WO1990015332A1

WO1990015332A1 - Sampling and analysing procedures and apparatus

Info

Publication number: WO1990015332A1
Application number: PCT/GB1990/000890
Authority: WO
Inventors: Alan Herbert Samuel; Ian Price Matthews; Colin Gibson
Original assignee: University Of Wales College Of Medicine
Priority date: 1989-06-08
Filing date: 1990-06-07
Publication date: 1990-12-13
Also published as: GB8913224D0; AU5674490A

Abstract

A sampling and analysing system sprays successive samples of analyte from a reservoir (10) onto a movable filter screen. A ''dose'' of labelled binding reagent (25) supplied from a container (26) is reacted with each sample and the sample is then analysed. The results of the analysis are supplied to a microprocessor (46) which computes the first and/or second order differentials from a series of repeated tests. Following analysis the samples may be rendered harmless e.g. by incineration.

Description

^* Sampling and Analysing Procedures and_ Apparatus

This invention relates to procedures and apparatus for use in detecting substances or analysing fluid and other environments.

The invention may have application therefore in the detection of antigens (such as, for example, micro¬ organisms, viruses or microbiological particles) in water supplies or in air streams, or in bio-engineering in general particularly where the antigen is in the nature of a virus or otherwise difficult to detect. Examples of typical applications include the monitoring of building water services for Bacillus Legionellum and the monitoring of critical environments for maintainence of asepsis, e.g. operating theatres, recovery rooms, Intensive Therapy Units, transplant wards, isolation wards, and children's wards for micro-organisms such as Meningococcal Bacillus. In general the invention may be applicable also to situations where the analyte fluid contains substances of value, which are to be extracted for useful purposes, in addition to situations where the antigen is toxic or dangerous, or for some reason undesirable. The invention may also be applicable to the detection of antigens in food.

Techniques exist for quantifying the population of an antigen in a particular environment at the time of sampling. For example, it is standard practice to culture micro-organisms on suitable growth media and after a controlled incubation period to count the number of colonies so formed either manually or by means of automatic instruments. It is assumed that the number of colonies is representative of the number of micro¬ organisms present in the sample at the time it was taken. However, the preparation of such a sample gives rise to significant errors in the quantitation due to several contributory factors. The first is that each raw sample is substantially diluted prior to incubation. This introduces a high degree of statistical error when determining the true environmental concentration of the micro-organisms, that is, the number of micro-organisms per unit volume. Secondly, at any time there will be an unknown proportion of the micro-organism population which are "resting", that is, they are not biologically active. Since "resting" micro-organisms do not replicate, their presence will not be indicated by the growth of a colony. Given that the"resting" proportion is unknown, varies with time and is affected by environmental conditions, the relationship between the number of colonies and the number of micro-organisms is itself subject to error. The latter is compounded by the current lack of knowledge regarding the relationship between the environmental conditions and the growth of micro-organisms. Our studies show that the absolute quantitation of the environmental population of an antigen is of little value in predicting the occurrence of disease due to the great variability in susceptibility of human beings arising from variations in, for example, age, state of health, lifestyle, climate, etc. However, our studies show that, in most cases, the precursor to an outbreak of disease in humans of all susceptibilities is a sudden and rapid increase in the environmental population of the offending antigen. Currently, we do not know what range of environmental conditions (temperature, pH etc.) lead to this spontaneous population "explosion" and so the explosion is not possible to predict by parametric monitoring. However we have found that by comparing successive observations of the antigen population and preferably, the first and/or second order differential with respect to time of the antigen population, it is possible to extract sufficient information to distinguish between random fluctuations in population and the early stages of an explosive rise which may lead to an outbreak of disease.

It is an object of the invention to provide a system, apparatus and procedure which overcome at least some of the drawbacks of the existing techniques and which may facilitate the accurate, reliable and timely prediction of an antigen population "explosion", allowing the prevention of the occurrence of disease.

Broadly stated from one aspect the invention consists in apparatus for conducting continuous or repeated tests to detect a substance of interest in a fluid stream or a changing fluid environment, comprising a sampling system for continuously or repeatedly extracting a sample or samples from the analyte, means for combining with the samples an im unological or other specific binding agent incorporating a "label" to form labelled complexes, the binding agent being specific to the substance of interest in the analyte fluid or a direct or indirect binding partner thereof, means for initiating a reaction designed to detect the presence or quantity of the labelled complexes, and means for detecting, measuring, analysing, operating upon, displaying or recording the results.

Conveniently the label includes a fluorescent substance and the detecting means includes an irradiating source and a receiver sensitive to the fluorescence. Alternatively the labelling substance may be bio- or che i-luminescent and the detector means includes a trigger device for activating the luminescent reaction. Tn yet another possible alternative the labelling compound includes an enzyme and the detecting means includes a trigger system to activate the enzyme.

In any case the apparatus preferably includes a continuous or repeating filtering system designed to separate the substance of interest from the analyte fluid and to pass the filtered products to the detection system. In one possible form of the invention the labelled specific binding reagent is mixed or added to the analyte before filtration and the specific binding property forms part of the filtration system, for example by allowing precipitation or other separation of the labelled complexes from the remainder of the sample. In another form of the invention the sample may be reacted with an antibody substance in solid phase, for example on the wall of a filter element, so that the antigen binds to the filter element at a first binding site whilst leaving another binding site exposed. The antigen now bound to the filter element may then be further reacted with a labelled specific binding reagent which binds with another binding site on the antigen, i.e. in the so-called indirect or sandwich test techniques.

In another embodiment of the invention, it may be advantageous to employ background correction of the results taken. For example, duplicate optics and/or duplicate sampling systems may be employed with suitable substrate treatment in order to compare signal and background. Background correction may be advantageous to compensate for masking of the signal or competitive binding.

Preferably the output of the detection system is fed to a computing system which incorporates means for computing or identifying the first order and/or the second order time differential and a means for registering, recording or operating upon these. The output of the detection system may be fed to a computing system which incorporates means for computing or identifying the transformation of the detected signal from the time domain to another mathematical domain, for example, the frequency domain and/or computing or identifying correlation functions, for example, the autocorrelation function of the detected signal. The computer system may include or be connected to an output system such as an alarm or an electro-mechanical controller or recorder arranged to be activated when the output from the detector changes in a specified or predetermined way, or changes in a specified way in relation to previous readings. In one possible arrangement the apparatus includes means for adjusting the size, or rate of sampling in accordance with the sensed or detected value of previous samples.

In one particular arrangement, the apparatus includes means for combining with the samples two or more immunological or other specific binding agents each incorporating a mutually distinguishable "label" whereby the apparatus may detect or otherwise process the results relating to two or more antigens substantially simultaneously.

The invention also consists in a method of continuously or repeatedly testing an analyte fluid to detect a substance of interest in which samples are automatically extracted from the analyte and combined with a specific binding agent which incorporates a

"label", the binding agent being specific to the substance of interest in the analyte or a direct or indirect binding partner thereof, a reaction is initiated to activate the label so as to detect presence of the substance of interest and the results are measured, displayed, recorded, analysed or operated upon.

Although the invention has been described above it includes any inventive combination of the features set out above or in the following description.

The invention may be performed in various ways and one example is illustrated diagrammatically in the attached drawing of a sampling and testing svstem intended for testing a drinking water supply.

In this example, the water supply or reservoir 10 may be an actual part of the water supply system or it may be a relatively small reservoir to which a stream of sampling fluid is diverted from the main water flow. Uater samples are extracted from the reservoir 10 via a short conduit 11 controlled by a valve 12, or by a otorised pump, operated by a control circuit 13. The samples of water are directed through a nozzle or spray 15 onto a movable filter screen 17 carried over spaced rollers 18,19 at least one of which is driven by a motor 20, controlled by a circuit 21. The part of the filter screen below the nozzle 15 is supported on a filter platen 22.

The system also includes means for chemically "labelling" the substance of interest with a fluorescent label, which in the present instance consists of a fluorescein labelled monoclonal antibody specific to the particular substance of interest in the analyte (thereafter referred to as the antigen) .These labelled antibodies are provided in liquid suspension 25 in a container 26 and are dispensed in measured quantity by a valve 27 controlled by a circuit 28, the "dose" being directed through a nozzle 29 on to the same sector of the filter screen in position over the platen 22. In addition the system includes a further "dosing" section comprising a container 30 holding a fluid medium 31 which may include other reagents for fixing, washing or activating the labelled antibodies. The contents of this container 30 are dispensed through a further valve 32 controlled by a circuit 33 and discharged through a nozzle 34 on to the same filter screen.

Thus the same discrete portion of the filter screen receives the filtered sample from the analyte reservoir 10 together with the filtered doses from the antibody container 26 and the reagent container 30. VJhile still in position above the platen the filtered constituents are irradiated by an ultra-violet source 40 acting through collimating and focusing optics 41 and a deflecting lens or prism system 42 so as to subject the filtered sample to a degree of radiation sufficient to cause fluoresence. This latter is detected by a photo- multiplier 45 whose output is connected as an input to a micro-processor 46. This is arranged to compute the first order and the second order time differential from a series of repeated tests and to compare each fresh computation with the rate of change and the rate of the rate of change of previous results so that the similarity or difference may be assessed. The output of the micro-processor is fed to and registered in a recorder or print-out unit 47. The unit 46 also has multiple outputs to the various control valves and the U.V. source 40 to control the whole system automatically.

Unlike the existing techniques discussed in the introduction, this method does not require absolute quantitation referred to "standards" but requires relative quantitation, i.e. determination of the growth rate (dN/dt) and the rate of growth rate (d N/dt ) of the antigen population (N) with respect to time (t) in the local circumstances to be monitored. In order to ensure the accuracy of the prediction, it is necessary to sample repeatedly and frequently with controlled precision, βven if it were feasible to achieve this using current techniques, which it is not, the inherent delay in obtaining the results due to manual procedure, incubation time etc. would delay the ability to make the prediction until it was too late to take preventive action. Other advantages of the method disclosed above are:-

1. There is no requirement to achieve absolute quantitation. The proposed measuring technique can be considered to be self-calibrating since the measured responses are the analogue of the variations in antigen population.

2. The automated system and the rapid and accurate procedure enables virtual "real time" analysis of the accumulated data, for example, the computation of the first order and second order time differential will enable the device to "learn" from comparisons with previous measurements whether or not an antigen population "explosion" is imminent.

3. The detection technique does not rely upon antigen replication, hence, the "resting" portion of the antigen population is counted along with active portion giving a measure of the total viable antigen burden.

4. The statistical errors arising from the sampling procedure are greatly reduced since the antigen content of the sample is concentrated prior to examination as opposed to being diluted.

In a modification (not shown) of the sampling and testing system in the attached drawing, the system is capable of substantially simultaneous determination of many different antigen populations. For example, the system may combine with the sample a series of doses each specific to a particular antigen and each having a mutually distinguishable "label" so that measurements can be taken and processed independently for each antigen under observation. Alternatively, a single dose may be applied which contains a mixture of mutually distinguishable labelled antibodies each specific to a particular antigen.

In one example, the mutually distinguishable labelled antibodies may incorporate fluorescent labels which emit photons with different frequencies, perhaps stimulated from multiple and different sources and detected by multiple and different detectors.

The illustrated example uses a photoraultiplier but other forms of photodetector arrays may be used, for exa ple photodiode arrays. In certain applications the light signal may be dispersed,e.g. by diffraction to different parts of the array for separate monitoring. This may be of use where different analytes are being monitored simul aneously.

The filtered constituents are irradiated in the illustrated example by a U.V. source. In some cases radiation of other wavelengths, e.g. the visible spectrum,may be used. Also, the irradiation and sensing of the filtered constituents may be carried out remote from the platen, for example at a separate irradiation and sensing module, depending on the various process time requirements.In the illustrated example the used filter screen and sample burden is simply collected on a roll. Another arrangement would incorporate an oven in which the used filter screen is incinerated. Alternatively, yet another arrangement would incorporate a cartridge of disinfectant or sterilant in which the filter screen is deposited thereby rendering the sample harmless.

Claims

1. Apparatus for conducting continuous or repeated tests to detect a substance of interest in a fluid stream or a changing fluid environment, comprising a sampling system for continuously or repeatedly extracting a sample or samples from the analyte, means for combining with the samples an immunological or other specific binding agent incorporating a "label" to form labelled complexes, the binding agent being specific to the substance of interest in the analyte fluid or a direct or indirect binding partner thereof, means for detecting the presence or quantity of the labelled complexes, and output means for detecting, measuring, analysing, operating upon, displaying or recording the results.

2. Apparatus according to Claim 1, wherein said means for detecting includes means for initiating a reaction designed to detect the presence or quantity of said labelled complexes.

3. Apparatus according to Claim 1 or Claim 2, wherein the label includes a fluorescent substance and the detecting means includes an irradiating source and a receiver sensitive to the fluorescence.

4. Apparatus according to Claim 2, wherein the label is bio- or chemi-luminescent and the detector means includes a trigger device for activating the luminescent reaction.

5. Apparatus according to Claim 2, wherein the label includes an enzyme and the detecting means includes a trigger system to activate the enzyme.

6. Apparatus according to any preceding claim, which further includes a continuous or repeating filtering system designed to separate the substance of interest from the analyte fluid and to pass the filtered products to the detection system.

7. Apparatus according to Claim 6, wherein the labelled specific binding reagent is mixed or added to the analyte before filtration and the specific binding property forms part; of the filtration system.

8. Apparatus according to Claim 7, wherein the specific binding property allows precipitation or other separation of the labelled complexes from the remainder of the sample.

9. Apparatus according to any of Claims 1 to 6, including a specific binding agent in solid phase.

10. Apparatus according to any preceding claim wherein said output means supplies data to a processor which incorporates means for computing or identifying the first order and/or the second order time differential and a means for registering, recording or operating upon these.

11. Apparatus according to any one of Claims 1 to 9, wherein said output means supplies data to a processor which incorporates means for computing or identifying the transformation of the detected signal from the time domain to another mathematical domain, for example, the frequency domain and/or computing or identifying correlation functions, for example, the autocorrelation function of the detected signal.

12. Apparatus according to Claim 10 or 11, wherein said processor includes or is connected to an alarm or an electro-mechanical controller or recorder arranged to be activated when the output from the detector changes in a specific or prdetermined way, or changes in a specified way in relation to previous readings.

13. Apparatus according to any preceding claim which includes means for adjusting the size or rate of sampling in accordance with the sensed or detected value of previous samples.

14. Apparatus according to any preceding claim, including means for combining with the samples two or more immunological or other specific binding agents each incorporating a mutually distinguishable "label" whereby the apparatus may detect or otherwise process the results relating to two or more antigens substantially simultaneously.

15. A method of continuously or repeatedly testing an analyte fluid to detect a substance of interest in which samples are automatically extracted from the analyte and combined with a specific binding agent which incorporates a "label" to form labelled complexes, the binding agent being specific to the substance of interest in the analyte or a direct or indirect binding partner thereof, the presence of quantity of the labelled complexes is detected to determine the presence or quantity of the substance of interest and the results are measured, displayed, recorded, analysed or operated upon.