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

 

(57) Abstract:

The invention relates to veterinary Virology, in particular to a method for the diagnosis of sheep pox and smallpox goats. The method involves sensitization of the solid phase monoclonal antibody clone 08,3, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 kDa virus sheep pox strain "niche". Detection of the formed complex of monoclonal antibody - antigen is HRP conjugate polyclonal antibodies isolated from hyperimmune serum of animals of convalescents. The method allows to increase the specificity and sensitivity of the method, and also allows rapid to detect the antigens of the virus sheep pox and smallpox goats and to differentiate them from the diseases that occur with clinically similar pattern. 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 antigen sandwich method of enzyme-linked immunosorbent assay (TF ELISA) based on monoclonal antibodies, and can be used in research institutes and veterinary laboratories.

Reaction diffusion precipitation has a low sensitivity and time-consuming.

The use of reaction both direct and indirect immunofluorescence using hyperimmune sera gives a high background of nonspecific staining.

The best is ELISA using monospecific serum proposed Carn V. M. (1995), which in contrast to the previously described S. Sharma et al. (1988) helped to get rid of nonspecific background staining.

The method proposed Carn V. M., consists in the following the immunization of a rabbit with purified virus. The next step is to contact the investigated antigen with antibodies of the substrate. Then to form a "sandwich" make monospecific serum obtained recombinant protein "merge" capripoxviruses on Guinea pigs. The detection of the formed complex of antibody - antigen - antibody spend individuum (anti - Guinea pig) conjugate.

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

This goal is achieved by the fact that for sensitization of the solid phase used monoclonal antibody clone 08,3, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 kDa virus sheep pox strain "niche". Detection of the formed complex of monoclonal antibody - antigen is HRP conjugate polyclonal antibodies isolated from hyperimmune serum of animals - convalescents.

Specific example

When setting the sandwich method TF ELISA used a 96-hole panel for microculture.

Pepper the beginning of the production method of the Institute of 0.01 M carbonate-bicarbonate pH 9.5 V for 18 hours at 4oC. After which the contents of the wells were removed, the wells washed three times wash buffer (SFR; 0.05% tween-20) and blocked the available sites of adsorption of polystyrene by incubation in the hole blocking buffer (SFR; 0.05% tween-20, 1% BSA) for 30 min at 37(0,5)oC. Then the contents of the wells were removed, twice washed with a wash buffer and added to wells of vertical rows of 0.2 cm3twofold dilutions of the control (normal and special), and the studied antigens in blocking buffer. After 1 h incubation at 37(0,5)oWith and subsequent three-time washing a wash buffer in the wells of the plate was made of 0.2 cm3specific conjugate working dilution in blocking buffer. The contents of the wells were incubated for 1 h at 37(0,5)oC. the seven Wells were washed in wash buffer, once SFR and contributed 0.2 cm3/well of chromogenic substrate ABTS solution. After 30 min incubation at room temperature was performed accounting and evaluation of results.

Accounting and valuation results the reaction is carried out photometrically.

When photometric accounting measure optical density chromogenic substrate solution at a wavelength of 405 nm. The reactions is evritania incubated specific control and test antigens, in 2 and more times higher than the optical density in the wells, which are pre-incubated control normal antigen.

In determining the sensitivity and specificity of this method was used homologous culture and organ antigens (subcutaneous tissue from smallpox lesions and hoani infected animals), heterologous antigens of virus diseases of small ruminants (Ovine catarrhal fever, plague of small ruminants, rift valley fever, AKABANE disease) and related antigens of viruses porcacchi cattle, contagious pustular dermatitis, ospowiki, chicken pox, myxomatosis. Results to determine the sensitivity and specificity of this method are presented in the table.

The research results presented in the table show that the proposed method tverdofaznogo enzyme immunoassay based on monoclonal antibodies can diagnose sheep pox and smallpox goats and to differentiate this disease from other representatives from the poxviridae family and heterologous pathogens causing infection with clinically similar pattern.

The proposed method has high sensitive is to be used in research institutes and veterinary laboratories.

Method for the diagnosis of sheep pox and smallpox goats, including the use of the sandwich method of enzyme-linked immunosorbent assay for the detection of specific antigens, characterized in that for sensitization of the solid phase using monoclonal antibodies clone 08.3, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 kDa virus sheep pox strain "niche".

 

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