A High-throughput Quantitative Immunoassay System Utilizing Super-paramagnetic Microbeads

A high-throughput quantitative immunoassay system was developed, in which commercially available Streptavidin MicroBeads with high-binding capacity were used as the solid matrix. Biotinylated IgGs linked to the beads via the Streptavidin molecules and then were quantitated colorimetrically at 405nm via alkaline phosphatase-conjugated monoclonal antibodies in the 96-well microplate. The system is supposed to be able to quantitate almost all the antigens and antibodies with clinical indicative uses. Miniaturized formats of this system, if practicable, can be used for developing protein chips and for large-scale drug screening.The researches and evaluation of the system were described in detail:1)Optimize some reaction conditions: First, the effects of pH and dilution on the Monoclonal Antibody to Rabbit IgG (Alkaline Phosphatase Labeled) were analyzed, results showed that the optimal pH of the Alkaline Phosphatase was 9.6 and the optimal dilution of the monoclonal antibody was 1:10, 000. Second, we determined the optimal ratio of biotin to protein for the biotinylation of antigens or antibodies. The best detection result was obtained at the biotin ratio of 8M biotin/M protein.2)Evaluate the quantitative analyses of the system: Using Rabbit IgG, Bovine IgG and Rat IgG as clinical samples, we evaluated the system. The result showed that the sigmoidal standard curve for Rabbit IgG displayed a characteristic shape and a linear dose response in the region between 2 and 20 u g/ml (80~800ng). The mean of all coefficient of variation values from eight triplicate standard concentrations was 10. 81% and the range ofCV values was 5.05-21.78%. The curves for Bovine IgG and Rat IgG also displayed a characteristic sigmoidal shape.3)Tentative researches on the miniaturization of the system: First, we examined the relationship between the amount of Streptavidin MicroBeads used and the maximum quantity which can be detected by the system. Using Rabbit IgG as a clinical sample, we found that the maximum detectable quantity of this system was almost in direct ratio to the amount of Streptavidin MicroBeads used. It suggested that the miniaturization of this system was possible. Second, we miniaturized this system by reducing the amount of Streptavidin MicroBeads to half. The result showed that the curve for Rabbit IgG displayed a linear dose response in the region between 2 and 12 u g/ml (80-480ng). The mean of all coefficient of variation values from eight triplicate standard concentrations was 4. 09% and the range of CV values was 0. 63-7. 65%. Compared with the former curve for Rabbit IgG generated before the miniaturization, the linear range of this curve reduced to approximately half. The detection limit or sensitivity, however, did not change, which remained 2 u g/ml(80ng). It suggested that the miniaturization of this system was practicable.Many manual washing procedures inevitably cause errors that greatly reduce the sensitivity of the system. And the relatively low sensitivity limits the extent to which the system can be miniaturized. Further studies on the miniaturization of the system are therefore needed.