Robert H. Cumming

Detection of protease activity in the wetted surface of gelatin-coated electrodes in air by AC impedance spectroscopy

As a step towards developing a biosensor which can detect airborne protease droplets, a biosensor which had previously been developed to detect protease in solution is shown to be capable of detecting different concentrations of protease in liquid films on the sensor surface in air. The biosensor measured impedance change due to proteolytic digestion of its gelatin coating. In saturated air there was a rise in impedance, with a loss in weight of the gelatin, in proportion to collagenase concentration. The addition of glycerol to the gelatin caused a lower impedance response and smaller loss in weight. A critical thickness of the gelatin layer prior to a more rapid change in the rate of impedance was noted, with and without the addition of glycerol. In low air humidity (40%), with gelatin, all collagenase concentrations produced a very similar rapid increase in impedance. However, with glycerol-enhanced gelatin, there was a clear distinction between the extent of impedance change with different collagenase concentrations. The application of these findings for use in the field of bioaerosol sampling is discussed.

A rapid fluorescence ELISA for ceftazidime

A simple and rapid ELISA with a fluorescent end-point has been developed for the measurement of the cephalosporin antibiotic, ceftazidime. The ELISA can be applied to the analysis of the drug in solutions that have been eluted from filters following the capture of air samples in the workplace and in biological fluids. The assay has good precision and shows insignificant interference from a range of other cephalosporins and penicillins. A comparison has been made between this assay and a similar ELISA method using spectrophotometric detection. The use of fluorescence detection improves the limit of detection from 3.5 × 10–9 mol dm–3 to 6.5 × 10–10 mol dm–3, and reduces the overall assay time from 45 to 25 min. The current method of choice is HPLC, which is slow and insensitive in comparison with the assay described.

Environmental analysis in the workplace; development of airborne monitoring systems for potentially harmful pharmaceuticals: An improved rapid ELISA for ceftazidime

Ceftazidime is a β-lactam antibiotic of the cephalosporin family. In common with other β-lactam antibiotics it is a respiratory sensitiser and occupational exposure limits have been set for workers who may inhale the drug in the workplace. Polyclonal antibodies have been raised in rabbits which specifically recognise this compound. These have been purified using immunoaffinity chromatography and the resulting anti-drug antibodies used with alkaline-phosphatase labelled anti-rabbit antibodies to generate a single reagent ELISA for ceftazidime which can be used in a rapid semi-quantitative mode to screen samples from conventional filters following monitoring of worker exposure. Presence of the drug in excess of the occupational exposure limit (OEL) (3 μg of captured ceftazidime eluted into 1 ml prior to assay) results in a lack of colour at the endpoint of the assay while samples with levels less than the OEL produce a yellow colour. The ELISA can process 45 samples in duplicate in 40 min. The sensitivity of the assay is 110 pg in a 50 μl sample volume. The assay has good precision with coefficients of variation less than 10% and it exhibits insignificant interference with a range of penicillin and cephalosporin antibiotics and other compounds tested. The assay is better suited than liquid chromatography for use on site by semi-skilled operatives immediately following the monitoring period when exposure of workers above the OEL can be rapidly ascertained and action taken.

Environmental analysis in the workplace; development of a rapid, sensitive ELISA for monitoring airborne alcalase

Abstract Alcalase, in common with other proteases is a respiratory sensitiser and long term and short term occupational exposure limits of 60 ng m−3 for periods of 8 h and 15 min respectively have been set for workers who may inhale these proteases in the work-place. Polyclonal antibodies have been raised in rabbits that specifically recognise alcalase. These have been purified using immunoaffinity chromatography and the resulting anti-alcalase antibodies used with alkaline-phosphatase labelled anti-rabbit antibodies to generate a single reagent for use in a competitive ELISA for alcalase that can be used in a rapid semi-quantitative mode to screen samples from conventional filters following monitoring of worker exposure. Presence of alcalase in excess of the occupational exposure standard (OES) results in a lack of colour at the endpoint of the assay while samples with levels less than the OES produce a yellow colour. The ELISA can process 45 samples in duplicate in 60 min. The limit of detection of the assay is 1 pg ml−1 in a 50 μl sample volume. The assay has good precision with coefficients of variation less than 10%. and is better suited than current colorimetric enzyme assays for use on site by semi-skilled operatives immediately following the monitoring period when exposure of workers above the OES can be rapidly ascertained and action taken.

Rapid and sensitive assay for some protease enzymes using a fluorosubstrate-immobilized bioreactor

Protease enzymes are commonly used in industry; exposure of the workforce to these enzymes may lead to respiratory sensitization. Assays are therefore needed which can be coupled to air sampling devices to produce real-time monitoring systems which are sufficiently sensitive and rapid to provide an early warning system that indicates that the airborne concentrations of enzyme have exceeded occupational exposure limits. Current methods based on spectrophotometric end-points are too slow and insensitive for this application. An assay is reported that is based on the release of fluorescein covalently attached to porcine thyroglobulin which is itself immobilized onto glass beads in a bioreactor. Exposure of the bioreactor to the enzymes, subtilisin, trypsin, alcalase and esperase produces a fluorescent peak in a flow through cell within 3–4 min. The resulting signal is linear (r= 0.999, n= 5) over the range 2–50 ng for subtilisin and the reproducibility of the assay (sr) at 5 ng is 0.8% for this enzyme.

Enzyme linked immunosorbent assay for ceftazidime in airborne samples

A simple and rapid enzyme linked immunosorbent assay (ELISA) method for the cephalosporin, ceftazidime, has been developed for the quantification of this antibiotic in solutions that have been eluted from filters following the capture of air samples in the workplace. The assay has the specificity, sensitivity and precision necessary for its use in determining airborne concentrations of ceftazidime in the workplace as part of an occupational health and hygiene programme.

Evaluation of a model for the detection of aerosol-containing protease in real-time using chromogenic substrates in the sampling fluid of a cyclone and a bubbler

Abstract A model previously developed for predicting the colour change in a liquid sampler containing a chromogenic substrate was tested for the detection of aerosols of the protease enzyme, alcalase. Aerosols were generated in a bioaerosol test chamber at 20°C and relative humidity of 40%. The detection system responded to changes in alcalase concentration in real time. The performance of a high flow rate Aerojet cyclone was contrasted with that of a low flow rate bubbler. The model was tested with both a constant aerosol concentration and also with the sudden release of an aerosol for a short time. It was found that the model needed correcting for fluid loss from both samplers. The model predicted the performance of the bubbler better than the cyclone. Possible reasons are discussed.

Monitoring proteolytic enzymes for health and safety in the manufacturing environment. A review

Abstract The widespread application of proteolytic enzymes in many industrial sectors brings considerable benefits to modern industrial manufacturing and to the quality of life for human beings. However, as the enzymes are known potent respiratory sensitising agents, there are potential risks to workers on site and to the general public off-site, if these compounds are released into the atmosphere during their manufacture and processing. To ensure adequate containment within the factory, to protect factory workers and the general public, and to comply with health and safety legislations, it is necessary to monitor airborne concentrations of the enzymes in the workplace atmosphere. At present, however, workplace monitoring of industrial enzymes can only provide exposure data for retrospective use. There is an urgent need to develop rapid and sensitive monitoring methods which provide continuous data on a near real-time basis and detect sudden release of the sensitising material. This paper reviews the existing monitoring methods for proteolytic enzymes in the industrial atmosphere and some recent developments in this important field. Possible strategies for developing integrated sampling and detecting systems for near real-time monitoring of industrial proteolytic enzymes in the manufacturing environment are discussed.

Monitoring of hazardous biochemicals in the work-place atmosphere

Abstract Regulatory bodies are increasingly expressing an interest in the monitoring of the work-place air for hazardous biochemicals but examples in the literature of this form of monitoring are not common. In contrast, there are many examples of sensitive assays being developed for the quantitation of very small quantities of biochemicals in fluids which use a multi-step process. An aerosol sensing system has two components: an aerosol collection device and the biochemical detector. The coupling of the two has rarely been investigated. The requirements for such a sensing system are reviewed, together with an appraisal of the types of detector devices that might be suitable. It is suggested that there is a need to develop one-step sensitive sensors for real-time aerosol monitoring.

A near real-time system for continuously monitoring airborne subtilisin-type enzymes in the industrial atmosphere.

We describe the development and validation of a portable system comprising an air sampler coupled to an automated flow injection analysis device. The system is able to monitor airborne concentrations of subtilisin-type enzymes in the workplace atmosphere on a continuous basis. Sampling is in two stages: using a sampling head that is designed to mimic human respiration at approx. 1 m s(-1) at a sampling rate of 600 l min(-1). In the second stage, the captured particles are deposited by impaction from the air stream onto the inner surface of a cyclone that is continuously washed with a jet of buffer solution. Deposited particles are then washed into a reservoir from which samples are taken every 5-6 min and injected automatically into a continuous flow injection analysis system. Proteolytic enzyme in the sample passes through a bioreactor maintained at about 40 degrees C. This contains a cellulose solid phase matrix on which is covalently immobilised Texas Red-labelled gelatin as substrate. The passing enzyme partially digests the substrate releasing fluorophore that is detected down stream in a flow cell coupled to a fluorimeter. The system is calibrated using enzyme standards and the intensity of the resulting peaks from the ex-air samples is converted to airborne concentrations using a mathematical model programmed into a PC. The system has a limit of detection of 4.8 ng m(-3) and a dynamic range of 5-60 ng m(-3). The within assay precision (RSD) is 6.3-9.6% over this range. The within batch precision is 20.3% at 20 ng m(-3) and the corresponding between batch value is 19.5%. The system has been run for periods up to 8 h in the laboratory and for up to 4 h at a factory site and the values obtained compared with time-averaged values obtained from a conventional Galley sampler and in-house analysis when reasonable agreement of the results was observed. The stability of the system over 21 days of continuous use with standards injected periodically was studied. Linearity was observed for all the standard plots throughout. At the end of 21 days, after a total exposure equivalent to 2395 ng ml(-1) of Savinase, the signal due to the 5.0 ng ml(-1) standard was still easily detectable.