Visualization of natural autoantibody polyreactivity by rotary metal-shadowing electron microscopy

Immune complexes formed by mouse polyspecific natural autoantibodies and various structurally different antigens, such as DNA, tubulin and myosin, were analysed by rotary-shadowing electron microscopy. Each of the four natural IgM autoantibodies studied (E7, D23, 3C3 and M2-9) recognized multiple epitopes on the myosin molecule. These results, confirmed by immunoblotting experiments using myosin subfragments as antigens, strikingly contrasted with those obtained with an induced myosin-specific IgG antibody which interacted with a single myosin antigenic site. Based on the measurements of the antibody position on the antigen, made on a series of electron micrographs, two negatively charged myosin peptides were prepared by solid phase synthesis. Polymeric forms of one of the two peptides interacted with the positively charged CDR part of E7 and inhibited the binding of E7 and M2-9 to myosin. The importance of charge in the observed cross-reactivities was further supported by enzyme immunoassays showing that most, but not all, antigen/natural autoantibody interactions were sensitive to increasing concentrations of NaCl.