The presence of antinuclear antibodies (Ab) is a major laboratory finding in systemic lupus erythematosus (SLE). Ab to nuclear ribonucleoprotein such as Sm, RNP, Ro/SSA and La/SSB have been found to be useful diagnostic markers. The genes encoding many of these autoantigens (auto- Ag) have been cloned. The recombinant proteins are useful in clinical investigations. The linkage between the major histocompatibility complex and responses to some of these auto-Ag has been established. Evidence suggests that many of these responses are Ag-driven. However, the inciting Ag and the role of the Ig gene complexes remains to be clarified. Thus the long-term objectives are to elucidate the molecular and genetic basis of autoantibody production in SLE.
Four specific aims are proposed. (1) to express ribonucleoprotein Ag in both prokaryotic and eukaryotic systems and to explore the various renaturation protocols to obtain bacterial recombinant proteins in a native state. Initial efforts will be centered on the 60kD Ro/SSA Ag. The protein products will be used to determine the extent of the anti-Ro/SSA response against the non-linear epitopes. (2) To determine the reactive epitopes of the myeloma proteins which are reactive with Ro/SSA and to determine their V region sequences to gain insights to the role of Ig V genes in this process. (3) To identify in vitro systems for anti-Ro/SSA Ab production and to use them to generate cell lines secreting anti-Ro/SSA Ab production and to use them to generate cell lines secreting anti-Ro/SSA Ab by EBV transformation and/or by heterohybridoma generation. The V region genes encoding for the autoantibodies and the relevant germline genes will be identified in order to determine the genetic element important in this response. (4) To screen phage epitope libraries with monoclonal Ab identified of generated in Aims 2 and 3 and affinity purified polyclonal antibodies from patients' plasma with antigens expressed in Aim 1. The sequences of the reactive epitopes will be used to search for proteins with homologous sequences in the gene bank data base. This approach may provide clues as to the inciting antigen(s). Results of these experiments will provide significant information regarding the pathogenesis of SLE.
