Enzyme-linked coagulation assay. IV. Sensitive sandwich enzyme-linked immunosorbent assays using Russell's viper venom factor X activator-antibody conjugates

We have applied the enzyme-linked coagulation assay (ELCA) system to the development of an amplified immunoassay using the clotting cascade to enhance sensitivity of detection of immune complexes. The factor X-activating enzyme of Russell's viper venom was detectable using ELCA in amounts as low as 0.25 fg per assay. Monoclonal antibodies to beta-hCG, placental alkaline phosphatase (PLAP), and the P-24 antigen of HTLV-III were labeled with this enzyme or peroxidase and used for "sandwich" immunoassays using another monoclonal antibody (beta-hCG, PLAP) or polyclonal patient IgG (P-24 antigen) bound to a polylysine-glutaraldehyde-coated plate as a "capture" reagent. After the immunobinding step, the plate was washed and substrate consisting of a mixture of factors X, V, and II in buffer containing calcium and lipid was incubated for various lengths of time. The mixture was transferred to another plate coated with fibrinogen and containing peroxidase-fibrinogen in EDTA solution to measure the amount of thrombin generated. Using this protocol, we were able to measure the presence of 2-10 pg/ml of beta-hCG and PLAP (5-30 amol per sample). All three model antigens were detectable at concentrations 2-3 orders of magnitude less using RVV-XA-labeled antibodies and ELCA than they were using peroxidase-labeled antibodies. The assay has considerable potential as a general immunoassay amplification system, yielding a "color test" for antigens of interest with a detection limit not readily attainable using other chromogenic methodologies.