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

Two monoclonal antibodies (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 two lines were selected and cloned. Both MAbs IG8 and IG10 subsequently demonstrated specificity for MIS by their ability to (a) inhibit biologically active MIS by precipitation with a second antibody, (b) directly block MIS activity in the organ culture assay, and (c) adsorb and elute active MIS when coupled to a solid support. SDS-polyacrylamide gel electrophoresis of affinity purified MIS demonstrated a major band at 140 kD in unreduced gels and two bands with approximate molecular weights of 70 and 74 KD following reduction. Protein bands were localized either directly by silver staining or on immunoblots developed with radiolabeled anti-MIS MA.