A milk clotting assay for antibody using a fusion protein between protein A and pepsinogen C

The proteolytic activity of a fusion protein between protein A and human pepsinogen C (PA-PGC) was measured by a modified milk clotting assay on 96-well microtiter plate. The assay ranges for 30 and 120 min-incubation were approximately 0.08-1.25 ng and 0.02-0.16 ng, respectively. Although the absorbance of milk solution decreased by the precipitation of clotted casein, the white precipitate was, if anything, convenient for an easy detection of reaction-positive wells. The borderline between the wells with or without precipitate was very clear and easily detected without the plate reader. This feature was thought to be suitable for the positive-negative judgment of a dilution test in enzyme immunoassay (EIA). Rabbit IgG adsorbed on a microtiter plate was then measured using PA-PGC and this milk assay (designated as PA-PGC assay). Although PA-PGC assay needed slightly longer incubation than the conventional color assay, the sensitivity of both systems was almost identical, and the reaction-positive wells containing white precipitate were easily detected at a very low range by the over night incubation without using the plate reader. PA-PGC assay possesses the unique and useful properties, and it can be used as a convenient and easy EIA technique.