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A dot-ELISA technique for the detection of Pseudomonas protease was developed using IgG of anti-Pseudomonas AFT-36 protease as capture antibody. The detection limit of protease in buffer or milk was 1.01 ng ml−1. The procedure was performed at room temperature, took about 2.5 h and was economical. Protease AFT-36 is immunologically related to five out of seven Pseudomonas spp. The results suggest that the assay could be used to detect proteases in dairy products.