Production of Monoclonal Antibodies for Affinity Purification of Bovine Mullerian Inhibiting Substance Activity

Abstract

Two monoclonal antibodies (MAb) (IG8 and IG10) specific for Mullerian inhibiting substance (MIS) were obtained from the fusion between myeloma cell line SP2/0 and spleen cells from an A/J mouse immunized with partially purified MIS. The resulting hybridomas were screened by a solid-phase RIA and 2 lines were selected and cloned. Both MAb IG8 and IG10 subsequently demonstrated specificity for MIS by their ability to inhibit biologically active MIS by precipitation with a 2nd antibody, directly block MIS activity in the organ culture assay, and adsorb and elute active MIS when coupled to a solid support. SDS[sodium dodecy sulfate]-polyacrylamide gel electrophoresis of affinity purified MIS demonstrated a major band at 140 kD [kilodaltons] in unreduced gels and 2 bands with approximate MW of 70 and 74 kD following reduction. Protein bands were localized either directly by Ag staining or on immunoblots developed with radiolabeled anti-MIS MA.