WO1981000725A1

WO1981000725A1 - Method of determining a substrate in a sample

Info

Publication number: WO1981000725A1
Application number: PCT/AU1980/000065
Authority: WO
Inventors: P Duffy
Original assignee: Charles Hospital Dev; P Duffy
Priority date: 1979-09-14
Filing date: 1980-09-11
Publication date: 1981-03-19
Also published as: EP0035521A4; EP0035521A1; JPS56501384A

Abstract

Method of determining a substrate in a sample which comprises converting the substrate to a product in a first stage of a cyclic reaction sequence and converting the product back to the substrate in a second reaction stage of the cyclic reaction sequence. At least one of the first and second reaction stages is enzyme catalyzed. Simultaneously with either the first reaction stage or the second reaction stage an indicating substrate is convened to an indicating product in a non-reversible indicator reaction. After this the loss of indicating substract or gain of indicating product may be determined. The method is particularly efficacious in enzyme immunoassay determinations which include homogeneous enzyme immunoassay and enzyme linked immunoassay wherein the free or bound enzyme-ligand or enzyme antibody conjugate is to be assayed by the method of the invention.

Description

"METHOD OF DETERMINING A SUBSTRATE IN A SAMPLE"

THIS INVENTION relates to a method of determining a substrate in a sample, and has particular but by no means exclusive use in the determination of very small quantities of a substrate. The present invention has particular application in quantitative enzyme immunoassay techniques such as the known homogeneous or non-homogenous enzyme immunoassay (EIA) and enzyme-linked immuncsorbent assay (ELISA) described for example in 'Quantitative Enzyme Immunoassay', Ed. E.Engvall and A.J. Pesce, Blackwood Scientific Publications (Scand. J. Immunol. 8, Suppl. 7,1978). Such assay techniques are based on the ability of antibodies to bind a specific antigen or ligand tightly and reversibly. By way of example the technique of non-homogenous enzyme immunoassay is based on the competition for the active site of an antibody between a ligand and a covalently coupled derivative of that same ligand to an enzyme. With separation of the bound and free enzyme phases, enzyme assay of either fraction can then be made. Plotting of the percentage of the enzyme-ligand bound to the solid phase against the concentration of ligand results in a typical immunoassay curve. The enzyme immunoassay system is also capable of simple modification to provide for the measurement of serum antibody levels by making enzyme-antibody derivatives and using a solid phase ligand to assist in the separation step.

It is one object of the present invention to provide a generalized system of non-homogeneous enzyme immuno-assay which can be demonstrated to be capable of measuring ligand concentrations in serum from a lower level of approximately 2 pico-moles/litre. The development has concentrated on the assay of serum digoxin as a working prototype. Therapeutic range of digoxin in serum is 0-6 nanomoles/litre and the working assay requires 50 microlitres of serum in a final volume of 500 microlitres. Results show that by simple modification the assav is capable of a 50-100 fold increase in sensitivity, and thus should be capable of measuring plasma ligands e.g. ACTH levels, in the range of 2-20 picomoles/litre.

It must be emphasised, however, that the present invention is not restricted to enzyme assay in non-homogeneous enzyme immuno-assays, and may equally be applied to enzyme assay in other known enzyme immuno-assay techniques. Further more, the method of the present invention also has application in fields entirely unrelated to enzyme immuno-assays. For convenience, however, the method of this invention will be described in detail with reference to enzyme assay in enzym immuno-assays.

In broad terms, the present invention is directed to increasing the sensitivity of an enzyme immuno-assay by method of determining a substrate produced as a product in the course of an enzyme immuno-assay by the following steps:

(1) Converting said substrate to a product in a first-stage of a cyclic reaction sequence, and converting said product back to said substrate in a second reaction stage of said cyclic reaction sequence, at least one of said first and said second reaction stages being enzyme-catalysed;

(2) Simultaneously with either said first reaction stage or said second reaction stage, converting an indicating substrate to an indicating product in a non-reversible indicator reaction; and

(3) Determining the loss of indicating substrate or gain of indicating product in said indicator reaction.

The present method may be schematically illustrated as follows: the present invention are discussed in more detail herein after.

(a) A first illustration of the application of the method of this invention is the β- galactosidase hydrolysis of a phenyl-β-galactoside, with the β-galactosidase covalent ly bound to an appropriate ligand as in, for example, digoxin-β-galactosidase. In assaying the free or bound enzyme-ligand phase in enzyme immuno-assay the phenol, or phenol derivative, produced by εnzymic action of the β-galactosidase is converted to o-catechol and o-quinone, catalysed by the enzyme tyrosinase, o-quinone may be reconverted to o-catechol in the cyclic reaction sequence, either non-enzymatically by use of a suitable redox acceptor such as NADH or an NADH analogue, or by use of a suitable enzyme such as lipoamide dehydrogenase , which uses NADH as cofactor. The indicator reaction may be determined by known means such as spectrophotometry or fluorometry. In addition, ether phenol derivatives that are substrates for the enzyme tyrosinase may similarly be used wi th system after forming an appropriate substituted phenyl-β-galactoside. Such derivatives include, for example, I-dopa and p-substit uted phenols with substituent, for example, -SCN, -COCH3, -CHO, -CN , -N02. The method of this invention is schematically illustrated as follows:

EIA enzyme Tyrosinase Tyrosinase

It will be appreciated by those skilled in the art that any enzyme or enzyme system capable of producing phenol could be similarly used as an enzyme-ligand derivative in an immunoassay using the above cyclising indicator system.

The details of the production of a working enzyme immuno-assay for digoxin including methods for the manufacture of ail the ingredients required for the assay are included in the appendix. INDICATING INDICATING SUBSTRATE ---------- PRODUCT

EIA SUBSTRATE

(In this illustration, dotted lines depicting the non reversible indicator reaction indicate that this reaction may be effected with either stage (1) or stage (2) of the main cyclic reaction sequence). The measurement of the coreaction may be made directly by nature of the inherent properties of the indicating substrate or indicating product e.g. in conversion of NADH to NAD, or it may require a subsequent measurement step e.g. in the measurement of ADP or ATP.

Thus, it will be seen from this illustration that a small amount of product resulting from, an initial enzyme immuno-assay is utilized as a substrate in the cyclising reaction to form a product which is itself reconverted to its original substrate form. The indicating coreaction, however, is continuously being changed to a new form and thus is an effective multiplier in concentration terms of the original substrate. The effect of this cyclic process is, therefore, to increase the sensitivity of an enzyme immuno-assay by substantially reducing the quantity of the enzymic component able to be detected, and thus required for the enzyme immunoassay. The participating substrate in the method of the present invention may be produced directly or indirectly by the free or bound enzyme-ligand phases which are to be assayed. Enhancement of this process is possible by utilizing the indicator substance used in the cycling process as a new substrate in a second cyclising reaction with a different indicating reaction.

A number of illustrations of this application of (b) A second illustration of the application of the present invention is the use of a ligand β-galactosidase to produce p-catechol or p-catechol derivative by enzymatic hydrolysis of a p-catechol or p-catechol derivative β-galactoside in an enzyme immuno-assay. Conversion of the p-catechol so formed to p-quinone is catalysed by the enzyme laccase at a pH of 7-8 with the p-quinone being reconverted to p-catechol directly by a suitable redox acceptor such as NADH or NADH analogue, or with addition of a suitable enzyme such as menadione reductase. Other derivative B-galactosides which produce substrates that are similarly catalysed by the laccase include β-galactosides appropriately formed with p-ρhenylenediamine, N ,N-dimethyl-p-phenylenediamine, N-phenyl-p-phenylenediamine, o-phenylenediamine, and p-aminophenol. The indicator reaction in which NADH is converted to NAD, the effect of which is multiplied due to the cyclic nature of the p-catechol/p-quinone reaction sequence, may be measured by known techniques, for example, a direct rate assay. The present invention is schematically illustrated as follows:

In a minor modification of the above system, the enzyme α -galactosidase may be covalently bound to a ligand and used to hydrolyse a p-OH-phenyl-α-galactoside and used with a fungal laccase at a pH of 4.5 in a similar manner. (c) A third illustration of the present invention is the use of a ligand-pyruvatekinase covalent derivative in an enzyme immuno-assay to catalyse the production of pyruvate from phosphoenolpyruvate with ADP as cofactor. The pyruvate so formed is converted to lactate at pH 7.4 by the enzyme lactate dehydrogenase (E.C. 1.1.1.27) which uses NADH as cofactor, and this lactate is reconverted to pyruvate at this same pH with, the use of the yeast cytochrome-c linked lactate dehydrogenase (E.C. 1,1,2,3). Thus the rate of the change in NADH may be used as the indicator reaction. For practical use of this system in enzyme immuno assay of biological products, prior removal of pyruvate and lactate from the system is necessary. This may be achieved by incubation of the cyclic reaction mixture together with the biological sample in the presence of the enzyme alanine dehydrogenase which is bound to a solid phase, and this will convert existing pyruvate and lactate to alanine. After an appropriate period this solid phase enzyme may be removed and the immuno-assay proceeded with.

This cyclising indicator system involving pyruvate kinase and the two lactate dehydrogenases may also be used as an additional second amplification assay involving ADP production or removal in a prior enzyme immuno-assay procedure. A suitable system is described in (d) below.

(d) A further illustration of the method of this invention is the use in an enzyme immuno-assay of a suitable glucose producing enzyme such as amylase covalently bound to an appropriate ligand. Glucose produced in an appropriate reaction is converted to glucose-6-phosphate catalysed by enzyme hexokinase. At the same time, the cofactor ATP is converted to ADP as the indicator reaction sequence. The glucose-6-phosphate so formed is reconverted to glucose with liberation of inorganic phosphate by use of an appropriate glucose-6-phosphatase enzyme. The overall effect of this cyclic sequence is the conversion of the indicating substrate ATP to ADP at molarities which are greatly increased over the glucose concentration formed in the enzyme immune-assay reaction. The ATP loss or ADP gain may be assayed by appropriate means, either simultaneously (as by the measurement of ADP in (c) above), or subsequently (as by the known determination of ATP using luciferase). In the enzyme immuno-assay of biological products which could contain glucose or giuccse-6-phosphate, prior incubation of the reaction mixture and biological sample with solid-phase glucose oxidase and solid-phase glucose-6- phosphate dehydrogenase would be required to remove these substrates before proceeding to the enzyme immuno-assay.

(e) In a further illustration of the present invention, the enzyme aidolase is covaiently bound to an appropriate ligand and used in an enzyme immuno-assay system together with the substrates dihydroxyacetone phosphate and gyceraldεhyde-3-phoεphate to produce fructose-1,6-diphosphate. The latter is converted to fructose-6-ρhosρhate and inorganic ohcsohate bv use of the enzyme fructose-1, 6-diphosphatase and the fructose-6-phosphate so formed reconverted to fructose-1, 6-dichosohate by use on an appropriate phospho-fructo-kinase.

Concurrentl with this reconvyersion, ATP is converted to ADP as the indicating reaction. It will be apparent that the overall effect of this cyclic reaction sequence is the conversion of high molarities of ATP to ADP in the presence of relatively small amounts of fructose-1,6-diphosphate formed in the enzyme immuno-assay step. The loss of ATP or gain of ADP is measured by techniques similar to those described in (d).

For use of this enzyme immuno-assay with products of biological origin, prior incubation with appropriate solid phase enzymes to remove pre-existing fructose-1,6-diphosphate and fructose-6-phosphate is required.

(f) Further, it will be understood, that many other systems may be selected for use in an enzyme immuno-assay with subsequent conversion of a substrate resulting from the enzyme immuno-assay to a product and back again under the same conditions of pH and temperature in a cyclic reaction sequence in accordance with this invention. By way of example, such further systems may include covalent ligand enzymes capable of producing substrates for use with the following: ascorbate oxidase (E.C. 1.10.3.3.) and oxidized asccrbate reductase (E.C. 1.5.1.4); and glutathione reductase (E.C. 1.6.4.2) and glutathione dehydrogenase (E.C. 1.8.5.1). It will be seen from the above illustrations, that within th.e broad concept of the present invention there is provided a method of enzyme immuno-assay which has been shown to be capable of use in the measurement of ligands present in serum in concentrations as low as about 2 picomoles/litre. This method of enzyme immuno-assay (including homogeneous and non-homogeneous enzyme immuno-assay and enzymelinked immunosorbent assay) is characterised in the free or bound enzyme-antigen or enzyme-antibody conjugate being assayed by a cyclising reaction amplification method. It is apparent that this cyclising reaction amplification method may have application apart from enzyme immuno-assay and may have general application for the measurement of a substrate in a sample. Examples of this would be the measurement of ADP in a biological sample or the determination of trace amounts of phenol or catechol derivatives as contaminants in surface waters.

APPENDIX 1. DIGOXIN ANTIBODY PRODUCTION 1.1 Coupling of digoxin to haemocyanin

To a suspension of digoxin (436 mg, 0.558 m mol) in absolute ethanol (20 ml), which was stirred at room temperature under N₂ was added dropwise a solution of 0.1 M sodium metaperiodate (20 ml, 0.2 m mol - also containing 2 ml of IN HCl) . After 15 min., excess periodate was destroyed by the addition of 1M. aqueous glycerol (0.6 ml). After another 5 min., the reaction mixture was added dropwise, with stirring to a solution of haemocyanin (250 mg, .00028 m mol) in water (20 ml), which had previously been adjusted to pH 9.5 by the addition of 5% aqueous potassium carbonate. The pH of the reaction mixture was maintained at pH 9.2 to 9. 5 by the addition of 5% K₂CO₃ as required.

After 45 mins., the pH was stable and sodium borohydride (0.3 g, 7.9 m mol) dissolved in water (20 ml) was added. After 3 hrs. at room temperature, the pH was adjusted to 6.5 with 50% aqueous acetic acid. After 3 mins ., the pH was raised to 8.5 with dilute ammonium hydroxide. The product was dialysed against water (7x31)and lyophvlised. The conjugate (128 mgl was examined spectrophotometrically and was found to contain 67 moles of digoxin per mole of haemocyanin. 1.2 Preparation of 3-0-succinyldigoxigenin

A mixture of digoxigenin (460 mg, 1.18 m mol) and succinic anhydride (448 mg, 4.48 m mol) in dry pyridine

(17 ml) was refluxed for 4 hrs. On cooling, water (20 ml) was added and the reaction mixture was carefully acidified by the dropwise addition of 50% aqueous HC1. The reaction mixture was extracted with ethyl acetate (4 x 20 ml), washed with water (2 x 10 ml) and dried (Na₂SO₄). Removal of solvent afforded a colourless foam (530 mg) which was purified by column chromatography on silica gel (50 g). Elution with ethyl acetate/methanol/ammonia (170/20/10) gave unchanged digoxigenin (130 mg). Elution with ethanol afforded two fractions:

(a) first fraction: white foam (350 mg) 3-0-succinyldigoxigenin.

(b) second fraction: pale yellow foam (90 mg) 3 , 12-0 , 0 ' -disuccinyldigoxigenin. 1.3

Immunisation

The animals used for the production of antibodies were goats and Droughtmaster cows. Both animals were amenable for obtaining large amounts of sera. The Droughtmaster cattle were chosen because they are a breed particularly resistant to ticks and tropical parasites. It was thought that this resistance was due to production of antibodies and therefore would be suitable for production of particular antibodies against a wide variety of immunogens. Digoxin haemocyanin was used as the immunogen in both goats and cattle. For goats 2mg/ml in phosphate buffered saline (PBS) was diluted 1/1 with Freund's adjuvant and homogenised. 2ml were injected into the animals intramuscularly and boosted with the same amount every month. A similar schedule was used with the cattle except the concentration of digoxin haemocyanin was increased to 10mg per animal.

1.4

Preparation of Digoxin Antibodies The digoxin specific antibodies were purified from goat and bovine serum using ammonium sulphate precipitation of the globulin fraction and preparation of the gamma globulin fraction was carried out using ion-exchange chromatography. The goat serum was found to contain titres of digoxin antibody such that 1 ml of serum contained sufficient antibody for 10,000 determinations under the conditions used in this laboratory. The serum was brought to 30% (w/v) with ammonium sulphate and the precipitated protein collected by centrifugation. The precipitate was dissolved in a minimum volume of 5 mM phosphate buffer pH 8.0 and dialysed exhaustively against this buffer. This material was applied to a DEAE cellulose (DE 52, Whatman) equilibrated against this buffer. The protein was eluted with a linear gradient from 5 mM to 100 mM phosphate buffer pH 8.0.

The gamma globulin fraction was eluted off in the first two peaks. Digoxin binding activity was found to be associated with these two peaks. The appropriate tubes were combined and the protein precipitated out with ammonium sulphate. The antibody was stored at -20ºC in this form and has been found to be extremely stable over a period of two years. This material has been used in routine digoxin assays in this laboratory in conjunction with an RIA method for some two years. For use in immunoassays the ammonium sulphate precipitate was dissolved in PBS, dialysed against this buffer and the appropriate dilutions required for the immunoassay prepared. The antibody stored lyophilised at -20ºC in this form was also found to be quite stable over a similar period. 1.5

Antibody Characteristics

The antidigoxin globulin prepared from both goats and cattle was found to have virtually identical characteristics. The binding constant for digoxin was found to be 1.8 x 10¹¹ litres/mole using a Scatchard plot.

The drugs listed in the following table were tested for cross reactivity with this antibody. Over a period of two years two major digoxin antibody preparations have been carried out. The first was produced using disuccinyl-digoxigenin -BSA as the immunogen. The second was produced using digoxin haemocyanin as the immunogen. Different specificities for some of the closely related chemicals were observed. In particular the second preparation was found to be suitable for digitoxin determinations. % CROSS REACTIVITY

COMPOUND FIRST SECOND

Digoxin 100 100

Proscillaridin 0 .12 2 . 29 Deslanoside 76 . 1 75 . 0 Digitoxin 5 . 3 59 . 1 Cholesterol 0 . 01 0 . 01 Cortisol 0 . 01 0 . 01 Frusemide 0 . 01 0 . 01 Spironolactone 0 . 01 0 . 01

Fab Derivative Preparation The Fab derivatives were prepared from the purified antidigoxin gamma globulin fraction by incubating the gamma globulin in 100 mM phosphate buffer pH 7 containing 10 mM cysteine, 2mM EDTA with papain (7% by wt. of gamma globulin). The papain digestion was carried out at 37º for 17 hours. Following this the digest was dialysed against PBS in the cold for two hours and then fractionated on a Sephadex G100 column equilibrated against this buffer. The tubes containing protein of molecular weight equivalent to 50,000 were pooled and the protein concentrated. 2. PREPARATION OF Fab ANTIBODY

The Fab derivative prepared from the cow gamma globulin fraction was used as an immunogen to produce antibodies in goats. The Fab (2 mg) in 1 ml of PBS was diluted 1/1 with Freund's adjuvant and homogenised. This material was injected intramuscularly into each goat.

The anti Fab globulin fraction was purified from goat serum by the methods described previously. 3. SOLID PHASE ANTIBODY PRODUCTION 3.1 Preparation of Sepharose - Ab Fab Sepharose 4B (10 ml) was washed with water (1000 ml) and the resultant washed Sepharose was suspended in water (10 ml) and stirred at 4 . Cyanogen bromide (3.0 g) was added to the Sepharose, and the pH rapidly adjusted to 11 with 1-2 drops of 5N NaOH solution. The pH was held at 10.5 to 11 with 1N NaOH solution at 4° for 15 mins. The suspension was rapidly filtered and washed with ice-cold 0.1M NaHCO₃ solution. The activated Sepharose was suspended in 0.1M NaHCO₃ solution (10 ml) at 5°. Fab antibody (C 62.2 mg previously dialysed against 0.1M NaHCO₃), was added to the suspension and was stirred at 4 for 8 hrs. The supernatant was filtered off, and the Sepharose was washed with 0.1M NaHCO₃ (20 ml) and was suspended in a solution of 0.1M NaHCO₃ , containing glycine (2 moles/1) and bovine serum albumen (50 mg/ml). The Sepharose was stirred at room temperature for 2 hrs. and stored overnight at 4º.

The resultant Sepharose - Ab Fab was washed successively with 0.1M NaHC0₃ (200 ml), 0.5M NaCl (200 ml) and PBS (200 ml). The Sepharose was suspended and stored in PBS (7.5 ml).

4. ENZYME LIGAND PRODUCTION 4.1 Preparation of β-gaiactosidase-digoxin

A solution of 3-0-succinyldigoxigenin (4.5 mg, .0092 m mol) in dry, N,N-dimethyIformamide (0.4 ml) was stirred under N₂ at 4º . N-methyImorpholine (5 ul, 4.6 mg, .0455 m mol) was added followed by i-butylchloroformate (5 ul, 5.05 mg, .0371 m mol). The mixture was stirred at 4º for 30 mins. The resultant solution was added dropwise to a preprepared solution of β-galactosidase (50 ul of a 5 mg/ml suspension in (NH₄ ) ₂ SO₄ , i.e. C .052 u mol of enzyme -NH₂) in 25 mM phosphate buffer, pH 8.0 (1.0 ml), the pH of which was adjusted to 9.3 with 0.1N NaHCO₃, and 1M NaOH solutions. After 30 mins., the reaction mixture was chromotographed on Sephadex G-25 and eluted with 0.01M Tris-acetate buffer, pH 7.45, containing 10 mM MgCl ₂ and 100 mM NaCl. After 33 ml of eluent had been collected, a fraction (6.8 ml) containing enzyme activity was obtained:

Protein = 134 nM Digoxin = 530 nM i.e. approximately 5.2 moles of digoxin per mole of enzyme.

By variation of reaction conditions β-galactosidase digoxin conjugates have been prepared with the molar ratio of digoxin to enzyme varying between 1 and 10. Properties of β-galactosidase-digoxin Stability: The enzyme preparation was stored in solution at 4 for 4 months in buffer but without ammonium sulphate or other protective agents and showed a slow loss of activity over the period being 60% of the original activity after this pe riod

Preparation have been lyophvlised and have shown no loss of activity on reconstitution.

Antibody With a solid phase anti -digoxin anti Binding : body 80-85% of enzyme activity is bound to the solid phase. Using Fab to bind the enzyme ligand and then sequentially adding sclid phase anti Fab antibody 85-90% of enzyme activity has been bound. Sensitivity: The minimum concentration of enzyme ligand able to be measured using 100 of sample to a total volume of 150 ul in the direct assay using p-nitrophenyl β-galactosice as substrate at 405 nm is 1 nM which is 100 f moles of enzyme digoxin in the cuvette. This results in an absorbance change of 0.01 per minute. Using the cyclising assay the minimum concentration of enzyme ligand using 100 ul of sample to a total volume of

150 ul at 340 nm is 0.35 x 10^-12 molar, which corresponds to 3.5 f moles of enzyme ligand in the cuvette, This results in an absorbance change of 0.01 per minute. DIGOXIN ENZYME IMMUNOASSAY Solid Phase Second Antibody

Principle: Enzyme-digoxin, serum digoxn, and antidigoxin antibody are incubated in buffer for thirty minutes at room temperature. 100 ul of solid chase precipitating antibody is then added, and the mixture incubated for 30 minutes. The mixture is then spun at 2000 rpm for 5 minutes on a bench centrifuge and the supernatant assayed for residual enzyme activity. Reagents: 1. Enzyme-ligand solution.

Digoxin β-galactcsidase solution (50 ul) contains 240 f moles digoxin, 20 f moles protein in PBS buffer.

2. Anti-ligand antibody.

Fab derivatives of anti-digoxin gamma globulin (100 ul) sufficient to give 60% binding of enzyme-ligand in assay in PBS buffer.

3. Solid-phase precipitating antibody. Sepharose Ab Fab diluted in PBS buffer to give

100% binding of Fab in assay.

4. Enzyme substrate solution.

Phenyl β-gaiactoside: 0.6 mg/ml 0.05 M phosphate pH 6.0 - twice recrystallized.

5. Second enzyme solution.

Tyrosinase : 1.8 mg/ml buffer, 0.05 M phosphate pH 6.0.

6. NADH/DOPA : 6 mg/ml NADH, 30 um DOPA in 0.05 M phosphate pH 6.0.

7. Buffer.

0.01 M phosphate pH 7.4, 0.15 M KCl containing 4 mg/ml bovine serum albumin (PBS buffer). 0.05 M phosphate pH 6.0.

8. Digoxin standards.

Serum containing 0,0.5, 1.0, 2.0, 4.0 u g/litre . Set up in conical glass tubes:- 50 ul buffer (0.01 M phosphate pH 7.4, 0.15 M KCl containing 4 mg/ml BSA). 50 ul β-galactosidase-digoxin (150 nM) diluted 1/400 with buffer. 50 ul serum/standard. 100 ul Fab diluted 1/100 with buffer (suff icient to bind about 60 % β-galactos idase digoxin) .

Stand R . T . (25° ). 60 mins . Add 100 ul stirred Sepharose Ab ^Fab

Shake 60 mins. R.T. (intermittent if necessary) .

Centrifuge (conical tubes necessary!) at 200 rpm for 5 mins.

Remove 100 ul supernatant. Add 10 ul NADH/DOPA

25 ul Tyrosinase

To initiate reaction add 50 ul phenyl β-galacto side.

Measure decrease in A₃₄₀ at 37º over 10 mins. Plot: Rate vs. concentration, or % bound vs. concentration from total by using 100 ul of buffer in place of solid phase antibody.

Applic- Ligands with serum concentrations 0-7 n ability: moles/litre. Comment: The previous comments on how to reduce the incubation times of the assay apply here as well. Times of 15 mins. have been used manually and this should be able to be reduced to 5-10 minutes with precise automated control.

Claims

1. A method of enzyme immunoassay which includes homogeneous enzyme immunoassay and enzyme-linked immunosorbent assay wherein the free or bound enzyme-ligand or enzyme antibody conjugate to be assayed is assayed by the following steps;

(i) producing a substrate directly or indirectly by enzymic action of said conjugate upon a suitable enzyme substrate; (ii) converting said substrate to a product in a first stage of a cyclic reaction sequence and converting said product back to said substrate in a second reaction stage of said cyclic reaction sequence, at least one of said first and second reaction stages being enzyme catalyzed, (iii) simultaneously with, either said first reaction stage or said second reaction stage converting an indicating substrate to an indicating product in a non-reversible indicator reaction; and (iv) determining the loss of indicating substrate or gain of indicating product in said indicator reaction.

2. A method as claimed in Claim 1 wherein β-galactosidase ligand or β-galactosidase antibody conjugate is assayed by β-galactosidase hydrolysis of a β-galactoside to produce a phenol or phenol derivative which is converted to a corresponding o-catechol by the enzyme tyrosinase, said o-catechol being converted to a corresponding o-quinone by the action of tyrosinase, said o-quinone being reconverted to said o-catechol with the conversion of NADH to NAD as the indicator reaction.

3 . A method as claimed in Claim 2 wherein the phenol derivative is selected from L-Dopa and p- substituted phenols where the substituent is selected from -SCN, -COCH₃ , -CHO, -CN and -NO₂.

4. A method as claimed in Claim 1 wherein β-galactosidase ligand or β-galactosidase antibody is assayed by p-galactosidase hydrolysis of a β-galactoside to produce a p-catechol which is converted to a p-quinone by the enzyme laccase with the p-quinone being reconverted back to p-catechol in the presence of NADH which is simultaneously converted to NAD as the indicator reaction.

5. A method as claimed in Claim 4 wherein fungal laccase at a pH of 4-5 is used to convert p-catechol to p-quinone which is subsequently reconverted to p-catechol, said p-catechol being derived from α-galactosidase-ligand hydrolysis of an α-galactoside.

6 . A method as claimed in Claim 1 wherein pyruvate kinase ligand or pyruvate kinase antibody conjugate is assayed by converting phosphoenol pyruvate with ADP as a cofactor to pyruvate which is subsequently converted to lactate in the presence of lactate dehydrogenase with the simultaneous conversion of NADH to NAD as the indicator reaction with the lactate being subsequently reconverted to pyruvate by cytochrome C linked lactate dehydrogenase.

7 . A method as claimed in Claim 1 wherein amylase ligand or amylase antibody conjugate is assayed by converting a glucose polymer to glucose which is subsequently converted to glucose-6-ρhosphate in the presence of the enzyme hexokinase simultaneously with the conversion of ATP to ADP as the indicator reaction, said glucose-6-phosphate being subsequently converted to glucose in the presence of glucose-6-ρhosρhatase.

8. A method as claimed in Claim 1 wherein aldolaseligand or aldolase antibody conjugate is assayed by forming fructose -1, 6-diphosphate from dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, said fructose 1, 6-diphosphat being converted to fructose 6 phosphate and inorganic phosphate in the presence of the enzyme fructose-1, 6-diphosphatase, said fructose 6-phosphate being reconverted back to the fructose-1, 6-diphosphate at the same time as ATP is converted to ADP by use of a phospho-fructokinase.

9. A method of determining a substrate in a sample which comprises:

(i) converting said substrate to a product in a first stage of a cyclic reaction sequence and converting said product back to said substrate in a second reaction stage of said cyclic reaction sequence, at least one of said first and said second reaction stages being enzyme catalyzed; (ii) simultaneously with either said first reaction stage or said second reaction stage, converting an indicating substrate to an indicating product in a non-reversible indicator reaction; and (iii) determining the loss of indicating substrate or gain of indicating product in said indicator reaction.