Study On The Microfluidics-based Paramagnetic Microsphere Immunoassay Reagents

ObjectiveCurrently, a number of CLIA products and supporting equipment from internationally known companies have entered China's market, but only some large hospitals use them. Due to the high cost of reagents, long testing time, complex operation, huge reagent consumption and high cost of analysis, these CLIA products cannot be widely applied in small and medium-sized hospitals, grass-roots medical institutions and wartime scene. Therefore, it's necessary for us to research and develop CLIA reagent products that can meet the domestic conditions and demands as well as have competitive power, to make China's immunoassay catch up with the development trend and pace of international immunoassay as soon as possible.Depending on integrating the microfluidic chip, immunomagnetic beads technology and chemiluminescence technology, This has laid a foundation for the research of multi-channel, integrated and small-sized immunoassay suitable for site or field conditions.Methods1. The carbon diimine crosslinking method is used to make the TSH antiboby and goat anti-rabbit IgG fixed on the magnetic polystyrene microspheres for the preparation of immunomagnetic beads, so a stable and efficient curing method for the immune magnetic microspheres is established. Depending on integrating the microfluidic chip, immunomagnetic beads technology and chemiluminescence technology, and making the paramagnetic microspheres as the solidified carrier of antigens and antibodies, as well as adding electromagnetic field in the chip system to gather the magnetic microspheres, this study separates the antigen (antibody) compounds from free antigens (antibodies) to position the immune responses, and makes the microfluidic chip can be repeatedly used.2. (1) Choose Luminol-hydrogen peroxide luminescent system as the chemiluminescence substrate, and study the influence of temperature, ph value, Luminol concentration and hydrogen peroxide concentration on chemiluminescence, as well as establish the chemiluminescence substrate reagents.(2) TSH and T₄ are taken as the research objects respectively, and suitable reagents in the microfluidic chip system to achieve the double antibody sandwich method and competition method are established, and their reliabilities are tested.(3) Collect clinical samples, and use self-designed testing process and a series of established immune reagents to test the clinical samples. The testing results will be used to conduct a relevance statistical analysis comparing with the clinic results of Roche E2010 electrochemiluminescence immunoassay that is commonly used in large hospitals now.Results1. The curing conditions of magnetic polystyrene microspheres are successfully explored. The TSH antibody and goat anti-rabbit IgG antibody's best work concentration are 1:200. After the cross-linking, it has been detected that the corss-linking rate between TSH antibody and magnetic polystyrene microspheres is 46.08%, and the corss-linking rate between goat anti-rabbit IgG antibody and magnetic polystyrene microspheres is 51.37%.2. The experimental results show that the optimum reagent for Luminol - hydrogen peroxide chemiluminescence system is the carbonate buffer solution with the ph value of 9.6; the optimum temperature is the room temperature or 37°C; the optimum concentration of hydrogen peroxide is 0.1mol/L; the optimum concentration of Luminol is 5×10-5mol/L; all consistent with the literature.3. Use microfluidic immunoassay chip control system, and self-crosslinking TSH immunomagnetic beads and goat anti-rabbit IgG antibody immunomagnetic beads to successfully have a double antibody sandwich method immunoassay for TSH, and competition method immunoassay for T₄. The analysis process is finished in 25 minutes, and the dosage of reagent is only 10μl. For the TSH, the intra-assay CV is 4.9%, and the inter-assay CV is 5.0%; for the T₄, the intra-assay CV is 4.9%, and the inter-assay CV is 4.7%. After the collection of clinical samples, there is no significant difference between the testing results by this method and the testing results by Roche E2010 electrochemiluminescence immunoassay.ConclusionsThe immune reagents and assay method developed by this study have been successfully applied in the micro-fluidic immunoassay chip control system, not only suitable for the double antibody sandwich method immunoassay, but also satisfying the requirements of competition method immunoassay. The assay method designed in this study is fast, accurate, stable, easy to operate and low-cost, so it has a significant advantage comparing with the currently and commonly used electrogenerated chemiluminescence method in clinic. This has laid a foundation for the research of multi-channel, integrated and small-sized immunoassay suitable for site or field conditions.