Establishment and Application of the AfuA-ELISA Assay for Haemophilus parasuis
In order to establish an ELISA assay with strong specificity and sensitivity for Haemophilus parasuis(HPS),and to provide technical support for diagnosis of HPS disease and detection of immune antibodies,2 immunogenic proteins(HbpA and AfuA)of HPS were firstly expressed for primarily screening,and AfuA was selected as the optimal coated protein.After optimizing the conditions of AfuA-ELISA reaction,the following optimal parameters were determined,including coated antigen concentration of 5 μg/mL,sealing solution of 5%skimmed milk,sample diluent of 2%BSA solution,incubation time of 30 min,sample dilution ratio of 1:25,dilution ratio of HRP-conjugated antibody solution of 1:20 000,incubation time of HRP-conjugated antibody solution of 30 min,and substrate incubation time of 10 min.The specificity test showed that the established AfuA-ELISA assay could not crossly react with the positive serum of other common pig pathogens,indicating a good specificity.For 300 clinical serum samples,177 positive and 123 negative samples were detected by AfuA-ELISA,respectively,the positive and negative coincidence rates with commercial kits were 90%and 87.5%,respectively,and the overall coincidence rate was 89%.In conclusion,the established AfuA-ELISA method could be used for clinical detection of HPS antibodies with its advantages of strong specificity,high sensitivity and high coincidence rate with commercial kits.
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维普
