基于pH敏感相分离技术的新型荧光免疫测定体系

In this paper, it was discovered that a novel pH-sensitive copolymer of N-isopropylacrylamide (NIP) and N-(3-dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pHtr) of P(NIP-DMAPM) polymer was found to be 7.4 at 37℃. The polymer was precipitated out of water above a critical pH=7.4 and re-dissolved below pH----7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP-DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pHtr of polymer to precipitate the polymer-immune complex,and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the FITC-labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100-1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pHtr of P(NIP-DMAPM), the damage to antigen-antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme-linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH-sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.

A Novel Fluorescence Immunoassay System Based on pH‐Sensitive Phase Separating Technique

In this paper, it was discovered that a novel pH‐sensitive copolymer of N‐isopropylacrylamide (NIP) and N‐(3‐dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pH_tr) of P(NIP‐DMAPM) polymer was found to be 7.4 at 37 °C. The polymer was precipitated out of water above a critical pH=7.4 and re‐dissolved below pH=7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP‐DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pH_tr of polymer to precipitate the polymer‐immune complex, and the polymer‐immune complex precipitate was separated and re‐dissolved by the adjustment of pH, finally the FITC‐labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100–1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pH_tr of P(NIP‐DMAPM), the damage to antigen‐antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme‐linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH‐sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.