Establishment And Application Evaluation Of Indirect ELISA For Detection Antibodies Against Pseudomonas Aeruginosa From Moschus Berezovskii

Pseudomonas aeruginosa could cause the purulent diseases of Moschus berezovskii. This disease has long disease course and couldn’t be easily found. It is difficult to cure even found, so it caused enormous losses to Moschus berezovskii’s breeding. To detect the levels of antibodies against Pseudomonas aeruginosa in Moschus berezovskii and offer help and guidance for prevention and treatment of the purulent diseases, this study aimed to develop a method of indirect ELISA with strong specificity, good repeatability and high sensitivity.The outer membrane protein(OMP), extracted from PA Sp-1 was used as coating antigen. There are seven to ten bands analyzed by SDS-PAGE, and molecular weight is from 15 kDa to 100 kDa. Based on this we knew they were main OMPs of Pseudomonas aeruginosa. It was also examined by western blotting and four bands were seen, whose molecular weight were 46 kDa,34 kDa,25 kDa and 19 kDa, which could be used as coating antigen of indirect ELISA.The optimum conditions for this indirect ELISA are as follows:the optimal coating concentration of OMP was 1.5 μ.g/ml, coating temperature and time was 37℃ and 4 hours; 5% skim milk was selected as blocking buffer, blocking condition was at 37℃ for 2 hours; the dilution of first antibody and HRP-SPA was respectively 1:1000 and 1:64000, the optimal working time for both first antibody and HRP-SPA is at 37℃ for 1.5 hours; the optimal working time for TMB is at 37℃ for 15 minutes.The serum with antibodies of E.coli, Salmonella, Acinetobacter and Staphylococcus aureus was respectively tested by this indirect ELISA, and the results were all negative. The Sp-1 positive serum treated with OMP was tested by the indirect ELISA, and the results showed that the OD value of treated group was significantly decreased compared with untreated group, and the blocking rate was ranged from 63.69% to 75.38%. The repeatability of this method was carried out by inter-batch and intra-batch reproducibility test.The results showed that both the coefficient of variation was ranged from 2% to 10%. The PA sp-1 positive serum double diluted was tested by this indirect ELISA. When the dilution was 1:32000, its P/N>2, which indicated positive.30 serum samples from mice, which were immuned with Sp-1 before, were detected by this indirect ELISA. The number of the titer reaching 1:12500,1:2500,1:500 and 1:100 was respectively 13,7,2 and 2 samples. There were also 6 serum samples whose titer was lower than 1:100. At the same time,32 serum samples from Moschus berezovskii in October and November 2014 were also detected,4 serum samples were positive and others were negative.This method showed good repeatability, strong specificity, and high sensitivity. It, could be used to detect the serum samples of Moschus berezovskii group after immuned, which provided a theoretical basis for making a reasonable immune procedure and offered help and guidance for prevention and treatment of the purulent diseases.