Establishment of Anti-TNP Antibody-producing Human Lymphoid Lines by Preselection for Hapten Binding followed by EBV Transformation

Abstract

Human lymphoblastoid cell lines that produce specific antibody against the haplen trinitrophenyl (TNP) have been established by selecting TNP-binding human B lymphocytes by TNP-rosetting and Ficoll-lsopaque separation, followed by Epstein-Barr virus (EBV) immortalization. Derived lines secreted polyclonal anti-TNP antibodies and contained relatively small numbers of specific rosette- and plaque-forming cells against TNP-RBC. Following rerosetting with TNP-RBC, the frequency of rosette-forming cells increased from 2% to 75%. In parallel, the frequency of plaque-forming cells increased from 0.4% to 30%. The antibody titres in the supernatants increased from 64 to 512 and from 48 to 192, as measured by TNP agglutination and haeniolytic assays, respectively. The antibodies were I9S, IgM. The specificity of the anti-TNP antibody was confirmed by the hapten inhibition test, in comparison and cross-reactivity tests with the supernatant of the previously established. EBV-transformed anti-4-hydroxy-3,5-dinitrophenacetic acid (NNP) antibody-producing cell line. Both antibodies were specific: the homologous hapten inhibited them but the heterologous hapten did not.