J. of Immunology, 132, 898-904, 1984.

9. Tyrcekova L., Borovkova O. Immunochemical analysis of larval antigens of Trichinella spiralis and T. pseudospiralis. Helmintologia, 34, 4: 241-243, 1997.

10. Wu Z., Nagano I. A panel of antigens of muscle larvae of Trichinilla spiralis and T. pseudospiralis as revealed by two-dimensional western blot and immunoelectron microscopy. Parasitology, 118 (Pt 6): 118, 1999.

Table number 1.
Investigation of diagnostic efficacy in IGF of somatic fractionated and excretory-secretory antigens of T.spiralis with blood serum of laboratory animals. Rabbit blood serum The number of animals in the group IGF Results fractionated somatic antigen excretory secretory antigen will put. neg. feels. spec. will put. neg. feels. spec. Experimentally infected with trichinosis 8 8 - one hundred% one hundred% 8 - one hundred% one hundred% Hyperimmunization. extract of T.spiralis larvae 5 5 - 5 - Immunized. excret / secret. AG T.spiralis + PAF 9 9 - 9 - Hyperimmunization. E. multilocularis extract (from protoscolexes) 2 - 2 - 2 Immunized. Ag E.multilocularis + PAF with sequ. infection with E. multilocularis 12 - 12 - 12 spontaneously infected with passaluris 2 - 2 - 2 Experimentally infected cryptosporidium culture 12 - 12 - 12 Spontaneously infected with pisiform cysticercosis 3 - 3 - 3 Healthy rabbits 5 - 5 - 5

Table No. 3.
Comparative analysis of the diagnostic effectiveness of antigens of the 2nd protein fraction of Trichinella in the reactions of ELISA and Western blot Blood serum quantity ELISA Results Western blot results positive negative Tested in Western blot positive negative Serums of people with confirmation. the diagnosis 3 3 - 3 3 0 Confirmed Trichinosis Serum Pigs 16 16 - 16 16 - Porcine sera from a trichinosis-poor farm 42 10 32 10 2 8

Table No. 4.
Study of the protective properties of Trichinella excretory-secretory antigens in an experiment on outbred mice Group number The number of mice in the group Immunizing agent Immunization Dose μg Protein The frequency of immunization The interval between immunizations (days) Dose of infection (number of larvae) The average number of larvae in 1 g of muscle tissue after transfers to IZHS The average indicator for the whole group (larvae) one 2 3 one thirty E / S AG T.spiralis + PAF (1: 1) thirty 2 fourteen one hundred 89.5 64.7 71.3 75,2 2 12 PAF + physical. rr - 2 fourteen one hundred 1576 1820 1470 1622 3 twenty Sterile physical rr - 2 fourteen one hundred 2038 2301 1989 2109.3

Table No. 5.
Study of the protective properties of Trichinella excretory-secretory antigens in an experiment on young rabbits of the breed "Soviet Chinchilla". Group number Immunizing agent Protein Immunizing Dose Multiple immunization Immunization Interval Dose of infection (number of larvae) The average number of larvae per 1 gram of muscle tissue Group average one 2 3 four 5 6 7 one E / S AG + PAF (1: 1) 175 2 fourteen 4000 25,4 111 67.8 7.9 0.6 26.3 46.8 38.8 2 Sterile physical rr - 2 fourteen 4000 1433.7 1021.3 1717.0 1390

Claims (1)

A method of producing an immunogenic antigen of Trichinella spiralis, including infection of laboratory animals, separation of muscle tissue and its proteolysis in artificial gastric juice, isolation of invasive larvae and their cultivation in an artificial nutrient medium, separation of the protein product, its purification and immunochemical screening, characterized in that proteolysis muscle tissue of a laboratory animal in artificial gastric juice is carried out at a temperature of 38.5-39.0 ° C, isolated invasive larvae of Trichinella are washed 5-7 times with a physiological saline solution, to which antibiotics with a different spectrum of action are added, after which cultivation of invasive larvae is carried out for 3-5 days at a temperature of 38.5 ° C, and after separation of the protein product by means of its dialysis and concentration, an excretory-secretory antigen is isolated, and the trichinella larvae remaining after separation of the protein product are crushed, the resulting homogenate is extracted, centrifuged from the somatic extract by fractionating it on a Sephadexo column m G-100 additionally receive fractionated somatic antigen.

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