Application of a novel immunization protocol to the production of monoclonal antibodies specific for macrophages in human placenta

Abstract

A monolayer depletion/adoptive immunization protocol that biased the immune response towards recognition of placental macrophage (pMO) antigens was established. BALB/c spleen cells immune to human pMO were adsorbed onto monolayers of the B-cell line QIMR-WIL. Monolayer-depleted or unfractionated cells were transferred to irradiated recipients, which subsequently were restimulated with pMO then killed for hybridoma production. Screening of hybridomas revealed an increased proportion of pMO-specific hybridomas following transfer and fusion of monolayer-depleted cells. Two monoclonal antibodies (mAb), L9 and L21, which were generated through application of this protocol, are described. L9 recognized an antigen on cells within the villi in sections of term placenta and freshly isolated pMO. With time in culture, expression of this antigen decreased markedly. Macrophages, but no other cell type, in placental cell suspensions expressed this antigen. L9 failed to react with any peripheral blood cells. Immunoprecipitation and SDS-PAGE analyses indicated that two proteins of molecular weight (MW) 40,000 and 43,000 were recognized by L9. Sections of term placenta and freshly isolated pMO failed to react with L21. After 2-3 days in culture, however, most macrophages expressed this antigen. L21 reacted weakly with peripheral monocytes and granulocytes but not other normal peripheral blood cells. Myeloid cell lines reacted strongly with this mAb only after activation with PMA. SDS-PAGE analyses of the L21 immunoprecipitate under non-reducing conditions revealed a single band of 61,000 MW, while two bands of 46,000 and 49,000 MW were detected under reducing conditions. Cellular distribution and molecular weight analyses indicated that the antigens recognized by these two mAb were apparently distinct from previously defined myeloid antigens.