Anti-basement membrane antibody: immunoenzymatic assay and specificity of antibodies

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Abstract
A sensitive enzyme linked immunosorbent assay has been developed for circulating anti-glomerular basement membrane antibodies and optimal conditions for each steps have been determined. Structurally different preparations of antigens from glomerular basement membrane are coated to the walls of polystyrene tubes. Sera containing either specific antibody or negative control are added and allowed to react. Immunoglobulins bound to the antigens on the tube are then detected using anti-immunoglobulin antibodies coupled to alkaline phosphatase. The enzymic activity is measured using the fluorogenic substrate methylumbellipheryl phosphate. The fluorescence recorded is proportional to the concentration of specific antibodies in the serum tested. The specificity of the assay has been determined. The two different structural components of glomerular basement membrane were used as antigens. Type IV collagen was prepared after pepsin digestion, while glycoprotein components were solubilized by digestion of glomerular basement membrane with collagenase. Sera from rabbits immunized with purified type IV collagen reacted with type IV collagen only, while serum from a rabbit immunized with a glycoprotein component of human glomerular basement membrane reacted with collagenase digests of glomerular basement membrane only. Sera from several patients with antibody mediated nephritis (Goodpasture's syndrome) reacted with glycoprotein components in collagenase digests, but not with type IV collagen prepared from human glomerular basement membrane.