Purify FHA From Bordetella Pertussis Culture, Preparation And Application Of Monoclonal Antibody Against FHA

[objective] To prepare pertussis filamentous hemagglutinin(FHA)from a Bordetella pertussis culture, and obtain monoclonal antibodies(McAbs)against FHA, develop a method for quantitative analysis of FHA. [Methods] With ammonium sulfate precipitation and ion exchange chromatography purify FHA from a Bordetella pertussis culture, the hybridoma technique was used to prepare McAbs, which were identified and purified. HRP was marked in polyclonal antibodies with Sodium iodide. Then an ELISA method for quantitative measurement of FHA was developed with the anti-FHA McAbs. [Results] FHA has been separated from a Bordetella pertussis culture, and four cell strains which ascites McAbs titer were 1:105～1:106 has been obtained. The isotype of the four McAbs is IgG1 or IgG2b. They were specifically recognized pertussis FHA without cross reaction to pertussis toxin and influenza virus hemagglutinin. Based on these results, a sandwich ELISA for measurement of FHA was established, the intra-and inter-assay coefficients of variation(CK)were 2.54%～8.13%and 3.42%-5.85%respectively. The linear detection range of this assay is 1.56～100ng/ml. The average recovery rates were 94.73%,108.67%,116.37% respectively. [Conclusion] FHA has been separated from a Bordetella pertussis culture successfully, and four cell strains which can last secreting antibodies against FHA has been obtained. An ELISA method for detecting quantitatively FHA was established and applied for quantitative analysis of pertussis FHA concentration.