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Anti basement membrane antibody immuno enzymatic assay and specificity of antibodies


, : Anti basement membrane antibody immuno enzymatic assay and specificity of antibodies. Scandinavian Journal of Clinical & Laboratory Investigation 41(8): 763-772

A sensitive enzyme linked immunosorbent assay was developed for circulating antiglomerular basement membrane antibodies; optimal conditions for each step were 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. Ig bound to the antigens on the tube are then detected using anti-Ig 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 assay was determined. The 2 different structural components of glomerular basement membrane were used as antigens. Type IV collagen was prepared after pepsin digestion; 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; 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.

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