Monoclonal antibodies against ovine IgA purified from lung lavage fluid.

ABSTRACT

Ovine secretory IgA (sIgA) has been purified to relative homogeneity by ammonium sulphate precipitation (to 40 per cent w/v) of lung lavage fluid from 3- or 12-month-old lambs, followed by molecular sieve chromatography on a Sephacryl S300 matrix. Three peaks A, B and C with molecular sizes corresponding to 550,000, 400,000 and 165,000 respectively were eluted from the column. Immunoelectrophoresis, radial immunodiffusion and enzyme-linked immunosorbent assays (ELISAs) with class specific antiserum confirmed that peak B contained only IgA. Polyacrylamide gel electrophoresis of peak B under reducing conditions resolved three subunits corresponding to secretory component, heavy and light chains. Hybridomas generated by fusing spleen cells from IgA-primed Balb/C mice with murine myeloma (Sp2/0) were screened for IgA-specific monoclonal antibodies against a panel of ovine IgG2, IgG1, IgA and IgM. One particular clone, F3-4B4, identified as murine IgG1, was monospecific against ovine IgA with no cross reactivity against bovine immunoglobulins. This hybridoma was successfully tested as a serological probe by ELISA profiling to locate IgA containing fractions in the course of immunoglobulin purification from biological fluids.