Development Of Monoclonal Antibody Against Rabies Virus And A Sandwich ELISA Kit

Rabies virus is a highly neurotropic agent,which causes acute and fatal infection of the central nervous system in warm blooded mammals. the mortality and the annual reported rabies cases keep at high level in china recently, The stablization and sensation of the clinical dignostic kits for rabies virus still need improvement.Six to eight week-old female BALB/C mice were immunized via foot pads using purified rabies viruses, fusion of mice myeloma cells(sp2/0) and spleen cells from immunized was reached under the action of PEG,3 positive hybridoma cell strains(1G9, 1E6, 3F3)against nucleoprotein of rabies virus were screened through subcloning 、 immunobloting and indirect immunofluorescent staining, All the antibodies could be classified to Ig G2 b,The antibodies were isolated from ascites by octanoic acid- ammonium sulfate precipitation, the titer of 3F3 was 1.6 x 104. its specificity and tensity under indirect immunofluorescence for brain smear samples were similar to that of the antibody against nucleoprotein abroadly.Purified 3F3 was employed for conjugated with horseradish peroxidase HRP),the labeled 3F3 is be applied to RABV infected BHK cell, its specificity was demonstrated by immunohistochemistry,then a procedure for rabies virus using double antibody sandwich enzyme-linked immunosorbent assay was established, the parameters of detection were also optimized.The coating concentration of polyclonal serum for rabbit was 1:100, 10% calf serum blocked the coated plate for 2 h,the incubated time for the detecing samples and the HRP labeled 3F3 was 1 h, the regression equation based on titrated rabies vaccine was that as:y = 1.7012 x- 0.3596, and its correlation coefficient was R2 = 0.9882,no significant results were observed between our labeled antibody and that of the abroad antibody for 10 samples.no false results also were shown for canine distemper viruses and canine parvoviruses.