The efficient production of stable, human monoclonal antibody-secreting hybridomas from EBV-transformed lymphocytes using the mouse myeloma X63-Ag8.653 as a fusion partner

Abstract

The mouse myeloma X63-Ag8.653 was fused to peripheral blood lymphocytes (PBL) from apparently healthy individuals, autoimmune patients and volunteers immunised with Rhesus (D) positive erythrocytes. Fusions were performed with or without prior transformation of PBL with Epstein-Barr virus (EBV). Using untransformed PBL, under the best conditions a mean fusion frequency of 8.4 × 10 −6 was obtained, with 22% of the resulting hybridomas secreting human immunoglobulin. Fusions with EBV-transformed cells gave fusion frequencies of 1.0 × 10 −4, with 85–90% of hybridomas secreting human immunoglobulin. The heterohybridomas formed in both cases cloned efficiently and had doubling times of 24–30 h. The heterohybridomas secreted human IgM, IgG and IgA of both kappa and lambda isotypes and culture supernatants contained up to 50 μg ml −1 of human immunoglobulin. Mouse immunoglobulin. M was not detected in the culture supernatants. 28 hybrids were selected for vigorous growth and antibody production by repeated cloning. Immunoglobulin synthesis was stabilised in 26 of these hybridomas after two or three cloning steps. The heterohybridomas have been successfully grown in large volumes for periods up to 15 months. It is concluded that the mouse myeloma X63-Ag8.653 is a suitable fusion partner with EBV-transformed B cells in the efficient production of human monoclonal antibodies.