GB2186078A - Method and apparatus for carrying out biochemical assay - Google Patents

Abstract

A method of performing a biochemical assay of a substance in a sample comprises providing a biochemical ligand bound to a first region of an absorbent substrate, providing an amount of an enzyme, bound to a second region of an absorbent substrate, carrying out an assay, involving the bound biochemical ligand, the substance, and a conjugate of the enzyme, with a biochemical ligand, so as to bind to the first region of the absorbent substrate an amount of the conjugate of the said enzyme dependent upon the amount of the substance originally present in the sample, causing the enzyme conjugate bound to the said first region, and the enzyme bound to the said second region, to take part in development reactions, to produce a determinable change dependent upon the amount of enzyme bound to the respective region, and comparing the change produced in the said first and second regions, to determine the amount of the said substance in the sample.

Description

SPECIFICATION Method and apparatus for carrying out biochemical assay This invention relates to methods of carrying out biochemical assays, in particular immunoassays, and to apparatus therefore. in particular, the invention relates to a method of carrying out a biochemical assay, in which one component or ligand in a specific binding reaction is bound to an absorbent substrate, for example a matrix of absorbent paper.

The use of absorbent substrates such as glass fibre filters or nylon membranes for immobilising antibodies has previously been proposed, for example in U.S. Patent No.

3888629. The method has the advantage that the capillarity of the substrate ensures a rapid and even dispersal of sample throughout the matrix. The close proximity of ligand and antiligand (normally antibody and antigen) in the absorbent substrate ensures that the binding reactions are very rapid. A disadvantage of the immunoassay method described in U.S.

Patent 3888629 is that there is no provision for standarisation of the assay with each device. For a quantitative or semi-quantitative assay it is thus necessary to carry out an assay using standards in further devices in order to compensate for the inevitable variations in temperature and time.

In accordance with a first aspect of this invention, a biochemical assay is carried out in which a biochemical ligand, for example an antibody, is bound to a first portion of an absorbent substrate, for example absorbent paper, nitrocellulose, a nylon sheet or an inorganic substrate such as silica or titania supported on an inert plastic sheet. A predetermined amount of an enzyme is provided, bound to a second portion of an absorbent substrate, preferably the same substrate to which the ligand is bound. An assay, for example an immunoassay, is then carried out, involving the said bound biochemical ligand, the substance it is desired to determine, and a conjugate of the said enzyme with a biochemi cal ligand.The assay may be of any desired format, for example a "sandwich", or a "competition" format, and is carried out so as to bind to the said first portion of the absorbent substrate an amount of the enzyme conjugate which is dependent upon the amount of the substance originally present in the sample.

The enzyme bound to the first region in the form of the conjugate, and the enzyme bound to the second region are then caused to take part in development reactions, so as to produce a determinable change, for example a colour change dependent upon the amount of enzyme bound to the respective region. The changes produced in the first and second regions are then compared, to determine the amount of the substance in the original sample.

The enzyme bound to the second region thus provides a standard, against which the result of the assay may be determined. Generally, the development reaction will result in a colour change, so that the sample region (the "first" region) may be compared visually with the "standard" region of the substrate, or measured by reflectance to enable the result to be determined. Alternatively electrodes may be incorporated in the absorbent matrix, and the development reaction may be determined by an electrochemical change on the electrodes, for example as disclosed in International Patent Application No. PCT/GB85/590.

In U.S. Patent No. 3888629, referred to -above, the glass fibre filter is applied to a pad composed of cotton fibre which is intended to draw the sample and the wash fluid through the absorbent substrate, thereby making the assay simple and robust in relatively unskilled hands. A disadvantage of this approach is that material of high molecular weight present in the sample such as cell debris in urine or fat globules in milk are retained by the top surface of the absorbent substrate and are not removed by the wash fluid passing vertically through the matrix of the substrate. Cell debris from mammalian tissues and bacterial cells contains large quantities of the enzymes alkaline phosphatase, peroxidase and, in some cases beta-galactosidase. Milk contains large quantities of alkaline phosphatase associated with the fat globules.Since these are the most commonly used enzymes in immunoassay their presence in the absorbent substrate as contaminants will lead to false positives in sandwich assays or false negatives in competitive type assays.

Therefore, in accordance with a preferred aspect of this invention, a washing step is incorporated in which a washing fluid is caused to flow by capillary action radially outwardly from a point on the surface of the absorbent substrate, into the surrounding absorbent substrate.

In case of particularly heavy contamination such as a cervical swab, milk with a substantial fat content or milk from a cow with mastitis, radial washing may not be sufficient and the provision of a second absorbent layer on top of the antibody coated substrate is desirable. This second layer acts as a filter to trap particulate matter and fat globules and may be removed and discarded before washing takes place.

The second absorbent layer may also be utilised to contain a component of the assay, for example a conjugate of an enzyme with a hapten.

Preferably, a plurality of the said second regions are provided, so as to provide a range of calibration points. These are desirably positioned symmetrically with regard to a point marked on the absorbent substrate, for example as symmetrical sectors (e.g. quadrants) of a circle, centered on the said marked point. In this way, reagents for the development reactions may be added simply to the single marked point, from which they spread out radially through the various second regions of the substrate. This construction makes it very easy to treat the various enzyme regions with a number of reagents serially, in a reproducible way.

After the- binding reaction of the conjugate in the immunoassay, excess enzyme conjugate may be removed from the said first portion of the aborbent substrate by continuously adding a washing solution to the substrate at a point thereon, and allowing the washing solution to spread through the substrate by capillary reaction.

It is a particularly advantageous feature of the present invention that, if the first and second regions are both provided on the same substrate material, both are subjected to the same regimes of temperature during the development reaction, so that the absolute value of the temperature is relatively unimportant.

It is also advantageous that the development step which produces the determinable change (e.g. the colour change or electrochemical change) is quite separate from the assay itself, so that, for example, a test kit can be provided which comprises strips of absorbent paper, each having thereon a first region having a biochemical ligand, (for example an antibody), bound to it, and a second region, having a bound enzyme. Reagents can be provided in appropriate dispensing containers to enable the assay to be carried out at any desired location, so as to bind to the first region an amount of enzyme conjugate, dependent upon the amount of the substance under investigation in the sample.

The immunoassay reaction may then be permanently stopped, and the paper strips taken back to the laboratory for the development reaction to be carried out. Because of this feature, the method of the invention is particularly suitable for the determination of hormones and the like in animal milk or urine samples on the farm, for example in the determination of progesterone in milk, to enable determinations of oestrus and pregnancy to be made.

Accordingly, in a further aspect of the invention, there is provided a biochemical test kit for determining a substance in a sample, comprising an absorbent substrate having a biochemical ligand, for example an antibody, and an enzyme, bound respectfully to first and second regions of the surface thereof, the biochemical ligand being capable of taking part in a specific binding reaction with a substance to be assayed, and the kit also including a conjugate of the said enzyme with a specific binding biochemical ligand.

The substrate may be a cellulosic material such as paper, an inorganic material such as silica or titania, nitrocellulose, a plastics material such as an absorbent nylon sheet or a woven fabric such as rayon, cotton, silk or terylene.

When the substance to be determined is a small molecule, for example a hapten such as progesterone, the assay will generally be a competition assay, and the enzyme conjugate may be provided in a separate container, equipped with means for dispensing the enzyme conjugate on to the substrate, for example a dropper.

Alternatively it may be provided within the absorbent matrix under conditions such as high pH, or in the presence of an inorganic solvent, that will inhibit the interaction of enzyme conjugate with antibody.

As a further alternative, the conjugate may be contained within a second layer of absorbent substrate to prevent its binding to antibody, exept when the first and second substrates are wetted by the sample. In this case, the conjugate will generally be a conjugate of the enzyme with the substance which it is desired to determine, such that, in the first region, the hapten conjugated to the enzyme competes with the free hapten for antibody binding sites on the substrate, and an amount of the enzyme becomes bound to the antibody which is inversely proportional to the amount of the hapten in the original sample.

When the substance to be determined is a larger antigenic substance, the assay format will generally be of the "sandwich" type. In this embodiment, the ligand bound to the substrate in the first region is a first monoclonal antibody for the antigen, and the conjugate is a conjugate of the enzyme with a second monoclonal antibody for the antigen. Because there is no reaction of the enzyme conjugate with the substrate, until the antigen is present, the enzyme conjugate may be provided within the absorbent substrate, such that no separate container for enzyme conjugate is required.

Thus, when the sample containing the antigen to be assayed is added to the substrate, the enzyme conjugate is solubilised, the antigen binds with both the enzyme conjugate and the immobilised antibody to form the desired sandwich.

In both assay formats, washing of the excess conjugate from the substrate is required, but in the case of the sandwich assay, a washing solution will in general be the only reagent which is needed.

The enzyme which is used as the standard, and in the enzyme conjugate, is preferably one which is capable of catalysing a reaction which results in the production of a trigger substance for a further reaction or series of chemical reactions, so as to enable the "amplification" of the response obtained, as disclosed in European Patent specification number 12345678. In a particularly preferred em bodiment, the enzyme is a phosphatase, for example alkaline phosphatase, and the development reactions consist of the reaction of alkaline phosphatase with nicotinamide dinucleotide phosphate (NADP+) to produce nicotinamide dinucleotide (NAD+) which acts as a trigger for the cyclic reaction of NAD+ to NADH, in the presence of ethanol, and an NAD±specific alcohol dehydrogenase, as disclosed in European Patent specification number 60123.The reaction may produce either a colour change by reduction of a tetrazolium salt or an electrochemical signal by reduction at a platinum electrode as disclosed in International Patent Application PCT/GB85/590.

A preferred embodiment of the invention is illustrated in the accompanying drawings, in which: Figure 1 is a schematic diagram of a test strip in accordance with the invention, and Figure 2 is a schematic representation of an electrode arrangement suitable for use with a variant of the strip shown in Fig. 1.

The strip in accordance with Fig. 1 takes the form of a sheet of chromatography paper 1 (Whatman Number 1), bonded to a "MY LAR" backing sheet, using a latex adhesive, to provide a support. A first region 2 of the strip 1 is coated with a monoclonal antibody against progesterone. The antibody is physi cally entrapped in the cellulose matrix of the paper, by mixing the antibody with acrylamide monomer, and polymerising in situ, in the form of a 7.5% (w/v) gel. The thus-treated paper was washed with a solution containing 0.1% (w/v) degraded gelatin, and 5.0% (w/v) lactose, and was dried in air.

The second region 3 on the strip 1 is treated so as to bind to each of the four regions 4, 5, 6, and 7 different standard concentrations of the enzyme alkaline phosphatase. The alkaline phosphatase is bound to the strip, using the gel polymerisation method described above, with the edges of the four quadrants of the circle, and the circumference of the circle, defined by a specially designed die press, which compresses the paper onto the mylar backing. This procedure prevents mixing of the alkaline phosphatase solutions, when applied to the absorbent substrate. The upper part 8 of the strip 1 is printed with a batch number and expiry date, and space for identification and the date, animal and sample used in the test.

Twenty such strips were bound together in a "book" format, with twenty strips to a book.

A test kit for the determination of progesterone in cows' milk was produced with the following components.

1. Substrate The substrate was in the form of a strip, as described above.

2. Conjugate The conjugate was a conjugate of progesterone with alkaline phosphatase, and was provided in a plastic dropper bottle, in a quantity sufficient to enable 20 fifty microlitre drops to be dispensed. The concentration of the conjugate in the solution was 50ng/ml in a 100mM-triethanolamine buffer pH 8.5 containing 0.1% (w/v) degraded gelatin, 400mM-ammonium sulphate, 5mM-magnesium chloride and 0.1% (w/v) sodium azide.

3. Washing Solution The washing solution contained 0.1 S (w/v) degraded gelatin, 400 mM-ammonium sulphate and 0.1% sodium azide in a plastic dropper bottle.

4. Enzyme Substrate A plastic dropper bottle containing 1 ml of 0.2 mM- NADP+ in 0.050M diethanolamine buffer pH 9.5 containing 0.1% (w/v) sodium azide.

5. Enzyme Amplifier 1 ml of a solution containing 200 micrograms of alcohol dehydrogenase, 200 micrograms of pig heart diaphorase, 4% (v/v) ethanol, 1.0 mM-INT violet, 0.02% (w/v) sodium azide in a 20 mM-sodium phosphate buffer at pH 7.0 in a dropper bottle.

6. Stopping Solution 1 ml of 0.3M sulphuric acid. When added to the substrate during the development reaction, the stopping solution prevents further colour development, and thus enables the colour to be viewed on subsequent days.

The invention is illustrated in the following Examples.

Example 1 Utilising a test kit as described above, a test for progesterone in milk in the filed may be carried out as follows.

A milk sample which it is desired to determine is introduced onto the test strip, so as to wet the first region (2). Because the strip is essentially self-metering, precise measurement of the quantity of milk added is not important, and thus the milk may be poured over the strip, added from a dropper bottle, or the strip may be dipped into milk.

Progesterone in the milk binds with monoclonal antibody in the first region, and the strip is left at room temperature for a minimum of one minute, for the reaction to proceed. The reaction stops rapidly after this time owing to the drying effect of the absorbent paper surrounding the antibody region.

One drop of the solution in the conjugate container is then added to the centre of the first region, and the progesterone conjugate fills the remaining free antibody binding sites. The strip is again left for a minimum of one minute and washing solution is then added dropwise from the washing solution bottle to the centre of the antibody-coated region. At least 5 fifty microlitre drops of washing solution are utilised.

After washing, one drop of the enzyme substrate is added to both the first region 2 (the sample region) and the second region 3 (the standards region) of the strip. After a futher minute one drop of the amplifier is added to region 2 and region 3 and the colour development is observed after a further period of one minute. The colour development is stopped after this period with one drop of the stopping solution on each of the two regions and the progesterone content of the sample was estimated by comparison of the colour intensity of the region 2 with that of the regions 4, 5, 6, and 7.

Example 2 A test strip was constructed in an essentially similar way to that shown in Fig. 1, except that the strip was provided with an overlying strip of absorbent paper (not shown), into which had been impregnated, the conjugate of progesterone with alkaline phosphatase by soaking in a solution containing 100 mg/ml conjugate and 0.1% (w/v) degraded gelatin and drying the paper in air.

In use, the milk sample was spotted onto the overlying paper layer, and thus a solution containing both the progesterone to be determined, and the enzyme conjugate is drawn through the overlying strip, and onto the strip 1, by capillary action. Thus, it is not necessary to add further enzyme conjugate, for the determination to proceed. After two minutes the overlying paper layer is removed to expose the underlying strip 1. The washing step and the development step are the same as in Example 1.

Example 3 A composite test strip was formulated for the electrochemical determination of progesterone in milk.

The test strip is produced by laminating to a test strip of the kind shown in Fig. 1 a backing strip of the kind shown in Fig. 2. The backing strip of Fig. 2 consists of a MYLAR sheet 10, to which are laminated a plurality of electrodes 11, 12, 13, 14, 15, 16.

Electrodes, 11, 12, 13, 14, 15, and 16 are arranged as segments of respective circles, electrodes 11 and 12 being, respectively, cathode and anode for measurements on region 2 of strip 1, and electrodes 13 and 14, and 15 and 16 being, respectively, anode and cathode for regions 4, and 5 of strip 1. Electrodes 11 to 16 are connected via conductors 17 laminated to sheet 10, to an edge connector 18. Similar electrodes are provided for regions 6 and 7 of strip 1.

A strip in accordance with Fig. 1 is laminated to the strip of Fig. 2, such that region 2 of strip 1 overlies electrodes 11 and 12, region 5 of strip 1 overlies regions 13 and 14, region 4 of strip 1 overlies regions 1 5 and 16 etc. In use, the immunoassay is carried out in essentially the same manner as described in Example 1 or Example 2. Determination of the bound enzyme however is made not by observation of colour, but by measuring apparatus (not shown) connected to the said electrodes 11 to 16, via connector 18. The measuring apparatus includes means for applying a desired voltage to a pair of electrodes, for example to electrodes 13 and 14 to measure the enzyme concentration in region 5, and for measuring the resulting current, which can be made to be dependent upon the amount of enzyme bound to region 5, as disclosed in International Patent Application No.

PCT/GB85/590.

As disclosed above, the whole of the strip assembly for use in such an electrochemical determination may be produced as one disposable element. In an alternative embodiment however, the electron strip 10 may be reusable, and may be provided with means for securing disposable strips to its surface.

Claims (32)

1. A method of performing a biochemical assay of a substance in a sample, which method comprises providing a biochemical ligand bound to a first region of an absorbent substrate, providing an amount of an enzyme, bound to a second region of an absorbent substrate, carrying out an assay, involving the said bound biochemical ligand, the said substance, and a conjugate of the said enzyme, with a biochemical ligand, so as to bind to the said first region of the absorbent substrate an amount of the conjugate of the said enzyme dependent upon the amount of the substance originally present in the sample, causing th enzyme conjugate bound to the said first region, and the enzyme bound to the said second region, to take part in development reactions, to produce a determinable change dependent upon the amount of enzyme bound to the respective region, and comparing the change produced in the said first and second regions, to determine the amount of the said substance in the sample.

2. A method as claimed in Claim 1 wherein a removable layer of absorbent substrate is provided covering the said first layer to act as a filter to remove particulate matter from the sample.

3. A method as claimed in Claim 2, wherein the removable second layer of absorbent substrate contains the enzyme conjugate.

4. A method as claimed in Claim 1, wherein excess conjugate is removed from the said first portion of the absorbent substrate by continuously adding a washing solution to a point on the substrate, and allowing the washing solution to spread through the substrate by capillary action radially outwardly from the said point.

5. Method as claimed in any one of the preceding claims, wherein the said development reactions are carried out by adding at least one development reagent to the respective substrates, the said reagents being added at a single point on the respective substrate for each of the said regions, whereby capillary flow is caused, outwardly of each of the said points, for each of the said regions.

6. A method as claimed in any one of the preceding claims, wherein a plurality of the said second regions are provided, the amount of enzyme per unit area of the absorbent substrate being different for each of the said second regions.

7. A method as claimed in Claim 6, wherein each of the said second regions is positioned symmetrically with respect to a point marked on the said absorbent substrate.

8. A method as claimed in Claim 7, wherein the said second regions ae arranged as symmetrical sectors of a circle, having its centre on the said marked point.

9. A method as claimed in any one of the preceding claims, wherein the substrate is absorbent paper, or paper-like material, nitrocellulose, nylon sheet, silican, titania or a woven fabric material.

10. A method as claimed in any one of the preceding claims, wherein the said first and second regions are provided in different areas of the same absorbent substrate.

11. A method as claimed in any one of the preceding claims, wherein the substance assayed is an antigen or a hapten.

12. A method as claimed in any one of the preceding claims, wherein the ligand bound to the first region of the substrate is an antibody.

13. A method as claimed in any one of the preceding claims, wherein the development reactions are such as to develop on the substrate a colour change or an electrochemical signal, dependent upon the amount of enzyme bound to the respective region.

14. A method as claimed in any one of the preceding claims, wherein the enzyme is one which is capable of catalysing a reaction, which results in the production of a trigger substance for a further reaction or series of reactions.

15. A method as claimed in Claim 14, wherein the trigger substance is a trigger for a cyclic series of reactions.

16. A method as claimed in Claim 14 or Claim 15, wherein the enzyme is a phosphatase.

17. A method as claimed in any one of the preceding claims, wherein the assay is one in which the said substance being determined binds with the said ligand, in competition with the said enzyme conjugate.

18. A method as claimed in any one of claims 1 to 16, wherein the assay is one in which a sandwich is formed between the said bound ligand, the substance being determined, and the enzyme conjugate.

19. A method of performing a biochemical assay, substantially as hereinbefore described with reference to the foregoing specific examples.

20. A test device for carrying out a biochemical assay, comprising an absorbent substrate having an amount of a biochemical ligand bound to a first region thereof, and an amount of an enzyme bound- to a second region thereof, whereby the said bound enzyme may be employed as a standard for a biochemical assay in which an amount of the said enzyme dependent upon the amount of a substance to be determined which is present in a sample becomes bound to the said first region.

21. A device as claimed in Claim 20 which includes an additional absorbent substrate overlying the main absorbent substrate, the additional absorbent substrate serving to filter the sample.

22. A device as claimed in Claim 21, wherein the additional absorbent substrate contains the enzyme conjugate.

23. A device as claimed in Claim 22, which additionally comprises a plurality of electrodes, positioned so as to facilitate the electrochemical measurement of the amount of enzyme bound to one of the said regions.

24. A device as claimed in any one of Claims 20 to 23, wherein a plurality of the said second regions are provided, the amount of enzyme per unit area of the absorbent substrate being different for each of the said second regions.

25. A device as claimed in Claim 24, wherein each of the said second regions is positioned symmetrically with respect to a point on the said absorbent substrate.

26. A device as claimed in any one of Claims 20 to 24, wherein a plurality of the said substrates are provided, joined to each other along an edge thereof.

27. A test device substantially as hereinbefore described with reference to and as illustrated by Fig. 1, or Fig. 1 and Fig. 2, of the accompanying drawings.

28. A test kit comprising a device as claimed in any one of Claims 20 to 26.

29. A test kit as claimed in Claim 28, which also includes a container of a solution of an enzyme conjugate, and means for dispensing the enzyme conjugate to the absorbent substrate.

30. A method as claimed in Claim 28 or Claim 29, which also includes a container of a washing solution, and means for dispensing the washing solution to the absorbent substrate.

31. A kit as claimed in any one of Claims 28 to 30, which also includes a container of at least .one chemical reagent, to carry out the said development reaction.

32. A test kit, substantially as hereinbefore described in any one of the foregoing specific Examples.