Production and characterization of mouse monoclonal antibodies recognizing multiple subclasses of human IgG

ABSTRACT

Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability if specific Monoclonal antibodies [MAbs]. In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes form Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins [Igs] of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 [n=4] and IgG1,2,3 [n=3]. Immunoblotting studies revealed recognition of linear [n=4] or conformational [n=3] epitopes by these MAbs. The most abundant cross-reactivity [71.4%] was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MABs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. Thes MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These Mabs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies