The selection and optimization of the detection system for self-contained multiplexed dot-immunoassay

This study performed a comparative estimation of the detection systems with the use of conjugates based on peroxidase, alkaline phosphatase, colloidal gold, and the amplification system "Super-CARD" for multiplex dot-immunoassay of antibodies. The results of the study show that the sensitivity of the detection system with colloidal gold was approximately 8 times higher than that of the system with amplification "Super-CARD", 30 times higher than that of the system with conjugate of alkaline phosphatase, and 250 times higher than the sensitivity of the system with the peroxidase conjugate. Gold immunosols limit the direct detection of human IgG of 10 pg with dynamic range of optical signal change from 5 ng to 10 pg. This limit corresponds to a range of concentrations of IgG from 2.5 μg/mL to 5 ng/mL and covers the range of specific IgG concentrations, to which a typical natural immune response leads. The most typical reasons for aggregate formation during obtainment of colloidal gold and the binding of colloidal gold with biocomponents were explored. The method of minimization of particle clusters formation was suggested as well as the method for increasing stability of diluted preparations of probe by means of sedimentation of aggregates from the ready-made product.