Expression of the fabs of human auto-antibodies in Escherichia coli: Optimization and determination of their fine binding characteristics and cross-reactivity

The Fabs of three human auto-antibodies (B3/33H11, anti-DNA; UK4, anti-phos pholipid) and six related hybrids have been cloned and expressed in Escheri chia coli, and their relative binding to single-stranded or double-stranded DNA or to cardiolipin has been assessed in the presence of modulators (sal ts and serum). We describe optimized conditions that have led to significan t improvement in the quality and quantity of the purified auto-antibodies. Protein expression of the assembled and Functionally active Fabs was achiev able with a yield of up to 5 to 9 mg/l of culture. The comparative DNA/card iolipin-binding analyses of the nine Fabs in the presence of modulators dem onstrated that B3 and 33H11 L chains possess both anti-DNA and anti-cardiol ipin activities. This is the first report of the demonstration that both an ti-DNA and anticardiolipin activities may lie on the same light chain of a human autoantibody. We provide evidence that the auto-antibodies that appea red to be similar, in that they bound DNA or cardiolipin in conventional EL ISA immunoassays, exhibited significant difference in their cross-reactivit y and binding to the antigen in the presence of modulators. Such auto-antig en specificity and/or cross-reactivity may dictate the potential of an auto -antibody to cause pathogenicity and may provide an explanation as to why a pparently similar auto-antibodies behave differently in vivo. (C) 2001 Acad emic Press.