The detection of specific antibodies to the virus sheep pox and smallpox goats method biphasic inhibition enzyme-linked immunosorbent assay based on monoclonal antibodies

 

The invention relates to the field of veterinary Virology, in particular to a method for the diagnosis of smallpox of sheep and goats. The method comprises the first step of the interaction of the studied serum with the antigen of the virus sheep pox. In the second phase, unreacted antigen interacts with monoclonal antibody clone 08.3. The formed complex of monoclonal antibody - antigen detected specific HRP conjugate polyclonal antibodies. The proposed method is more sensitive and specific, and allows rapid carry out a study of blood sera of animals. 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 virousspecificakih antibodies by the method of two-phase inhibition enzyme-linked immunosorbent assay (TF ELISA) based on monoclonal antibodies, and can be used in research institutes and veterinary laboratories.

Currently, the most commonly used laboratory methods for the detection virousspecificakih antibodies to sheep pox and smallpox goats are: the reaction of neutralization (Davies F. G. description characterist pox in the Sudan// Bull. Anim. Health Prod. Afr. - 1979. - v. 27. - p.105-112), reaction diffusion precipitation (Bhambani C. D., Krishnamurthi D. An immunodiffusion test for laboratory diagnosis of sheeppox and goatpox// J. Tech. Path. - 1963. - v.73. - p.349); the reaction of indirect immune fluorescence (Davies F. G., Atema C. The antibody response in sheep infected with a Kenyan sheep and goats pox virus// J. Comp. Path. - 1978. - v.88. - p. 205-210; Sarkar P., Singh, S. P., Pandey A. K. et al. Application of the fluorescent antibody test in the diagnosis of sheep-pox, and study of sheep-pox virus multiplication in cell-culture// Indian J. Anim. Sci. - 1980. - v.50. - p.428-433); enzyme-linked immunosorbent assay (ELISA) (Sharma, S., Negi C. S., M. R. Yadav Application of ELISA for detection of antigen and antibodies to the virus of smallpox goats// Acta Virol. - 1988. - v.32. - p. 65-69; Cam V. M., R. P. Kitching, J. M. Hammond et al. Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus// J. Virol. Meth. - 1994. - v.49. - p.285-294).

The reaction of neutralization along with high sensitivity requires special conditions and has no expressnet. The final accounting of the results of reactions carried out in 9 days from the moment of its formulation.

Using reaction diffusion precipitation for the diagnosis of sheep pox and smallpox goats is limited to the lack of specificity due to possible cross-reactions with antibodies to the virus contagious pustular dermatitis.

The use of reaction by indirect immune fluorescence using polyclonal sera by the subject methods are ELISA. Developed Sharma S. et al. (1988) a method of inhibiting TF ELISA, combining sensitivity and performance, did not allow to differentiate capripoxviruses antibodies from antibodies to the virus contagious pustular dermatitis.

This problem has been solved Cam V. M. et al. (1994), who developed an indirect ELISA for detection of antibodies to the virus porcacchi cattle using recombinant protein P 32 capripoxviruses as a specific antigen and individualo conjugate.

The essence of the method consists in the following. Polystyrene is used as the solid phase, sensibiliser purified recombinant protein P 32, expressed in a plasmid vector. The expression product is a structural polypeptide, characteristic for all capripoxviruses. In the next step reaction of the antibodies studied serum interact with immobilized onto the solid phase antigen. Identification of the formed complexes antigen-antibody exercise individuum (antiochien, anticosti or antimycin) peroxidase conjugate.

The disadvantage of the described method, which is the prototype of the present invention, it is possible to consider that the technology immunoreagents Troyes several nucleotides, encoding the amino acid sequence of this protein can cause loss of functional activity.

The aim of the present invention is to develop a method of two-phase inhibition enzyme-linked immunosorbent assay based on monoclonal antibodies suitable for the detection of specific antibodies to the virus sheep pox and smallpox goats in the blood sera of patients and animals recover and differentiate them from antibodies to heterologous pathogens diseases of small ruminants.

This goal is achieved by the fact that at the first stage of the reaction taking place in a separate panel, antibodies investigated serum interact with specific cultural antigen of the virus sheep pox.

Detection of unreacted antigen occurs in the second stage, where as "exciting" fundamentals are immobilized on a solid phase monoclonal antibody clone 08.3, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 KD virus sheep pox strain "niche". Identification of complex monoclonal antibody - specific antigen is HRP conjugate polyclonal antibodies isolated from chipdumping inhibition enzyme-linked immunosorbent assay (TF ELISA) used 96-well tray to microculture.

a) Preparation of test panels Before setting method of preparing test panels 1 and 2. To block free sites of adsorption of polystyrene in the hole of the panel 1 made of 0.4 cm3blocking buffer (phosphate buffered solution pH 7,2-7,4 (STR); 0.05% tween-20 and 1% bovine serum albumin) and incubated for 30 min at 37(0,5)oC. the Contents of the wells were removed, the wells washed twice a wash buffer (SFR; 0.05% tween-20).

Hole panel 2 was senzibilizirani monoclonal antibody working dilution in 0.01 M carbonate-bicarbonate buffer (pH 9,5) for 18 hours at 4 (0,5)oC.

b) Setting reaction In the wells of the bottom row of panel 1 made by 0.15 cm3control (normal and special), and the samples of blood serum and prepared two-fold dilution in blocking buffer. Then in the wells of all ranks made specific antigen of the virus sheep pox strain "niche" in the working dilution in blocking buffer in a volume of 0.15 cm3and incubated for 1.5 h at 37(0,5)oC.

After an hour incubation panel 1 content-hole panel 2 was removed, the wells washed three times wash bubel 0.2 cm3at 37(0,5)oC. After 30 min, the contents of the wells were removed and washed them twice a wash buffer. The mixture is then serum-antigen of the holes of the panel 1 in the amount of 0.15 cm3transferred in the same hole panel 2. After 1 hour incubation at 37(0,5)oWith and subsequent triple wash a wash buffer in the wells of the panel contributed to 0.15 cm3specific polyclonal HRP conjugate working dilution in blocking buffer. The contents of the wells were incubated for 1 h at 37(0,5)oC. Wells sevenfold laundered wash buffer, once SFR and contributed to 0.15 cm3/well of chromogenic substrate ABTS solution. After 30 min inkubirovanija at room temperature was performed accounting and evaluation of results.

Photometric reaction accounting by photometric accounting measure optical density chromogenic substrate solution at a wavelength of 405 nm. The reaction is considered positive if the optical density of a chromogenic substrate solution into the wells, which are pre-incubated with normal control serum, 2 or more times higher optical density chromogenic is optimum whey.

In determining the sensitivity and specificity of this method was used homologous (normal and special), and specific heterologous serum for virus bluetongue, contagious pustular dermatitis, the plague of small ruminants, rift valley fever, AKABANE disease, illness Nairobi. Results to determine the sensitivity and specificity of this method are presented in the table.

The research results presented in the table indicate that the proposed method TF ELISA based on monoclonal antibodies can detect serum antibodies specific to antigens of the virus sheep pox and smallpox goats, and differentiate those from antibodies generated to diseases of small ruminants, flowing with clinically similar pattern. The method is easy to use and can be applied in research institutes and veterinary laboratories.

Claims

The detection of specific antibodies to the virus sheep pox and smallpox goats method of inhibition enzyme-linked immunosorbent assay, involving the interaction of the studied serum with anti the specific antigen polyclonal peroxidase conjugate, characterized in that the first stage of the reaction is the interaction of the test serum from the culture antigen of the virus sheep pox, in the second stage, unreacted antigen interacts with immobilizerturning on a solid phase monoclonal antibody clone 08.3, having specificity for antigenic determinants of a polypeptide of molecular weight 36-40 KD virus sheep pox strain "niche", and the formed complex of monoclonal antibody - antigen to detect specific HRP conjugate polyclonal antibodies isolated from hyperimmune serum of animals-convalescents.

 

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