Screening and identification of monoclonal antibodies against human neutrophil and detection of inhibiting opsonic phagocytic activity
Xu, L.; Gao, J.; Wang, B.
Hua Xi Yi Ke da Xue Xue Bao 22(1): 21-23
1991
ISSN/ISBN: 0257-7712 PMID: 1774029 Document Number: 384668
Using Ficoll-Hypaque gradient centrifugation, we have successfully obtained the neutrophils from normal human donors, which can be used as particle antigens for immunizing BALB/c mice. Hybridomas were produced through the fusion of SP2/0 myeloma cells and splenocytes from immunized BALB/c mice. The ratio of fusion was 1:5. The cells were fused with 30% polyethylene glycol (MW 4000). The rate of fusion was 90%. The antibody producing colonies against the membrane of neutrophils in HAT medium were selected by indirect immunofluorescence assay. Antibody positive rate was 60% (102/170). Limiting dilution was used for colonization of the antibody-producing hybridoma cells. After colonization for three times, one hybridoma cell line was obtained, which could inhibit complement-mediated phagocytosis. Culture supernatant was used against class- and subclass- specific rabbit antisera (rabbit anti-mouse IgM, IgG, IgG1, IgGd22, IgG2b, IgG3) in an agar immunodiffusion system, it was shown to be IgG22.