Enzyme-linked immunosorbent assay for anti-DNA antibodies using fluorogenic and colorigenic substrates.
An enzyme-linked immunosorbent assay is described for the assay of anti-DNA antibodies. The method employs plastic surface for immobilization of the antigen and alkaline phosphatase-linked rabbit anti-human IgG for the detection of immune complex using PNP-P and 4MU-P as substrates. The sensitivity of the assay increased by as much as 16-fold when fluorogenic substrate was used instead of conventional PNP-P and could therefore be employed for the detection of low avidity antibodies. Using PNP-P as substrate 57% of SLE patients were positive for DNA antibody, but if 4MU-P was introduced as substrate, 71% gave a positive response. Moreover, using a fluorogenic substrate, it was possible to minimise the amount of antigen (2 nM bp). The technique is simple, reproducible and of high sensitivity.
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000974:Antibodies, Antinuclear / Q000032:analysis*; D002417:Cattle; D002863:Chromogenic Compounds; D004247:DNA / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006923:Hymecromone; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D009596:Nitrophenols; D011817:Rabbits; D013379:Substrate Specificity; D014468:Umbelliferones
D000974:Antibodies, Antinuclear; D002863:Chromogenic Compounds; D009596:Nitrophenols; D014468:Umbelliferones; C024719:4-nitrophenyl; D006923:Hymecromone; D004247:DNA; D000469:Alkaline Phosphatase