[Preparation and characterization of mouse monoclonal antibody against outer membrane protein P6 of Haemophilus influenzae].

ABSTRACT

OBJECTIVE: To prepare and identify monoclonal antibody against Haemophilus influenzae(Hi) outer membrane protein P6. METHODS: Recombinant protein P6 as an immunogen was administered intraperitoneally to BALB/c mice. The splenocytes of the mouse were isolated from spleen and hybridized with Sp2/0 myeloma cells. Indirect ELISA was used for screening hybridoma and the number of chromosomes in hybridoma cells was determined by karyotype analysis. The titers and specificity of monoclonal antibodies in their culture supernatant were detected by indirect ELISA. The immunoglobulin class, subclasses and type of the monoclonal antibody were identified with colloidal gold labeled IsoQuick(TM) strips. RESULTS: Two hybridoma cell lines designated α2G3 and γ2C4 were obtained. Karyotype analysis showed that the chromosome numbers of α2G3 and γ2C4 were 103 and 95, respectively. The highest titers of antibodies in their culture supernatant were 1256 and 1512, respectively. Both monoclonal antibodies only reacted with standard or clinical isolated strains of Hi, and they both did not react with other bacteria. A2G3 was IgG2b, and γ2C4 was IgM, both of which were kappa light chains. They could recognize different antigen epitope of protein P6. CONCLUSION: Two hybridoma cell lines producing the monoclonal antibodies against protein P6 of Hi outer membrane are obtained.