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 capture the analyte during workplace monitoring for health and safety purposes. 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 incubation in which the captured enzyme is dissolved and then digests an alkaline-phosphatase-labelled antibody immobilised as a small dot on the surface of the filter; and second, is a 10 min incubation with substrate solution, which follows an in situ wash under a vacuum. During the incubation colour develops on the spot at the location of the immobilised enzyme antibody conjugate. The intensity of the spot can be assessed visually within the sampler to ascertain the presence or absence of captured enzyme, or alternatively quantitative results can be obtained using an optical scanner. The limit of detection is 5 ng per filter for subtilisin (20 ng for visual discrimination between this standard and the zero). The assay is stable to the effects of ambient air sampling at 31 min(-1) for 18 h.

Direct on-filter immunoassay of some beta-lactam antibiotics for rapid analysis of drug captured from the workplace atmosphere.

A simple competitive enzyme-linked immunoassay for the antibiotic ceftazidime and structurally similar beta-lactam antibiotics has been developed which can be performed directly on the surface of a cellulose nitrate filter used to capture the airborne drug during workplace monitoring for health and safety purposes. Post sampling analysis is performed on the filter retained within the air sampler. It involves two steps; the first a 10 min incubation in which the captured drug is dissolved and competes with drug immobilised within a protein conjugate on the surface of the filter for an enzyme-labelled antibody reagent, and the second, following washing under vacuum in situ, a 5 min incubation of substrate solution when colour develops on the spot at the location of the immobilised drug-protein conjugate. The intensity of the spot can be assessed visually within the sampler to ascertain the presence or absence of captured drug, or quantitative results can be obtained using an optical scanner. The intensity of the spots in linear from 10 ng to 1 microgram (r2 = 0.9996, n = 3) and the limit of detection is 1.9 ng of captured drug (10 ng for visual discrimination between this standard and the zero). The assay is precise with between-assay RSD values of < 4% over the linear range of the assay.