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Production of Monoclonal Antibodies Against Banana Bunchy Top Virus and Their Use in Enzyme‐Linked Immunosorbent Assay
Author(s) -
Wu R. Y.,
Su H. J.
Publication year - 1990
Publication title -
journal of phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.53
H-Index - 60
eISSN - 1439-0434
pISSN - 0931-1785
DOI - 10.1111/j.1439-0434.1990.tb04266.x
Subject(s) - monoclonal antibody , biology , antibody , clone (java method) , antigen , virology , isotype , microbiology and biotechnology , immunology , biochemistry , gene
When purified banana bunchy top virus (BBTV) was used to immunize Balbc mice, all 480 wells from one fusion contained hybridomas and culture fluid from 92% of them showed a positive reaction to BBTV using plate trapped antigen (PTA) ELISA (also known as indirect ELISA). After a third screening, 84 hybridoma cell lines retained their ability to produce BBTV‐specific antibodies. Fitry‐eight monoclonal cell lines with the ability to produce BBTV‐specific antibodies. were obtained when these hybridomas were cloned by limiting dilution to single cells. The antibodies produced by these monoclonal hybridomas were mostly isotype IgG2a. The titres of antibody in culture supernatant and ascitie fluid of one clone were 10 4 and 10 4 , respectively, while those of another clone were as high as 10 4 and 10 4 . Hybridomas produced 40 to 62 times more anti‐BBTV specific antibodies in mouse ascites than in culture. The minimum active concentration of purified immunoglobulin produced by three hybridomas ranged from 0.03 to 0.06 μg/ml, while the minimum detectable concentration of BBTV antigen was 0.26 μg/ml with PTA ELISA. Antibody trapped antigen (ATA) ELISA (also known as direct ELISA) was 16 times more sensitive than PTA ELISA in detecting BBTV. Although PTA ELISA could not detect BBTV in crude extract of diseased tissues, ATA ELISA was able to detect the virus even when the extract was diluted 1/512.