Direct on-filter assay of subtilisin-type proteolytic enzymes for rapid analysis of analyte captured from the workplace atmosphere

A simple assay for some proteolytic enzymes has been developed which can be performed directly on the surface of a cellulose nitrate filter used to ca pture the analyte during workplace monitoring for health and safety purpose s. Following air sampling the analysis is performed on the filter which is retained within the air sampler. This involves two steps: first, a 15 min i ncubation in which the captured enzyme is dissolved and then digests an alk aline-phosphatase-labelled antibody immobilised as a small dot on the surfa ce of the filter; and second, is a 10 min incubation with substrate solutio n, which follows an in situ wash under a vacuum. During the incubation colo ur develops on the spot at the location of the immobilised enzyme-antibody conjugate. The intensity of the spot can be assessed visually within the sa mpler to ascertain the presence or absence of captured enzyme, or alternati vely quantitative results can be obtained using an optical scanner. The lim it of detection is 5 ng per filter for subtilisin (20 ng for visual discrim ination between this standard and the zero). The assay is stable to the eff ects of ambient air sampling at 3 l min(-1) for 18 h.