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

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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.