Partial Amino Acid Sequence Analysis and Variable Subgroup Determination (VH and VL) of a Monoclonal Rheumatoid Factor Derived from a Rheumatoid Arthritis Patient

Abstract

Continuous cell lines secreting monoclonal rheumatoid factors (RF) were derived from rheumatoid arthritis (RA) patients by cloning Epstein-Barr virus (EBV) transformed B cells and by hybridoma techniques. We studied five different clones with stable RF secretion. All were IgM, 4 κ and λ A. One of these clones, RFM was extensively studied, and the partial amino acid sequences of the variable regions of both heavy and light chains were determined. After affinity purification, the IgM λ RF antibody derived from the EBV clone was run under reducing conditions on SDS-polyacrylamide gel electrophoresis. The separated heavy and light chains were blotted and then sequenced by a gas-phase sequenator. The N-terminal sequence of the λ light chain corresponded to that of the V _λ III subgroup. The heavy chain of the same IgM RF clone had a blocked N-terminus, but a cyanogen bromide peptide starting after methionine at position 82 showed a sequence typical of the V_H III subgroup. Heavy and light chains were also prepared by gel filtration after reduction and carboxymethylation from the same EBV clone made into a hybridoma. After this preparation, the heavy chain was not blocked and the N-terminal sequence confirmed that the heavy chain variable region belonged to the V_H III subgroup. We believe this to be the first amino acid sequence study of a monoclonal RF derived from the repertoire of an RA patient.