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Artifactual Plaque Formation in Vitro and in Vivo Due to Passive Transfer of Specific Antibody

From the Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014

Abstract

The localized hemolysis in gel (LHG) assay for antibody-secreting cells was used to evaluate antibody production in heterologous erythrocyte-immunized cultures of human peripheral blood lymphocytes supplemented with 10% human plasma. Antigen-specific plaques were detected in such cultures and the number of plaques varied with different lymphocyte donors and were inhibited by heat killing the cultures before assay. However, plaque number varied directly with the number of immunizing erythrocytes added to the cultures and inversely with the number of lymphocytes added. Maximum plaque production occurred within 10 min of initiation of the cultures and occurred in the absence of any lymphoid cells. Absorption of the plasma used as culture supplement with the immunizing erythrocyte resulted in complete abrogation of subsequent specific plaque formation. Mice previously passively immunized with high titered anti-erythrocyte antibody had large numbers of plaques detected 30 min after i.v. immunization with the appropriate erythrocyte. These plaques detected by LHG are the result of carry-over of aggregates of antibody-coated erythrocytes and subsequent release of this antibody in the LHG assay and are not the result of active antibody synthesis. This "pseudoplaque" production may lead to the false interpretation of plaque formation as indicating active synthesis of antibody in vivo and in vitro where such experiments are carried out in immune animals or with antibody containing serum culture supplements.