Monoclonal Anti-Rat MHC (H-1) Alloantibodies

Abstract

We wish to report the isolation of 6 clones of hybrid myelomas secreting rat antibodies with specificity for alloantigens of the rat major histocompatibility complex H-1 (AgB). An account of the isolation of the first such clone including details of the immunization and fusion techniques has been published (1). Briefly two AO (H-1^w, AgB2) rats were hyperimmunized against DA (H-1^a, AgB4) lymphoid cells and finally boosted with an intravenous injection of DA cells 2.5 days or 4 days before fusion. Spleen cells were fused with P3-X63-Ag8 BALB/c mouse myeloma cells in the presence of Polyethylene Glycol (PEG) 1500. We used the intravenous route to boost before fusing to favour localization of the antibody response in the spleen (2). After fusion, aliquots initially containing 2 x 10⁶ spleen cells were grown in the wells of 6 x 4 Linbro trays in HAT medium for two weeks. Active growth of presumptive hybrids was seen in all wells. Supernatants obtained at this stage were assayed for specific antibody by ⁵¹Cr release from AO and DA RBC in a complement dependent lytic assay. 106/113 wells contained significant lytic activity against DA RBC while no autoantibody activity against AO RBC was detected.