Method for the diagnosis of sheep pox and smallpox goats histochemical enzyme linked immunosorbent assay based on monoclonal antibodies

 

(57) Abstract:

The invention relates to the field of veterinary Virology. Proposed method for the diagnosis of sheep pox and smallpox goats. The proposed method provides for histochemical enzyme immunoassay using peroxidase conjugate based on monoclonal antibodies clone 010.2. Antibodies of this clone have specificity for antigenic determinants of a polypeptide of molecular weight 36-40 KD virus sheep pox and smallpox goats. While the detection of antigens of the virus sheep pox and goat takes place in the cytoplasm of infected primary cells or transplantable cell cultures of sheep origin. The proposed method is more simple, sensitive, specific, allows to reliably detect the antigens of the virus sheep pox and smallpox goats and to differentiate them from the diseases that occur with clinically similar pattern. The method can be used in research institutes and veterinary laboratories. table 1.

The invention relates to the field of veterinary Virology, in particular to a method for the diagnosis of sheep pox and smallpox goats by identifying specific antigens above sosbud the clonal antibodies, and can be used in research institutes and veterinary laboratories.

One of the conditions of production of reliable diagnosis of various infectious viral diseases, including when sheep pox and smallpox goats, is the allocation of the pathogen with the use of sensitive biological systems - susceptible animals or permissive primary and transplantable cell culture followed by identification virousspecificakih antigens in a variety of serological reactions.

Taking into account the fact that when virus isolation is the gradual adaptation of pathogens to the biological system, resulting in the first passages there is minimal accumulation of the virus, for early diagnosis requires the use of more sensitive methods. Such methods, which allows to detect the antigens of the virus sheep pox and smallpox goats in the cytoplasm of infected cells and used in the diagnosis of sheep pox and smallpox goats are direct immunofluorescence reaction, the reaction of indirect immunofluorescence and immunohistochemical method enzyme immunoassay.

The reaction of both direct and indirect immunofluorescence (Sarkn. J. Anim. Sci. - 1980. - V. 50, no. 5. - P. 428-433; OIE Manual of standards for diagnostic tests and vaccines. - 1996. - P. 119-127) provides for the use of test drugs, representing a fixed acetone swabs-prints of edematous fluid winced. For detection of antigens of the virus sheep pox use immunoglobulins isolated from hyperimmune serum or conjugated with fluorescein-isothiocyanato for the reaction of direct immunofluorescence or isolated antibodies from hyperimmune serum with subsequent detection of immune complexes using individualo (anti-goat) fluorescin-isothiocyanato conjugate in the case of the reaction of indirect immunofluorescence.

The disadvantages of these methods is the necessity of using a fluorescent microscope to record the results of the reaction and the lack of specificity due to the use of specific immunoglobulins isolated from polyclonal hyperimmune sera for detection of immune complexes.

A fundamentally different method immunohistochemical assay for the diagnosis of sheep pox was proposed Gulbahara M. Y. et al. (Gulbahara M. Y., Calabar M., Gul y, Icen H. Immunohistochemical detection of antigen in lamb tissues naturally infected with sheep-pox virus // J. Vet. Med. B. - 200 which I in the cytoplasm of cells of organs and tissues of sheep, stricken with smallpox, carried out directly in the selected samples. For this stricken with smallpox organs and tissues of sheep fixed with formalin and prepared paraffin blocks. After preparation, slices of tissues and organs and their dewaxing spend the detection of antigens. With this purpose, initially on the surface of the slice are applied specific to antigens of the virus sheep pox immunoglobulins isolated from hyperimmune rabbit serum. Then to enhance the positive signal on the surface of the slice are applied biotinylated anti-rabbit antibodies. The detection of the formed complex antigen - specific antibody - biotinylated anti-rabbit antibodies spend streptavidin peroxidase conjugate.

This method along with positive aspects - expressnet and the relative simplicity of the setting reaction has a number of drawbacks. These include the use of polyclonal immunoglobulins isolated from hyperimmune sera, which take a cross-reactions with antigens of the virus contagious pustular dermatitis of sheep and goats and can cause nonspecific background staining, due to the use of nizkozhirnyh antigens in obtaining hyperim is possible, using monoclonal antibodies having specificity for antigenic determinants of the virus sheep pox and goat. Way to obtain is suitable for these purposes, monoclonal antibodies and their characteristics described earlier (Magnin A. C., , Summary A. A., Strizhakova O. M., Novikov M. B., Sereda A. D. Development of diagnostics sheep pox on the basis of monoclonal antibodies // Diagnosis, prevention and control measures particularly dangerous, exotic and zooantroponoznyh animal diseases. Sat. articles international. scientific-practical. Conf. - Cover. - 2000. - S. 141-143).

The aim of the present invention is to develop a method for the diagnosis of sheep pox and smallpox goats histochemical enzyme linked immunosorbent assay based on monoclonal antibodies.

This goal is achieved by the fact that the detection of the antigens of the virus sheep pox and smallpox goats are histochemical enzyme linked immunosorbent assay involving the use of peroxidase conjugate based on monoclonal antibodies clone 010.2, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 KD virus sheep pox and smallpox goats.

Specific example

When setting histochemical immunopharmaceutical lamb (TAN) and transplantable cell culture kidney sheep (ON), grown on cover glasses in test tubes, after removal of the culture medium and three times washing of the monolayer medium for cultivation without serum was added to the clarified suspension two-fold dilution of the control culture (normal and special), and analyzed organotypic antigens. Contact of cells with the test materials was carried out for 1 h at 37(1)C. then the supernatant was removed, cells washed twice with media for cultivation without serum. Further cultivation of the cells was carried out with addition of 2% sheep serum and antibiotics (100 U/cm3penicillin, 100 mg/cm3streptomycin) at 37(1)for 5-6 days. Then the cell monolayer grown on slides were fixed for 10 min at room temperature 100% acetone, cooled to minus 20C. The glass was dried in air and further used as test drugs.

Before setting reaction test drugs enshrined in the matches, washed for 5 min wash buffer (phosphate buffered solution pH 7,2-7,4 (STR); 0.05% tween-20). Then, on the edge of the slide caused by the drop of specific virus sheep pox and smallpox goats peroxide glass fixed monolayer down. Conjugate were incubated for 1 h at 37(1)in a humid chamber. Nepovsemestnoy conjugate is washed twice 3-5 min in wash buffer and once SFR. Then, the slides were immersed in a chromogenic substrate solution and after 30 min incubation at room temperature, cover glasses were placed in a drop of glycerin, placed on a glass slide, and conducted analysis of the reaction by a light inverted microscope.

On the specific interaction peroxidase conjugate based on monoclonal antibodies clone 010.2 with antigens of the virus sheep pox and smallpox goats in infected cells was judged by the presence of characteristic dark brown staining of cells in the location virousspecificakih antigens.

In determining the sensitivity and specificity of this method used the test drugs, representing fixed intact and infected cell culture THEE and homologous viruses isolated from subcutaneous clutchette from smallpox lesions, Joan and a lesion in the lung of infected animals, as well as heterologous viruses of small ruminants (virus contagious pustular dermatitis of sheep is knosti of this method are presented in the table.

The research results presented in the table show that the proposed method enzyme immunoassay based on monoclonal antibodies can diagnose sheep pox and smallpox goats and to differentiate this disease from other diseases of small ruminants, flowing with clinically similar pattern.

The proposed method has higher sensitivity and specificity compared with existing methods. It is accessible, easy to use and can be applied in research institutes and veterinary laboratories.

Method for the diagnosis of sheep pox and smallpox goats, including the use of histochemical enzyme immunoassay for the detection of specific antigens of the virus sheep pox and smallpox goats, characterized in that the detection of the antigens of the virus sheep pox and smallpox goats are in the cytoplasm of infected primary cells or transplantable cell cultures sheep origin using peroxidase conjugate based on monoclonal antibodies clone 010.2, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 KD virus sheep pox and smallpox goats.

 

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