A phase-separation immunoassay method for Schistosomia japonicum antibody (SjAb) was developed using a pH-sensitive fluorescent polymer prepared by polymerization of N-isopropylacrylamide (NIP), N-(3-dimethylaminopropyl)methacrylamide (DMAPM) and 4-methoxy-N-(2-N’,N’-dimethylaminoethyl-N’-allyl)naphthalimide chloride quaternary ammonium salt (DMNAA) as a carrier and indicator. The polymer P(NIP-DMAPM-DMNAA) possesses relatively strong fluorescence and can be precipitated out of water above a critical pH 7.2 and re-dissolved below pH 7.2. The characteristic of this “smart” polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting immunoreaction complexes from the reaction mixture. After the polymerization of acryloyl-Schistosomia japonicum antigen (acryloyl-SjAg) with aforementioned monomers,
P(NIP-DMAPM-DMNAA)-SjAg conjugates were gotten. In a fluorescence immunoassay procedure, analyte SjAb was reacted with the polymer-SjAg forming P(NIP-DMAPM- DMNAA)-SjAg-SjAb complexes, then the pH of solution was adjusted above the pHt 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 antibody binding to the polymer-immune complex was absorbed by protein A entrapped in sol-gel matrixes and quantified by fluorescence measurement. The calibration graph for SjAb was linear over the range of 1～1500 ng/mL with a detection limit of 1.3 ng/mL. Owing to neutral pH and fluorescence response signal of P(NIP-DMAPM-DMNAA) itself, the damage to antigen-antibody immune complex was greatly decreased in the course of separation and the additional labeling was not needed for fluorescence measurement for immunoassay. The proposed method has been successfully used for SjAb analysis of the cow serum samples with satisfactory results.

Key words: pH-sensitive fluorescent polymer, polymer-labeling, phase separation, fluorescence immunoassay, Schistosomia japonicum