An object of the present invention is to provide a method of detecting an analyte (antigen) in a sample by an antigen-antibody reaction with an antibody fragment including an antigen binding region for the analyte antigen (hereinafter “an antibody fragment comprising an antigen binding region for an analyte antigen” will simply be referred to as “an antibody fragment against antigen”), the method suppressing nonspecific reaction that is caused by antibody fragments. More specifically, provided is a method of detecting an analyte antigen in a sample by an antigen-antibody reaction with an antibody fragment against antigen, the method comprising the steps of: a) bringing a sample into contact with a denatured antibody fragment; and b) bringing the sample into contact with the antibody fragment immobilized on an insoluble carrier after the step of a), the method suppressing nonspecific reaction.