Bioluminescent immunosorbent for rapid immunoassays.
We describe a bioluminescent immunoassay procedure which does not require a separation step to remove excess free label. A luminescent immunosorbent constituted of bacterial luciferase, FMN oxidoreductase, and an antibody coimmobilized on Sepharose is used to determine specifically the label enzyme (glucose-6-phosphate dehydrogenase, coupled to an antigen) bound by a specific antibody. The immunosorbent confines the bioluminescent reaction in a small volume, and the bound label produces NADH, which is directly used by the nearby luciferase FMN oxidoreductase enzyme system. On the contrary NADH produced by dehydrogenases in solution is directly oxidized without emitting light. Dehydrogenases contained in the biological sample do not interfere with the assay, which can be performed directly on 25 microliter of serum. In this paper we describe the general procedure and we analyze the different parameters that must be optimized.
D000818:Animals; D000936:Antigen-Antibody Complex; D004800:Enzymes, Immobilized; D038181:FMN Reductase; D005954:Glucosephosphate Dehydrogenase; D007124:Immunoenzyme Techniques; D007164:Immunosorbents; D007770:L-Lactate Dehydrogenase; D008156:Luciferases; D008163:Luminescent Measurements; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D011374:Progesterone; D011817:Rabbits
D000936:Antigen-Antibody Complex; D004800:Enzymes, Immobilized; D007164:Immunosorbents; D009243:NAD; D011374:Progesterone; D007770:L-Lactate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D008156:Luciferases; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases