WO1989006802A1

WO1989006802A1 - Enzyme labelled biochemical assay for two analytes

Info

Publication number: WO1989006802A1
Application number: PCT/GB1989/000058
Authority: WO
Inventors: David Leslie Bates; Warwick Roy Bailey
Original assignee: Novo Biolabs Limited
Priority date: 1988-01-20
Filing date: 1989-01-19
Publication date: 1989-07-27
Also published as: DK173690D0; EP0398920A1; AU2948789A; GB8801199D0; DK173690A

Abstract

A method of carrying out a biochemical assay for first and second analytes in a sample, comprises: carrying out an assay using alkaline phosphatase as a first enzyme label and beta-galactosidase as a second enzyme label. The development reaction of at least the beta-galactosidase is carried out in the presence of ethanediol in an amount sufficient to raise the pH at which the beta-galactosidase development reaction takes place, to about 7.9, thereby rendering it compatible with an amplified alkaline phosphatase development reaction, employing an NAD/NADH cycle. Detection may be electrochemical or spectrophotometric.

Description

ENZYME LABELLED BIOCHEMICAL ASSAY FOR TWO ANALYTES

This invention relates to biochemical assays, and has particular application in the field of immunoassays, although it also has application in other biochemical assays, for example DNA probes and the like. Biochemical assays are available to test for a wide range of biological substances, e.g. antigens, hormones, cancer markers etc. To date, substantially all commercial assays of this kind have been capable of estimating only a single analyte. It is a very attractive proposition to be able to determine simultaneously more than a single analyte. For example, in the field of immunoassays it is often desirable to test simultaneously for, say, hCG and prolactin. Hepatitis and Aids, Chlamydia and Herpes, B12 and Folate. Simultaneous tests for such analytes offer the possibility of substantial decreases in the time taken to carry out tests, as well as savings in materials and the like from which test kits are produced.

Radiochemical assays exist for measurement of two analytes, in which the two analytes are distinguished by employing as labels two different radioisotopes which have different characteristic emissions.

Radioimmunoassays are however difficult to carry out and require complex detection apparatus, and detailed safety precautions.

There also exist proposals for carrying out dual-analyte assays, using enzyme labels. For example, European Patent Application No. 0238353 discloses a method in which alkaline phosphatase and beta-galactosidase are employed as labels for the respective analytes. Great difficulties arise with such dual analyte enzyme assays however, because the optimum conditions required by the enzyme labels utilised will usually be very different, to the extent that it is difficult to obtain a sufficient rate of reaction of the two enzyme simultaneously under any reaction conditions. Examples of ways in which enzymes may be incompatible are pH, temperature and ionic strength dependence, or the presence or absence of substances required to make the enzyme reaction proceed. In particular, the examples given above of beta-galactosidase and alkaline phosphatase are incompatible as regards the pH required for the enzyme development reaction, alkaline phosphatase requiring a much higher pH (9 to 11.5) than does beta-galactosidase (typically around 7.1).

A further example of a dual analyte enzyme assay employing these two labels is described in Clin. Chem (28/7, (1469-1473) 1982)) (Blake et al). This article discloses an immunoassay, which is described as a "simultaneous" assay, in which beta-galactosidase and alkaline phosphatase are both bound to a support by an assay procedure, and are then the subject of separate development reactions, to enable two analytes to be determined in the same vessel. The procedure disclosed by Blake et al is, however, not a true "simultaneous" assay, in that, as disclosed at page 1472, column 2 of the article, the incubation of the enzyme is carried out in two stages, such that a first incubation stage is used to estimate the first enzyme, and a second incubation stage, at a different pH, to determine the second enzyme.

The reason for this is that, as disclosed in the Blake article, the two enzymes used, alkaline phosphatase and beta-galactosidase, are incompatible as regards their allowable pH range. Thus, the assays disclosed are so-called "stopped" assays, in which the enzyme development is carried out for a predetermined period of time, and the development of colour after that period of time is compared with a standard. It is a consequence of the method of Blake et al, because development reactions are not carried out simultaneously, that at least one of the development reactions must be a "stopped" reaction. Stopped assays are inherently inferior to so-called "kinetic" assays, in which the rate of signal development is observed over a period of time. In particular, kinetic determinations are in general much more accurate, have a substantially greater dynamic range and are less prone to certain types of measurement errors than stopped assays.

Blake et al realise the desirability of being able to carry out assays in which the observed development reaction for the two products (i.e. the colour development reactions) are simultaneous, but because of the incompatibility of the two enzymes which they employ, acknowledge that this was not possible in practice. The problem encountered by Blake et al is not unique to the combination of enzymes beta-galactosidase and alkaline phosphatase. In any combination of enzyme markers, it is likely that the conditions required for development of one will effectively prohibit the development of the second, for example by reason of preferred pH, temperature, ionic strength, the requirement for certain substances which would inhibit the reaction of the other enzyme or the requirement by one enzyme reaction of effectors (for example metal ions) which can in other circumstances act as enzyme inhibitors. A specific example is peroxidase, which requires in its development the presence of peroxide, which in general will inhibit other enzyme reactions. EP-A-0238353, referred to above, indicates that it is possible to obtain simultaneous colour developments using alkaline phosphatase and beta-galactosidase labels, by carrying out the development reactions in the presence of high concentrations (e.g. 0.25 to 1M) of diethanolamine (DEA). This is said to lower the pH optimum of alkaline phosphatase activity from 9.5 to around 8.6, at which pH the beta-galactosidase is said still to have substantial activity. However, as indicated in the reference, the high levels of DEA employed inhibit the beta-galactosidase reaction in a competitive fashion, such that it is necessary to employ high concentrations of the enzyme substrate (o-NPG). This approach gives rise to a number of difficulties. One of these is the low activity of the inhibited beta-galactosidase and a second is the fact that the phenolphthalein which is the coloured product determined has a low extinction coefficient at the low pH employed. The system is therefore very insensitive, and sensitivity can only be increased by raising the pH.

In accordance with a first aspect of this invention, we have discovered that a development reaction for a beta-galactosidase enzyme label in a biochemical assay can be carried out at substantially higher pH that hitherto, increasing the compatibility of the enzyme reaction with alkaline phosphatase, by carrying out the develpment reaction in the presence of ethanediol. Acσordingly, in a first aspect of the invention, there is provided a method of carrying out a biochemiσal assay for first and second analytes in a sample, which method comprises:- carrying out an assay so as to localise a first enzyme which is alkaline phosphatase in an amount which is dependent upon the amount of the first analyte present in the sample, and a second enzyme which is beta-galaσtosidase in an amount whiσh is dependent upon the amount of the second analyte present in the sample, causing the alkaline phosphatase and beta-galactosidase to take part in respective development reactions to produce, for each development reaction, a determinable change, and observing the determinable change for each of the two development reactions, to determine the presence in the sample of the two analytes, characterised in that at least the development reaction of the beta-galactosidase is carried out in the presence of ethanediol in an amount sufficient to raise the pH at which the development reaction of beta-galactosidase takes place. The ethanediol is also able to act as an activity enhancer for beta-galactosidase.

We have also discovered that it is possible to effect simultaneous development of two otherwise incompatible enzymes in a biochemical assay, thereby permitting kinetic measurements to be carried out on both development reactions, by causing the first enzyme to take part in a chemical reaction to produce an intermediate substance, in the substantial absence of reaction of the second enzyme, such that the amount of the intermediate substance produced is dependent upon the amount of the first enzyme in the sample, and thereafter causing the second enzyme and the intermediate substance to take part simultaneously in development reactions to produce, for each reaction, a determinable change. This effect is particularly applicable to the beta-galactosidase/alkaline phosphatase system described above, but is also of value in other dual enzyme assay systems, in which the enzyme labels are incompatible as regards their requirements in their development reaction.

In accordance with a second aspect of the invention therefore, there is provided a method of carrying out a biochemical assay for two analytes in a sample, which method comprises:- carrying out an assay so as to localise an amount of a first enzyme dependent upon the amount of the first analyte present in the sample, and an amount of a second enzyme dependent upon the amount of the second analyte present in the sample, causing the first enzyme to take part in a chemical reaction to produce an intermediate substance, in the substantial absence of reaction of the second enzyme, such that the amount of the intermediate substance produced is dependent upon the amount of the first enzyme in the sample, thereafter causing the second enzyme and the intermediate substance to take part simultaneously in development reactions to produce, for each reaction, a determinable change, and observing the determinable change for each of the two development reactions, to determine the presence in the sample of the two analytes, wherein at least one of the said determinations is carried out kinetically.

It is preferred that the determination of both of the development reactions is carried out kinetically, since by this method substantially increased dynamic range and accuracy is achieved for the determination of both of the analytes. However, it may be in certain circumstances that, because of the nature of the analytes under investigation, and the concentration in the sample, high sensitivity and dynamic range is not required in respect of one of the analytes, although it is in respect of the other. In these circumstances, it may be satisfactory to carry out the determination of one of the enzyme labels as a "stopped" determination, whereas the other label is determined kinetically.

The amount of ethanediol employed is preferably such as to raise the pH optimum of the beta-galactosidase development reaction by at least 0.5, and is typically such as to shift the pH optimum by about 0.8 for example from 7.1 to 7.9.

It is important when discussing pH optimum in the present context to distinguish between the optimum pH for the reaction with which the enzyme is itself concerned, and the overall optimum pH for the colour development reaction, of which that enzyme reaction forms a part. Because the colour development step may itself be pH dependent, the pH optimum of the overall colour development reaction (including the enzyme reaction) may be substantially different from that of the enzyme reaction alone. For the present purposes, the feature of interest is the pH at which the overall development reaction of beta-galactosidase takes place, to produce something which is measurable (typically a colour development reaction, although in certain embodiments, a product capable of electrochemical determination).

The determinable change produced by each of the development reactions may in one embodiment be a colour change, in which the two development reactions produce substances which give rise to light absorption at differing wavelengths.

Alternatively, the determinable change may be an electrical change, for example a current change in which either or both of the enzymes produce a substance which may be determined amperometrically, for example as disclosed in PCT/W086/03837. In particular diaphorase may be employed as a component of an enzyme system as disclosed in WO86/03837, and utilised to reduce ferricyanide to ferrocyanide which can be determined amperometrically. As an alternative, phenyl phosphate may be employed as a substrate for alkaline phosphatase, (Wehemeyer, Clin Chem, 31/9, 1546-1549 [1985]). Electrochemical determination of beta-galactosidase may be carried out, for example, by utilising the galactosidase to cleave an electrochemically inactive substance such as phenyl beta-galactoside or a derivative thereof and produce an electrochemically active one.

The development reaction of the second enzyme may be carried out under conditions which inhibit normal development reactions for the first enzyme. For example, the reaction of the first enzyme to produce the intermediate substance may be carried out at different conditions of temperature, pH, ionic strength, or in the presence or absence of substances, which inhibit the development reaction of the second enzyme.

The method of the invention is particularly suited to application in the field of immunoassays, and in particular to immunoassays in which the two enzyme labels are bound to a surface, in amounts dependent upon the amounts of, respectively, the two analytes present in the sample. Such immunoassay techniques are very well known, and. will not be described in detail. Conventional sandwich and/or competition methods may be utilised.

Typical pairs of analytes which may be determined by the method of the present invention are the following:- T4 & TSH T3 & T4 FSH & LH hCG & prolactin Hepatitis & AIDS (antigens) Chlamydia & Herpes B12 & Folate CEA & AFP PSA & PAP beta-microglobulin & ferritin oestrad iol & progesterone phenytoin & phenobarbital insulin & glucagon insulin & proinsulin C-peptide CMV & Rubella viable and non-viable pathogens (eg moulds and fungi) pathogens and their toxins (eg Botulinus). Further examples are native and glycated proteins, e.g. native and glycated haemoglobin, albumin or IgG. The present method is particularly suitable for such pairs, because the result very often needs to be expressed only as % glycation. The method can thus be independent of sample volume.

The intermediate substance produced by the first enzyme may, in one embodiment of the invention, be a substance which is quantitively converted in a development reaction, in order to produce a determinable change. However, in a preferred embodiment, the intermediate substance produced in the reaction of the first enzyme is a trigger substance capable of taking part in, or of producing a substance capable of taking part in, a cyclic chemical reaction. thereby "amplifying" the signal produced by the first enzyme, for example as disclosed in European Patent Specification Nos. 19606, 37036, 58635 and 60123. In a particularly preferred embodiment, the intermediate substance is nicotinamide adenine dinucleotide (NAD), or a derivative thereof (for example dihydro nicotinamide adenine dinucleotide (NADH)).

In a further particularly preferred embodiment, the first enzyme is alkaline phosphatase, the second enzyme is beta-galactosidase, and the intermediate substance is NAD. In this example, it has been discovered that, the pH optimum of the beta-galactosidase colour development reaction can be shifted by 0.8, for example from 7.1 to 7.9, by the use of ethanediol. The reaction of alkaline phosphatase normally requires a pH of from 9 to 11.5. By first utilising bound alkaline phosphatase to generate NAD from NADP (nicotinamide adenine dinucleotide phosphate), in a first reaction step, at a pH of from 9 to 9.5, and thereafter lowering the pH to, say, 8, it is possible to determine beta-galactosidase by a development reaction resulting in the production of a coloured product (e.g. the production of o-nitrophenol from o-nitrophenylgalactoside) and similary to determine NAD colorimetrically, (e.g. by the production of the coloured product INT-formazan from INT-violet).

As indicated above, the method in accordance with the second aspect of the invention is of utility when the development reactions for the two enzymes used as labels are incompatible in a number of ways, for example when they are incompatible in pH, temperature, ionic strength, or in substrates and co-factors required.

For example, most enzymes operate over a fairly narrow pH range, reflecting the ionisation state of the functional groups of the active site. Losses in activity due to pH change are therefore predictable, approximately 90% activity being lost for each pH unit moved from the optimum. Enzymes used in immunoassays cover a wide range of pH optima (for example the optimum pH of peroxidase is in the region 5-7, arid that of alkaline phosphatase is in the range 9.5 to 10.5) and in many cases this represents an unbridgable gap. As far as alkaline phosphatase is concerned, there is no other enzyme commonly used in immunoassays that functions well (or at all) at the alkaline pH required. Incompatibility in pH optima is the major obstacle to simultaneous enzyme detection.

As regards temperature, most enzyme immunoassays are run at ambient temperature (20 to 25ºC) some 10 to 15°C below the temperature optimum of the enzyme commonly used. Temperature change is not therefore normally a major factor likely to render two enzyme reactions incompatible, although it may in some circumstances be desirable to carry out two development reactions at different temperatures. As regards ionic strength, enzymes differ markedly in the ionic environment that promotes activity. This can be a complex phenomenon which also depends on the pH, the nature of the ions involved, and varies with different substrates. The choice of buffer is an important factor therefore, and there is considerable scope for incompatibility of development reactions, because of different ionic strengths required . There is also considerable scope for incompatabilities to arise between the substrate or cofactor of one enzyme system, and one of the components required in a second enzyme system. Examples of such incompatabilities are as follows:- 1. the substrate for one enzyme reaction is unstable in the reaction system for the second

2. the substrate for the first reaction system reacts with the substrate for the second

3. the substrate for the first reaction system competes with the substrate for the second, and

4. the substrate for the first reaction system inhibits the second enzyme. As indicated above, a prime example of this kind of incompatability is peroxidase, which uses hydrogen peroxide as a substrate, which will react with other enzymes or substrates.

In addition to the types of incompatabilities listed above, certain enzyme reactions have specific requirements for the presence of effectors (for example the presence of metal ions and the like), which, in other systems, can act an enzyme inhibitors, or cause other adverse reactions. For example, beta-galactosidase is reported to require high concentrations of 2-mercaptoethanol, whiσh will interfere with any enzyme-catalysed redox reaction. The method in accordance with the second aspect of the invention is capable of ameliorating all of the above difficulties, since the actual reaction of the two enzymes is not carried out simultaneously, but instead the first enzyme reaction produces an intermediate substance, and the intermediate substance is then developed in the presence of, and simultaneously with the development of, the second enzyme.

The detection of two otherwise incompatable enzymes, alkaline phosphatase, and beta-galactosidase, is illustrated in the following Examples. Figures 1 to 5 of the accompanying drawings are graphical representations of experimental results.

Example 1 Mixtures of the two enzymes alkaline phosphatase and beta-galactosidase are prepared as follows. A 5×5 matrix is prepared of solutions containing four concentrations (1.6 pg, 3.2 pg, 4.8 pg, and 6.8 pg, each per 10 microlitres) of alkaline phosphatase and a blank (zero alkaline phosphatase), and four concentrations (10 ng, 20 ng , 30 ng, and 40 ng, each per 10 microlitres) of beta-galactosidase, and a blank. The solutions of the matrix (except those derived from one or both of the blanks) thus contain both enzymes in varying concentrations. Each sample has a total volume of 10 microl itres .

The mixtures are pipetted in triplicate into the wells of a 96 well NUNC microplate. Replicate controls are also provided on the plate, containing no enzyme, alkaline phosphatase alone (6.4 pg in 10 microlitres) and beta-galactosidase alone (40 ng in 10 microlites).

The two enzymes are determined simultaneously, using the following detection systems. A first reagent solution is prepared containing the following components: nicotinamide adenine 0.1mM dinucleotide (NADP) diethanolamine buffer 50mM (pH 9.5) magnesium chloride 1mM ethanol 4% sodium azide 0.02%

Similarly, a second development reagent solution is prepared containing the following components. diaphorase/ alcohol dehydrogenase(ADH) 1.5 u/ml (1) sodium phosphate 30mM (pH 7.2) ethanediol 8% iodonitrotetrazolium-violet 1mM (INT-violet) o-nitrophenylgalactoside 1mg/ml sodium azide 0.02%.

(1) as defined by Johannsson et al (Journal of Immunological Methods, Volume 87, pages 7 - 11, 1986).

Reagent 1 100ml is pipetted into each well of the miσroplate, and the plate is incubated at 20ºC for ten minutes.

The second development reagent is then added to each of the wells of the microplate 200ml, and the plate maintained at 20º C, whilst absorbance is measured spectrophotometrically, for ten minutes at two wavelengths (414 and 492 nm). The linear rate of increase is determined for each well at both wavelengths. The results are shown in Table 1. Data Analysis Because of the spectral overlap of the two dyes ( o-nitrophenol and INT-forma zan ) produced in this system , both enzymes contribute to the absorbance measured at each of the wavelengths indicated . It is however possible to resolve the two contributions as follows:-

V₁ = V_1a + V_1b V₂ = V_2a + V_2b, where V₁ and V₂ are the measured absorbance increase rates at wavelengths 1 and 2 respectively, and V_1a, V_1b, V_2a, and V_2b represent respectively the contributions of the two enzymes a and b to the absorbance at those two wavelengths.

The signal from enzyme a is mostly at wavelength 1, and from enzyme b most at wavelength 2. The overlap of each enzyme at the second wavelength can be defined as a fraction of the signal at the other.

Hence:- ( K_a and K_b are less than 1 ) .

K_a and K_b can be determined from control wells containing only one enzyme. The measured velocities can therefore be rewritten as :

V₁ = V_1a + K_b ·V_2b V₂ = K_a ·V_1a + V_2b

The ind ividual rates V_1a and V_2b can then be resolved as : -

Results The raw data (velocity increase at 414 and 492nm) for the enzyme mixes and controls are shown in Table 1. The control wells show the following values (standard deviations bracketed):-

492 nm 414 nm reagent blank 5.232 (0.205) 2.148 (0.268) B-gal 20.75 (0.35) 112.7 (1.4) alk phos 109.9 (3.8) 34.93 (1.07)

The spectral overlaps are calculated as follows:- K_a (alk phos at 414nm) = 34.93-2.148/(109.9-5.232)=0.3133 K_b (B-gal at 492 nm) = 20.75-5.232/(112.7-2.148)=0.1403 Using these values, the individual rates are resolved by solving the above equations for each well, and the results are shown in Table 2.

Figures 1 and 2 are respectively a graphical representation of the raw data and resolved rates for alkaline phosphatase, and Figures 3 and 4 show similar representations for beta-galactosidase.

Table 1

Combined Rates of at Two Wavelengths (mAu/min)

492 nm Alkaline Phosphatase pg 0 1.6 3.2 4.8 6.4

B-gal ng 0 5.570 31.96 58.47 85.82 113.3

10 9.505 36.73 63.71 92.99 119.1

20 13.99 42.10 68.81 95.89 127.6

30 18.57 45.97 74.98 103.3 133.1

40 22.67 50.59 79.57 108.0 136.4

414 nm Alkaline Phosphatase pg 0 1.6 3.2 4.8 6.4

B-gal ng 0 2.060 10.89 19.56 28.82 35.50

10 31.89 40. 83 50.00 56.73 64.84

20 60.20 71.67 77.74 84.53 97.43

30 92.36 99.93 108.8 113.0 123. 3

40 120.1 129.7 135.8 141.2 152.0 Table 2 Resolved Rates at Two Wavelengths (mAu/min)

492 nm Alkaline Phosphatase pg 0 1.6 3.2 4.8 6.4

B-gal ng 0 5.52 31.83 58.29 '85.54 113.3

10 5.26 32.43 59.30 88.94 115.1

20 5.80 33.52 60.57 87.89 119.2

30 5.87 33.42 62.46 90.42 121.1

40 6.09 33.88 63.30 92.24 120.4 mean 5.71 33.02 60.78 89.01 117.8

SD 0.32 0.85 2.10 2.54 3.43

CV% (5.6) 2.6 3.5 2.8 2.9

Linear regression r 0.99995 A 5.23 B 17.51

414 nm Alkaline Phosphatase pg 0 1.6 3.2 4.8 6.4 mean SD CV %

B-gal ng 0 0.33 0.92 1.30 2.02 0.06 0.93 0.78 (84)

10 30.24 30.67 31.42 28.87 28.79 30.00 1.15 3.8

20 58.38 61.17 58.77 56.99 60.10 59.08 1.61 2.7

30 90.52 89.46 89.23 84.67 85.35 87.85 2.65 3.0

40 118.2 119.1 116.0 112.3 114.3 116.0 2.79 2.4

Linear regression r 0.99998 A 1.17 B. 2.880 By resolving the rates as described, the two enzymes can be determined independently, and the rates so obtained show good precision (CVs of 2-4%), with only a slight perturbation of one enzyme by the other (about 3%).

The data sets for each enzyme produce good linear calibration curves (r greater than 0.99995).

Thus in each of the 25 mixtures, the enzymes alkaline phosphatase and beta-galactosidase are each determined in the presence of the other by a kinetic colour development monitored simultaneously at different characteristic wavelengths.

Example 2 Eight sample solutions are prepared having varying pH's in the range 6.5 to 9.5, and each containing 10 mM phosphate, 5 mM aminomethylpropanediol buffer, and 1mM of o-nitrophenylgalactoside (o-NPG) substrate. Two samples are prepared at each pH, one of which contains, and the other of which does not contain 6% ethanediol.

70ng beta-galactosidase standards are added to each sample, and the rate of colour development is determined at 405 nm. The samples in which ethanediol are present show an apparent pH optimum .for the development reaction of 8.5, and the solutions containing no ethanediol show a pH optimum of 8.1. Corrections for the variations in extinction coefficient with pH show a pH optimum for the enzyme reaction alone of 7.9 (with ethanediol) and 7.1 (without ethanediol). As well as the pH shift noted, an increase in the maximum signal observed from 110 to 175 mAu/min is observed, in the presence of ethanediol.

The practical implementation of the observed effect in an assay is described in Example 1. Example 3

Dual Antibody-Enzyme Conjugate Assay for Glycated Haemoglobin, HbAlc

Glycated haemoglobin is measured by a variety of analyical techniques (HPLC, electrophoresis etc) as an index of mean blood glucose concentration and control in diabetics over the 4-8 weeks preceeding the sample measurement. Values are usually expressed as the % HbAlc of total haemoglobin. The immunoassay described below uses the method described in Examples 1 and 2 to determine both haemoglobin and glycated haemoglobin HbAlc.

IgG subclass antibodies to haemoglobin and HbAlc are raised in mice and conjugated to alkaline phosphtase and beta-glactosidase enzymes respectively using the method of Ishikawa et al (Journal of

Immunoassay, 4 (1983) pp 209-232). The conjugates are purified by HPLC and then both diluted to working concentration into a single buffer solution containing: imidazole buffer 100mM, pH 7.5 ammonium sulphate 100mM magnesium chloride 1mM zinc chloride 0.1mM bovine serum albumin 2.5% sodium azide 0.02% This is referred to below as the "Dual Conjugate Solution". Each individual conjugate is also diluted to working concentration in the absence of the other as controls.

Three calibrator haemolysates of HbAlc (3.9%, 7.4%, 10.5% HbAlc) are prepared by reconstituting freeze dried haemolysate and diluting 50 microlitre of this solution into 1ml of 3.3mM potassium ferricyanide solution and standing at room temperature for 10 minutes. 500 microlitre of diluted haemolysate is then added to 5.5ml of a buffer solution of 100mM sodium citrate pH 4.5. 100 microlitre of this haemolysate/citrate solution is then added to the wells of a microtitre plate and incubated at 25ºC for 15 minutes. During this time haemoglobin and HbAlc is passively adsorbed to the plastic surface of the microtitre well. Following the incubation the microtitre wells are washed 3 times with 250 microlitre of a simple washing buffer and then 100 microlitre of the dual conjugate solution is added to the wells. Individual conjugate controls are also added to some wells without the addition of dual conjugate solution. These control wells allow calculation of spectral overlap factors as required by the data analysis in Example 1 . Conj ugates are incubated in the wells for 2 hours at 25 º C and then washed 3 times with 250 microl itre of a simple washing buffer .

100 microlitre of a solution containing NADPH as alkal ine phosphatase substrate is then added in the following buffer : aminomethyl propane diol 10mM pH 9.5 magnesium chloride 1mM ethanol 4% sodium azide 0. 02%

NADPH 0. 1mM. 10 minutes incubation at 25º C is used to allow dephosphorylation of NADPH to the intermed iate NADH .

Following the dephosphorylation step 100 microlitre of an enzyme amplif ier solution is added containing phosphate buffer as phosphatase inhibitor , o-nitrophenylgalactoside (o-NPG) as galactosidase substrate and INT-violet as d iaphorase substrate : sodium dihydrogen phosphate 20mM pH 7.4 magnesium chloride 1mM ethanediol 12% o-NPG substrate 5mM INT violet 1mM sodium azide 0.02% diaphorase/ADH 1.5u/ml as defined by

Johannsson et al. Addition of this solution to ten microtitre wells gives a final pH of 8.5 so that beta-galactosidase and NADH can be monitored simultaneously. Colour development arising from beta-galactosidase and the NADH intermediate is measured kinetically every minute for 10 minutes at two wavelengths (405 and 490nM respectively) and the rate of colour development determined by regression analysis through the linear absorbance vs time plot for colour development at each wavelength.

Data reduction to resolve the contribution from each enzyme is carried in an analogous fashion to that in Example 1 using the control wells to determine the spectral overlap factors, ka, kb. Finally a plot of signal due to alkaline phosphatase divided by signal due to beta-galactosidase against calibrated HbAlc values is constructed to give a calibration curve for HbAlc. The result is shown in Figure 5. Example 4 Dual Enzyme Assay using Electrochemical Detection An analogous system to that described in Example 3 is employed, utilising electrochemical rather than spectropKometric detection. Electrochemical detection of alkaline phosophatase is carried out via an NAD( H) intermed iate as described by Cardosi et al (Journal of Immunological Methods , 112 ( 1988 ) pp 153-161) and detection of beta-galactosidase is carried out using the general method of Wehemeyer et al ( Clinical Chemistry 31/9 ( 1985) pp 1546-1549 ) .

The spectrophometric diaphorase substrate , INT of Example 1 is replaced with potassium ferricyanide such that the reduced product , ferrocyanide , may be detected electrochemically. The spectrophotometric galactosidase substrate is replaced with a beta-galactoside (for example , amino-phenyl galactoside or naphthyl galactoside) , which is electrochemically inactive at low potentials but on hydrolysis releases an electrochemically active product (e .g . amino-phenol or naphthol) which shows irreversible electrochemistry at low potentials . Monitoring of the NADH and beta-galactosidase products takes place at two characteristic potentials replacing the use of two characteristic wavelengths in the spectrophotometric system described in Example 1. Any of the generally known biochemical assay formats may be utilised in the methods in accordance with the invention. For example the assay may be a sandwich assay or a competition assay, in which the two enzymes are conjugated with different monoclonal antibodies, each of which binds specifically with different antigens which it is desired to determine simultaneously, for. example T4 and TSH, T3 and T4, or one of the other antigen pairs listed above. The assay may be carried out by any of the many known procedures. Generally, the assay is carried out so as to bind to a solid support, such as the wall of a microplate, amounts of two antigens to be determined, dependent upon the amounts of the antigens present in the sample under test. Kinetic determination of the two enzymes is then carried out utilising the method just described.

It will of course be appreciated that a wide range of other specific configurations are possible, in addition to those specifically described above.

Claims

1. A method of carrying out a biochemical assay for first and second analytes in a sample, which method comprises:- carrying out an assay so as to localise a first enzyme which is alkaline phosphatase in an amount which is dependent upon the amount of the first analyte present in the sample, and a second enzyme which is beta-galactosidase in an amount which is dependent upon the amount of the second analyte present in the sample, causing the alkaline phosphatase and beta-galactosidase to take part in respective development reactions to produce, for each reaction, a determinable change, and observing the determinable change for each of the two development reactions, to determine the presence in the sample of the two analytes, characterised in that at least the development reaction of the beta-galactosidase is carried out in the presence of ethanediol in an amount sufficient to raise the pH at which the development reaction of beta-galactosidase takes place.

2. A method as claimed in Claim 1, wherein the amount of ethanediol employed is such as to raise the said pH by at least 0.5.

3. A method as claimed in Claim 1 or Claim 2, wherein at least one of the said determinations is carried out kinetically.

4. A method as claimed in any one of the preceding claims wherein the development reaction of alkaline phosphatase is carried out so as to produce an intermediate substance in the substantial absence of reaction of the beta-galactosidase, such that the amount of the intermediate substance produced is dependent upon the amount of the first enzyme in the sample.

5. A method of carrying out a biochemical assay for two analytes in a sample, which method comprises:- carrying out an assay so as to localise an amount of a first enzyme dependent upon the amount of the first analyte present in the sample, and an amount of a second enzyme dependent upon the amount of the second analyte present in the sample, causing the first enzyme to take part in a chemical reaction to produce an intermediate substance, in the substantial absence of reaction of the second enzyme, such that the amount of the intermediate substance produced is dependent upon the amount of the first enzyme in the sample, thereafter causing the second enzyme and the intermediate substance to take part simultaneously in development reactions to produce, for each reaction, a determinable change, and observing the determinable change for each of the two development reactions, to determine the presence in the sample of the two analytes, wherein at least one of the said determinations is carried out kinetically.

6. A method as claimed in Claim 5, wherein the first enzyme is alkaline phosphatase and/or the second enzyme is beta-galactosidase.

7. A method as claimed in any one of the preceding claims where the determination of both of the development reactions is carried out kinetically.

8. A method as claimed in Claim 1 or Claim 2, wherein either or both of the said development reactions is such as to produce a colour change, and the corresponding observation is carried out spectrophotometrically, or wherein either or both of the said development reactions is such as to produce an electrochemical change and the corresponding observation is carried out electrochemically.

9. A method as claimed in any one of the preceding claims, wherein the development reaction undergone by the second enzyme is carried out under conditions of temperature, pH, ionic strength, or in the presence or absence of a substance, which inhibits the reaction of the first enzyme.

10. A method as claimed in Claim 4 or Claim 5, wherein the intermediate substance is nicotinamide adenine dinucleotide, or a derivative thereof.

11. A method as claimed in any one of Claims 4 to 10, wherein the first enzyme is alkaline phosphatase, the intermediate substance is nicotinamide adenine dinucleotide or a derivative thereof, the first enzyme is caused to take part in a chemical reaction to produce the said nicotinamide adenine dinucleotide at a pH of from 9.0 to 10.0, and the development reactions are carried out at a pH of from 8.0 to 9.0.