Both poly(N-isopropylacrylamide) and poly(N-isopropylacrylamide)-antibody (PINP-Ab)-labelled enzyme adhered quickly and tightly to cellulose acetate/nitrate membrane either below (less efficiently) or above (more efficiently) the lower critical solution temperature, and the retention of PINP-Ab on the membrane increased over 30-fold when compared with the unconjugated Ab. These characteristics were used to develop a novel polymer-enzyme-linked immunoassay method: homogeneous antigen-antibody immune-complexation reaction and a heterogeneous separation process. By using a simple horseradish-peroxidase-labelled antibody as a probe, we applied this method to the detection of human serum hepatitis B surface antigen (HBsAg). This immunoassay system can detect as little as 1 ng/ml of HBsAg. The advantages of this method are: (a) fast homogeneous immune complexation; (b) a rapid heterogeneous separation process; (c) high sensitivity; and (d) low non-specific background.