RU2219551C1 - Method for obtaining diagnostic antigen in cestodes - Google Patents

Abstract

FIELD: veterinary science. SUBSTANCE: the present innovation deals with immunological diagnostics of larval cestodoses, echinococcoses, in particular. The suggested method deals with simultaneous preparing primary cell culture out of protoscoleces of cestodes and cultivating protoscoleces' cells. Then one should obtain passaged cell culture and select diagnostically active series of cellular metabolites by applying immunoenzymatic reaction. Also, the method deals with possibility for cryoconservation of passaged culture cells followed the stage of its obtaining. The method in question enables to obtain antigen at 87.09- 95.30% sensitivity and 81.9-90.2% specificity. It could be applied in medicine for obtaining highly sensitive and highly specific antigen for predicting echonococcoses. EFFECT: higher efficiency. 4 ex, 2 tbl

Description

 The invention relates to veterinary medicine, in particular to the immunological diagnosis of larval cestodoses, namely echinococcosis. It can be used for the same purpose in medicine with human echinococcosis.

 The purpose of the invention is to use a biotechnological method for producing cestode antigens.

 Known methods for producing echinococcus antigens using traditional physicochemical methods, including the acquisition of a source of antigenic material (cystic fluid, mature or larval forms of a parasite), extraction of antigen from helminth material and its purification from host proteins and non-specific protein impurities that reduce the specificity of diagnostic antigens, by molecular filtration followed by gel filtration on 4B Sepharose and isolation of a fraction with a molecular weight of 600 kDa (N. N. Ballad and other "Method for the diagnosis of alveococcosis" AS 766583, 1980). They also carry out a combination of gel filtration on Sephadex (from G-50 to G-200) and ion-exchange chromatography on DEAE-Sephadex A-50, which allows maximum purification of antigenic material from host proteins and nonspecific impurities and significantly increases the specificity of the diagnostic antigen (Klimenko V.V. et al., AC 1363564 dated 1.IX-1987).

 The disadvantage of these methods is that each time to prepare a diagnostic antigen, it is necessary to have a large amount of the original helminth material, the preparation of which is very difficult because it requires a large number of infected animals. A significant drawback when using cystic fluid as a source of antigen is that it contains host proteins, the presence of which negatively affects the specificity of antigenic preparations, therefore, it requires additional purification from these proteins and other non-specific impurities.

 Closest to the claimed technical solution is to obtain an excretory-secretory antigen of echinococcus, representing the three-day metabolic products of protoscolex parasites placed in an artificial nutrient medium (Worbes N., 1989; Worbes H. et al., 1989).

 The disadvantages of this method, as in the first case, are the difficulty in obtaining a large number of viable protoscolexes of the parasite (due to the need for infected animals), as well as their short-term viability in artificial nutrient media (no more than 3-5 days).

The proposed method for producing echinococcus antigens is based on the use of cell technology; the antigen is a diagnostically active metabolite of protoscolex cells cultured in artificial nutrient media with the addition of the necessary growth factors for active cell proliferation in culture. The method includes the following steps:
1) preparation of the primary cell culture of protoscolexes;
2) cultivation of protoscolex cells;
3) obtaining transplantable cell culture;
4) selection of diagnostically active series of cellular metabolites ("cellular" antigen).

 Preliminary preparation of the necessary glassware, instruments, solutions and their sterilization was carried out, according to the methods used in virology when working with cultures of animal cells and tissues ("Methods of cell cultivation" edited by GP Pinaev, 1988; "Biotechnology of animal cells" under Edited by Spiera et al., 1989; teaching aid “Culture of animal cells and tissues.” A. P. Dyakonov et al., 1978, 1980; “Cell biology in culture” edited by A.S. Corresponding Member A.S. Troshina, 1984). An assortment of disposable plastic utensils was used directly for cell cultivation (Flow, Belgium, Costar, USA). Cultivation was carried out in artificial nutrient media RPMI-1640, DMEM, Igla-MEM.

 1. Preparation of primary cell culture.

Example 1
Protoscolex Echinococcus granulosus obtained from echinococcal cysts from infected sheep in a sterile box was used as the material for the preparation of the primary cell culture. Helminth material was placed in a Petri dish and washed with saline with antibiotics at the rate of 180-200 IU / ml of penicillin and 0.8-1.0 mg / ml of streptomycin at least 3-5 times and twice with sterile saline. The washed material was ground with scissors, filtered through mill gas (cell diameter 1 mm) and subjected to gentle homogenization for 5-7 minutes in a manual homogenizer with the addition of 0.5-1.0 ml of Hanks sterile solution until a homogeneous mass was obtained. The resulting homogeneous mass was passed through filters with a cell diameter of 220, 110 and 50 μm. The process of homogenization and cell isolation was repeated 3-5 times. The quality of homogenization and the determination of the viability of the isolated cells was carried out in a Goryaev chamber after staining with a 0.4% trypan blue solution with a 20X10 microscope.

После подсчета доводили их концентрацию в суспензии до 600-800 тыс./мл и вносили в ростовую среду.The resulting cell suspension was placed in sterile centrifuge tubes, saline with antibiotics (500-1000 U / ml penicillin and 0.2-0.5 mg / ml streptomycin) was added and centrifuged at 1000-1200 rpm for 5-7 minutes . The procedure for washing cells by centrifugation was repeated at least 5-7 times. The output of the cells after all the methodological procedures was carried out on the basis that at least 38 • 10 ⁶ cells are obtained from 1 g of raw helminth material. Viable cells were counted in a Goryaev chamber according to the formula:

After counting, their concentration in suspension was adjusted to 600-800 thousand / ml and introduced into the growth medium.

2. Cultivation of protoscolex cells
Protoscolex cells were cultured in a growth medium representing a mixture of RPMI-1640 synthetic nutrient medium, 7-10% fetal serum of cattle or horse serum, 2 ml of alpha-glutamine, 50 μl of mercaptoethanol and 8 mg of gentamicin per 100 ml of medium. The cell culture was placed in a CO ₂ incubator with predetermined temperature (35-37 ^° C) and gas (CO ₂ - 5%; O ₂ - 95%) at 70% humidity in the culture chamber.

3. Obtaining transplantable cell culture
Subject to the entire procedure for processing helminth material and obtaining viable cells on the 7-10th day of cultivation, with the active proliferation of cells in the culture, the formation of a monolayer of cells was observed. During this period, the products of vital activity (metabolites) of the cells were selected and the cells were reseeded in a fresh portion of the growth medium. The cells remaining after subculture were subjected to cryopreservation in liquid nitrogen. If necessary, a portion of the frozen cells was thawed, their viability was checked, and used to cultivate and obtain a “cellular” antigen.

4. The selection of diagnostically active series of cellular metabolites ("cellular" antigen)
The cell metabolites of E. granulosus protoscolexes active in immunodiagnostic reactions were selected using active sera obtained from animals or people suffering from echinococcosis (positive control), sera of clinically healthy animals or human donors (negative control). An analysis of selected series of cell metabolites was performed by an enzyme immunoassay. Evaluation of the reaction was carried out on an automatic reader, the activity of the tested series of cellular metabolites was evaluated by optical density. A series of cellular metabolites in which the optical density with positive control sera was in the range of 0.464 and higher, and with negative 0.180-0.230, was classified as antigenactive. A series of metabolites with established antigenic activity were collected and used in further studies to determine their diagnostic effectiveness. The selected active series of cell metabolites ("cellular" antigen) were stored at -20 ^o C.

5. Example 2
The steps for obtaining the “cellular” antigen are the same as in Example 1, but secondary larvocysts of E. multilocularis isolated from experimentally infected white rats or white mice were used as helminth material. Infection of laboratory animals was performed intraperitoneally using an injection needle with a diameter of 0.5-0.8 mm in a dose of 400-600 acephalocyst E.multilocularis for rats and 200-250 for mice. Secondary larvocysts were obtained from infected animals after 4-6 months, from which they subsequently prepared a primary cell culture, cultured, obtained a transplantable cell culture, and selected diagnostically active series of cell metabolites (“cell” antigen) as described in Example 1.

Examples of specific performance
6. Example 1. The diagnostic efficacy of the selected antigenic series of cellular metabolites (“cellular” antigen) of protoscolexes E.granulosus was evaluated with 114 samples of pig sera, including 31 samples infected with E. granulosus, 18 with tenuicol cysticercosis, 15 with ascariosis, and trichinosis - 14 and clinically healthy - 36. The results of the study are presented in table 1.

 Example 2. The diagnostic efficacy of the "cell" antigen of protoscolexes of E. granulosus was evaluated with 125 human sera, including infected E. granulosus - 43, T.canis "larva migrans" - 10, T.spiralis - 5, C. cellulosae - 2 donor serum - 65. The results of the studies are presented in table 2.

 An analysis of the data on the diagnostic effectiveness of the “cellular” antigen of protocholecata echinococcus (E.granulosus and E.multilocularis) in the enzyme immunoassay indicates a sufficiently high sensitivity and specificity of the studied antigen, which makes it possible to use it as a universal diagnosticum for echinococcosis of any animals and humans.

Sources of information
1. Ballad N.E. and others. "A method for the diagnosis of alveococcosis." AC 766583, class A 61 B 10/00, 1980.

 2. Klimenko V.V., Belozerov S.N., Shekhovtsov N.V. "A method of obtaining an echinococcal diagnostic antigen for enzyme immunoassay."

 3. Methods of cell cultivation. (Collection of scientific papers), Leningrad, publishing house "Science" .- 1988.- 320 p.

 4. Spier R.E., Adams GD, Griffiths J.B. Biotechnology of animal cells. T2 (translation from English by V.M. Tarasenko, edited by G.A.Safonov) .- 1989.-520 p.

 5. Cell biology in culture. Leningrad, publishing house "Science" .- 1984.-274 p.

 6. Dyakonov L.P., Pozdnyakov A.A., Pankova G.E. and others. "Guidelines for the production, cultivation and use in production veterinary laboratories of primary and diploid cultures of animal origin." - M., 1978.-8 p.

 7. Dyakonov L.P., Pozdnyakov A.A., Pankova G.E. and others. "Guidelines for cryopreservation of cell cultures of animal origin in liquid nitrogen" .- M., - 1978.-9 p.

 8. Dyakonov L.P., Glukhov V.F., Pozdnyakov A.A. and other Culture of cells and tissues of animals: educational-methodical manual. / Stavropol, 1980.-9 p.

9. Worbes H. Echinococcus granulosus control in Erfurt region (GDR) using ELISA to diagnose canine echinococccosis.//13 ^th Conf.WAAVP, August 7-11.-1989, Berlin, GDR. Progr. and Abstracts.-Berlin.-1989.

 10. Worbes H., Thompson R.C.A., Eckert J. Occurence of the cattle strain Echinococcus granulosus in the German Democratic Republic.//Parasitology Research.-1989.-V.75.-N 6.-P.495-497.

Claims (1)

A method for producing a diagnostic cestode antigen, which involves the simultaneous preparation of a primary cell culture from cestode protoscolexes, culturing protoscolex cells, obtaining a transplantable cell culture, and selecting diagnostically active series of cell metabolites using an enzyme-linked immunosorbent reaction and with the possibility of cryopreservation of transplanted culture cells after the preparation stage.

Images