Ehrenstein, Michael Randolph; (1995) Production and analysis of human monoclonal IgG anti-DNA antibodies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The hypothesis underlying the work described in this dissertation is that anti-deoxyribonucleic acid (DNA) antibodies of the immunoglobulin G class are pathogenic in the disease systemic lupus erythematosus (SLE), particularly in patients with renal involvement. The diversity of circulating antibodies in SLE prevents their precise study without producing monoclonal antibodies. Thus the objective was to generate and analyse human monoclonal IgG anti-DNA antibodies in terms of their fine specificity, idiotypes and biological properties. Initial studies using a number of techniques demonstrated that a heteromyeloma cell line, designated CB-F7, when fused with the peripheral blood lymphocytes of lupus patients yielded the highest number of monoclonal IgG antibodies. Using this technique, five monoclonal IgG anti-DNA antibodies from one patient with active lupus were generated. A comparison with five monoclonal IgM anti-DNA antibodies, derived from the same patient when her disease was inactive, demonstrated a change to the IgG isotype associated with active disease and increased binding affinity to dsDNA. Two IgG anti-DNA antibodies, designated B3 and D5, were selected for further study. To analyse the structure and distribution of the idiotypes related to these antibodies in the serum and tissue of patients with SLE and other diseases anti-idiotypic reagents were produced against B3 and D5. The idiotype associated with 83 was at or near the binding site for DNA, and may be partly encoded by two adjacent arginines in the complementarity determining region (CDR) 1 of the light chain. The expression of this idiotype in serum was associated with active arthritis in lupus patients. A polyclonal and monoclonal anti-idiotype were produced which identified idiotypes on D5 (D5-RId and D5-MId respectively) and although generated from different species each identified overlapping structures on D5 which were at or close to the binding site for DNA. D5-MId, but not D5-Rld, was found deposited in renal lesions of some SLE patients. The biological properties of anti-DNA antibodies were investigated in terms of their ability to bind to murine renal and other tissue antigens in vivo. This was achieved by transferring hybridomas secreting the antibodies into mice with severe combined immunodeficiency. B3, but not D5, bound to cell surfaces in a variety of organs including the kidney and induced proteinuria. One anti-DNA antibody derived from another SLE patient deposited exclusively in the glomeruli of the mice but none of the antibodies caused glomerulonephritis. In conclusion, human monoclonal IgG anti-DNA antibodies were produced which are representative of a proportion of antibodies found in the serum of SLE patients with active disease. The expression of an anti-DNA antibody associated idiotype, which was located in CDR1 region of the light chain, in the serum of SLE patients was associated with active arthritis. Only a proportion of IgG anti-DNA antibodies bound to tissue structures in vivo.

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