GB2329247A

GB2329247A - Method for monitoring airborne chemicals

Info

Publication number: GB2329247A
Application number: GB9817124A
Authority: GB
Inventors: Frederick John Rowell; Zhi-Feng Miao
Original assignee: University of Sunderland
Current assignee: University of Sunderland
Priority date: 1997-08-12
Filing date: 1998-08-07
Publication date: 1999-03-17
Also published as: WO1999008111A1; AU8737598A; GB9817124D0; GB9716929D0

Abstract

A method for monitoring for the presence of airborne chemicals in the ambient atmosphere and/or determining the concentration of such airborne chemicals entails immobilising upon a sampling surface either a sample of the chemical or a derivative thereof, or alternatively a reagent for the analyte, and collecting on that surface a deposited sample of the airborne chemical being monitored. The immobilised and deposited samples are both contacted with a solution of an enzyme-labelled reactant, namely either a labelled form of the analyte if the immobilised sample was of a reagent for the analyte or a labelled form of such a reagent if the immobilised sample was of the analyte or a derivative thereof, and unreacted solution is subsequently removed. By the subsequent application of a substrate for the enzyme to the sampling surface, the extent to which the enzyme-labelled material has reacted with the immobilised material may be determined, as a measure of the quantity of the immobilised material.

Description

2329247 METHOD FOR MONITORING AIRBORNE CHEMICALS The present invention is

concerned with monitoring for the possible presence of airborne chemicals in the ambient atmosphere and/or determining the concentration of such airborne chemicals. In particular, the invention comprises a method for carrying out such monitoring and/or determination.' Chemicals such as drugs, proteins and other natural or synthetic products f eature as reactants or products in a number of industries, including in particular the chemical and pharmaceutical industries. When such chemicals are released into the atmosphere they can be inhaled by persons in the surrounding area, in particular by workers in the industry. Since inhalation or dermal contact with these substances can give rise to deleterious effects on the health of the person concerned, for example respiratory sensitisation in the case of some antibiotics or feminisation of male workers with oestrogenic compounds, it is important to monitor for the presence of such substances in the ambient atmosphere, to ensure that prescribed exposure limits are not exceeded.

Available methods for monitoring potentially hazardous substances in the atmosphere entail a number of sequential operations which may take days to complete. Such operations typically entail collecting an air sample, extracting solid material from the sample, eluting the relevant solid substance into solution and then analysing the resulting solution, for example by a separative method such as high performance liquid chromatography. The collecting of the solid airborne material from the air sample usually entails a physical filtration step or the deposition of solid material upon an adhesive surface.

The operations following sampling are usually carried out in a laboratory away from the sampling site and may therefore delay the results for several days. Clearly such methods are of limited value for regular onsite monitoring of airborne chemicals and may give results which are of little current relevance.

It is therefore an object of the present invention to provide an improved method for monitoring airborne chemicals, by means of which some at least of the disadvantages of currently available methods for that purpose are reduced or avoided.

The method according to the present invention comprises immobilising upon a sampling surface either a sample of the material to be monitored (the llanalytell) or a derivative thereof or immobilising upon a said surface a reagent for said analyte, exposing said sampling surface to an air sample whereby to collect upon said surface a deposited sample of the airborne material being monitored, contacting both said immobilised sample and said deposited sample with a solution of either an enzyme- labelled form of the analyte (if the immobilised sample is a reagent for the analyte) or an enzymelabelled reagent for the analyte (if the immobilised sample is of the analyte or a derivative thereof), subsequently removing unreacted said solution, and finally applying to said sampling surface a predetermined quantity of a substrate for the enzyme whereby to determine the extent to which said enzyme-labelled analyte or reagent has reacted with the immobilised material.

The foregoing method is based upon creating a competitive demand for the analyte-responsive reagent between the analyte in the collected air sample on the one hand and the analyte in the reacted immobilised material on the other. By monitoring the extent to which said reagent remains unreacted, it is possible to determine whether, and to what extent, the analyte was present in the collected air sample.

The method according to the present invention may be applied to the monitoring of any airborne chemical or biochemical for which an antibody specific for that product is available or can be devised. However it is of particular value as applied to the monitoring of such materials as are of potentially deleterious effect on the human body, in particular where predetermined exposure limits are to be borne in mind or are required to be observed. Thus it may advantageously be applied in industrial or other settings in which it is required to ensure that acceptable levels of airborne drugs and similar chemical or biochemical materials are not exceeded. By way of example, the method may be applied with particular advantage to the monitoring of synthetic steroids, antibiotic drugs and proteins. Among such proteins are serum albumin, subtilisin, alpha-2 macroglobulin and tumour necrosis factor alpha (TNFalpha).

The sampling surface upon which the air sample is collected may take the form of a filter through which the air sample is drawn or may, less preferably, be an adhesive surface to which the airborne solid material adheres. The method of the present invention is particularly suitable for carrying out either without removal of the sampling surface from within the sampler or at least at the workplace upon removal of the sampling surface from the sampler, thereby avoiding the hitherto necessary delay entailed in transferring the sampling surface to the laboratory. The sampler itself may conveniently be worn by a worker within the environment being monitored or may alternatively be a static monitor located within that area.

As described above, the method may be practised in either of two alternative forms. In the first method, which is preferred, a sample of the analyte or of a derivative thereof may itself be immobilised upon the sampling surface and, following collection of an air sample for monitoring, the collected and immobilised analyte materials are contacted with an enzyme-labelled reagent for the analyte, whereby the two analyte samples compete for reaction with the labelled reagent. According to the second method, the material immobilised upon the sampling surface is a reagent for the analyte and, when the air sample has been collected, the collected and immobilised materials are contacted with an enzyme- labelled form of the analyte and the collected analyte and the labelled analyte compete in solution for the limited predetermined amount of the immobilised reagent.

Following the competitive reaction, the solution is removed and the reaction is thereby terminated. A substrate for the enzyme is now applied to the surface to enable the extent to which the enzyme-labelled material has reacted to be determined, thereby indicating the possible presence or the quantity of the analyte in the collected sample. If that quantity was high, then the quantity of enzyme remaining for determination is low. Thus the concentration of enzyme may be used as a measure of the quantity of analyte in the collected air sample. That concentration may be determined analytically by means of suitable instrumentation. As an alternative, by using a colour-responsive substrate, the analyte concentration may be displayed as the colour intensity of the treated surface.

The method according to the present invention will now be further described and illustrated, by way of example only, by means of the following two Examples, which describe the application of the method to the detection and/or determination of an antibiotic drug and of a synthetic steroid respectively.

Example 1

A conjugate of the drug ceftazidime, which is a thirdgeneration cephalosporin, with porcine thyroglobulin was immobilised on the surface of a cellulose nitrate filter and the filter was fitted into a personal air sampler worn in the workplace. After the required air sampling period, a reagent in the form of a solution of an anti-ceftazidime serum I conjugated with the enzyme alkaline phosphatase was added to the filter. After 10 minutes, during which time the ceftazidime captured during the air sampling and the ceftazidime conjugate immobilised on the sampling surface reacted competitively with the enzyme-labelled reagent, the residual unreacted reagent was removed by vacuum and washing.

A substrate f or the enzyme was now added in the form of a solution prepared from a mixture of a solution of 5-bromo-4chloro-3-indolyl phosphate in dimethyl formamide with nitro blue tetrazolium and with carbonate buffer, in the ratio of 1:1:100. Reaction of the residual enzyme with this substrate generated a blue colour on the filter, the intensity of which reflected the concentration of residual enzyme. Thus if the ceftazidime quantity in the collected sample is low, then the blue colour on the filter is high and vice versa.

By comparison of the intensity of the generated colour with a series of standard samples, or by monitoring the colour signal with appropriate instrumentation, the quantity of the ceftazidime captured on the filter during the measured sampling period may readily be determined.

Exanole 2 This further Example illustrates the use of the method according to the present invention for monitoring the amount of the synthetic steroid ethynyl estradiol captured on air filters.

The filters were cellulose nitrate membrane filters, each of diameter 25mm and having a pore size of one micron. To each filter a 5 microlitre sample of 0.2 mg/ml estradiol-BTG (drugprotein) conjugate was applied as a spot at its centre. The coated filters were then each washed three times with 3 ml of the phosphate buffer PBST and air dried after incubating at 37'C in 100% humidity for 60 minutes.

To each of these filters, a 15 microlitre sample of ethynyl estradiol in a concentration from 10-6 to 10-8 mole/litre was applied to form a second spot and the filter was subsequently dried. Each such second spot represented a deposit of the steroid notionally formed by sampling from one of a range of air samples.

To each dried air filter, 600 microlitres of estradiol antiserum diluted 1:80,000 with PBST phosphate buffer was added. Following shaking at 200 motion/minute for 15 minutes, the f ilters were washed three times with 3 ml of distilled water.

600 microlitres of goat anti-rabbit antibodies labelled with alkaline phosphate diluted 1:2000 with PBST was added to each filter. After shaking at 200 motion/minute for 10 minutes, the filters were washed three times with 3 ml of distilled water.

300 microlitres of BCIP/NBT substrate specific for alkaline phosphatase was added to each filter. BCIP is 5-bromo-4chloro-3-indolyl phosphate and NBT is nitroblue tetrazolium. The filters were shaken at 200 motion/minute for 10 minutes and then washed three times with 3 ml of distilled water.

The filters, after drying in air, were stuck on plain paper and scanned by an optical scanner connected to a computer. The intensities of the colour spots on the filters were monitored by this means.

It was found that the intensity of the colour spot on each filter was dependent on the concentration of the ethynyl estradiol sample captured on the filter.

Claims

I. A method for monitoring airborne chemicals, which method comprises immobilising upon a sampling surface either a sample of the material to be monitored (the Ilanalytell) or a derivative thereof or immobilising upon a said surface a reagent for said analyte, exposing said sampling surface to an air sample whereby to collect upon said surface a deposited sample of the airborne material being monitored, contacting both said immobilised sample and said deposited sample with a solution of either an enzyme-labelled form of the analyte (if the immobilised sample is a reagent for the analyte) or an enzymelabelled reagent for the analyte (if the immobilised sample is of the analyte or a derivative thereof), subsequently removing unreacted said solution, and finally applying to said sampling surface a predetermined quantity of a substrate for the enzyme whereby to determine the extent to which said enzyme-labelled analyte or reagent has reacted with the immobilised material.
2. A method as claimed in Claim 1, wherein the analyte is a synthetic steroid, an antibiotic drug or a protein.
3. A method as claimed in either of the preceding claims, wherein the sampling surface is a filter through which the air sample is drawn.
4. A method as claimed in Claim 1 or Claim 2, wherein the sampling surface is an adhesive surface.
5. A method as claimed in any of the preceding claims, wherein a sample of the analyte or of a derivative thereof is immobilised upon the sampling surface and, following collection of an air sample for monitoring, the collected and immobilised analyte materials are contacted with an enzyme-labelled reagent for the analyte.
6. A method as claimed in any of Claims 1 to 4, wherein a reagent for the analyte is immobilised upon the sampling surface and, following collection of an air sample for monitoring, the collected and immobilised materials are contacted with an enzyme-labelled form of the analyte.
7. A method as claimed in any of the preceding claims, wherein the extent to which the enzyme-labelled analyte or reagent has reacted with the immobilised material is determined by instrumental analysis of the residual quantity of the enzyme.
8. A method as claimed in any of Claims 1 to 6, wherein the extent to which the enzyme-labelled analyte or reagent has reacted with the immobilised material is determined by adding a colour- responsive substrate for the enzyme and monitoring the intensity of the colour so generated.
9. A method for monitoring airborne chemicals, which method is substantially as hereinbefore described in either of the foregoing Examples.