Antisecretory Factor (AF) is expressed in most tissues and can be demonstrated in plasma and other body fluids. Most of the AF in plasma is in an inactive form and activation of AF occurs after exposure to bacterial toxins or after intake of various dietary components. Patients with chronic diseases involving disturbances in inflammatory and secretory processes may benefit from an AF-inducing diet. The aim of the present study was to develop an in vitro assay for the analysis of AF-activity in human plasma. Monoclonal antibodies were raised against a native form of AF prepared from human placenta. Nine clones of the monoclonal antibodies recognizing AF and AF peptides were identified. With the aid of these antibodies, we developed a sensitive ELISA method for direct detection of AF-activity in human plasma. The AF activity in plasma from five healthy volunteers was low, 0.112+/-0.022 (absorbance at 405 nm), before intake of the AF-inducing diet with the SPC-Flakes, and increased significantly (p<0.05) to 0.444+/-0.068 after >or=6 weeks on the diet. A comparison of the plasma-AF values, obtained by the bioassay and the immunogenic assay (indirect ELISA), shows that there is a significant correlation (r=0.85) between the values from the two methods. The results indicate that the ELISA measures AF-activity and has the potential to be an important tool for the analysis of AF-activity in further clinical studies on AF-therapy.