GB2102946A

GB2102946A - Enzyme immunoassay

Info

Publication number: GB2102946A
Application number: GB8218641A
Authority: GB
Inventors: Richard Julian Stuart Duncan; Peter David Weston
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1981-06-30
Filing date: 1982-06-28
Publication date: 1983-02-09
Also published as: GB2102946B; JPS587561A; FR2508486B1; FR2508486A1; DE3224217A1

Abstract

The present invention comprises a method for determining the presence of, or quantifying, a substance in a medium suspected of containing the substance which comprises: a) mixing together the medium with a conjugate consisting of the substance linked to an antibody capable of activating a substantially inactive enzyme, with an antibody or binding protein specific to the substance and with excess of the substantially inactive enzyme, the constituents being added in such an order that the enzyme is not admixed with the conjugate until any substance in the medium has had the opportunity to react with the antibody or binding specific to the substance, and b) adding substrate for the enzyme and determining the activity of the enzyme; a conjugate as defined hereinbefore and a test kit for carrying out the method of the invention.

Description

SPECIFICATION Enzyme immunoassay The present invention relates to an enzyme immunoassay method for determining the presence of, or quantitating, a substance in a medium suspected of containing the substance.

A variety of enzyme immunoassay techniques are known which include methods wherein an enzyme is bound to an antibody or antigen, the presence of which is to be detected. Enzyme immunoassay methods, and in particular homogeneous enzyme immunoassay methods, are reviewed in U.K. Patent Application Number 2,043,245A. This particular patent application relates to a homogeneous enzyme immunoassay wherein an enzyme and an irreversible inhibitor to that enzyme are present, the irreversible enzyme inhibitor being conjugated to an analogue of the ligand to be detected and the conjugate being capable of reacting with the enzyme by forming covalent bonds which alter the structure of the enzyme thereby irreversibly inhibiting the enzyme activity.A binding protein bindable to the ligand to be detected and the ligand conjugate is also present, the conjugate being inactivated when bound to the binding protein. The amount of ligand to be detected affects the binding between the conjugate and the binding protein therefore effecting the action that the conjugate has on the activity of the enzyme. The enzyme activity is then measured, compared with the activity when no ligand is present and the amount of ligand present quantified.

FEBS letters, vol. 16, July 1980, p. 285-288, also describes such a system but in addition suggests several alternative homogeneous enzyme immunoassay systems working on similar principles. These are not exemplified, however.

One such system, it is suggested could involve the use of an enzyme activator, such as an antibody to the wild-type E. coli B-galactosidase.

The present invention relates to a novel homogeneous enzyme immunoassay technique for the quantification of a substance believed to be present which is based on the activation of an inactive enzyme, the degree of activation of the enzyme being dependent on the amount of the substance present.

Accordingly, the present invention provides a method for determining the presence of, or quantifying, a substance in a medium suspected of containing the substance which comprises: a) mixing together the medium with a conjugate consisting of the substance linked to an antibody capable of activating a substantially inactive enzyme, with an antibody or binding protein specific to the substance and with excess of the substantially inactive enzyme, the constituents being added in such an order that the enzyme is not admixed with the conjugate until any substance in the medium has had the opportunity to react with the antibody or binding protein specific to the substance, b) adding substrate for the enzyme and determining the activity of the enzyme.

In one preferred embodiment, the present invention provides a method for determining the presence of, or quantifying, a substance in a medium suspected of containing the substance which comprises: a) adding the medium to a mixture of an antibody or binding protein specific to the substance and excess of the substantially inactive enzyme, b) adding a conjugate consisting of the substance linked to an antibody capable of activating the substantially inactive enzyme, and c) adding substrate for the enzyme and determining the activity of the enzyme.

In a further preferred embodiment the present invention provides a method for determining the presence of, or quantifying, a substance in a medium suspected of containing the substance which comprises: (a) mixing together the medium with a conjugate consisting of the substance linked to an antibody capable of activating a substantially inactive enzyme, (b) adding antibody or binding protein specific to the substance, (c) adding excess of the substantially inactive enzyme, and (d) adding substrate for the enzyme and determining the activity of the enzyme.

Preferably, the antibody capable of activating the substantially inactive enzyme is a monoclonal antibody.

The antibody or binding protein specific to the substance is capable of binding to the substance when this is unconjugated or when this is conjugated to the activating antibody. When the antibody or binding protein specific to the substance is bound to the conjugate then this decreases the level of enzyme activation that is observed. Suitably, the antibody would have been purified by standard techniques, for example by fractionation involving precipitation with ammonium sulphate and subsequent column chromatography on ion exchange cellulose.

Suitably the purified antibody will be substantially free of other proteins, i.e. will contain less than 10% of other proteins.

Suitably, a further antibody capable of activating the substantially inactive enzyme is mixed with the enzyme before this is added to any of the other ingredients.

By substantially inactive enzyme is meant an enzyme, such as an inactive mutant of pgalactosidase which has less than 10%, suitably less than 5% and preferably less than 1% of the activity of the same enzyme when activated.

Suitably such a mutant form of ,B-galactosidase is prepared by the cultivation of a mutant strain of E.coli.

It has been found that a number of types of substantially inactive p-galactosidase produced by cultivation of mutant strains of E.coli are unstable. It has also been found that a number of types of substantially inactive /3-galactosidase produce a high background noise, i.e. produce a colour reaction, when mixed with a substrate without the presence of an activating antibody.

Accordingly the substantially inactive ,B- galactosidases suitable for inclusion within the method of the present are those that are stable (i.e. they retain their ability to be activated over a period of several months and catalyze a reaction that is linear for many minutes) and do not produce a significant background noise. A particularly suitable type of p-galactosidase is that produced from strain (WM298) of E. coli, Allele; lac aba 627 obtained from Dr. W. Messer, Max Planck Institute, 1 Berlin 33 (DAHLEM), Ihnestrasse 63-73.

The activating monoclonal antibody used in the technique is suitably intact immunoglobulin although fragments of immunoglobulin may also be used. The antibody or binding protein specific to the substance being tested for is preferably a high titre polyclonal antibody or a monoclonal antibody specific to this substance.

The present invention relates to methods particularly suitable for determining substances in biological fluids such as serum, plasma, spinal fluid and urine. Suitable substances for detection and quantitation by the method of the present invention include hormones, proteins, vitamins, poisons, drugs or drug metabolites. The methods of the present invention are particularly suitable for the detection of substances of medical importance. Such substances include steroids, vitamins, proteins and fragments of proteins, folic acid, serotonin, prostaglandins, adrenalin, noradrenalin, opiates, theophylline, dilantin, barbiturates, aminoglycoside antibiotics, acyclovir, and antiviral agents. Preferably the substance is an anti-epileptic drug, an antibiotic, an antiviral agent, a hormone or a protein.It has been found that the method is particularly convenient for the quantitation of diphenyl hydantoin, cortisol, acyclovir, fibrinogen and fibrinogen degradation product D. It is believed that it is particularly surprising and advantageous that the methods of the present invention are suitable for the detection of substances of high molecular weight, i.e. between 10,000 and 1,000,000 such as proteins and fragments of proteins.

The substance to be quantitated will normally be present at a concentration in excess of 1 nanomolar, suitably at a concentration in excess of 40 nanomolar in the case of cortisol, at a concentration in exess of 250 nanomolar in the case of acyclovir at a concentration in excess of 10 micromolar in the case of diphenyl hydantoin and at a concentration in excess of 60 nanomolar in the case of fibrinogen and fibrinogen degradation product D.

The conjugate consisting of the substance and the antibody capable of activating the substantially inactive enzyme will be prepared by conventional methods well known to those skilled in the art. The substance will be linked to the antibody through conventional bridging groups.

For both cortisol, acyclovir and diphenyl hydantoin, a particularly suitable method has been to introduce a carboxylic acid function into these ligands followed by conversion to the Nhydroxy succinimide ester which readiiy links to appropriate functions of the monoclonal antibodies; for fibrinogen degradation product D a particularly suitable method has been to introduce protected thiol groups into the D fragment and the maleimide residues into the monoclonal antibody then removing the protecting group from the thiol residues and mixing the two proteins to facilitate coupling. Other suitable conjugation methods are reviewed by Kabakoff in Enzyme Immunoassay, C.R.C. Press, Editor E. T. Maggio, 1980,71-104.

The assay of the present invention will normally be carried out in an aqueous liquid medium in the pH range 5 to 10 and preferably at about pH 7.4. The assay is conveniently stopped by raising the pH to about pH 10 and by adding a chelating agent for magnesium, such as EDTA, and a lipid solubilising agent, such as sodium cholate or sodium deoxycholate. Preferably an alkanol or polyol is present in the aqueous liquid medium. Methanol, ethanediol, propan-1,2-diol, mannitol, sorbitol and D-arabitol are particularly suitable for inclusion in the aqueous medium.

Surprisingly it has been found that D-arabitol both increases the sensitivity of the assay and decreases the length of time required to carry out the assay, by a mechanism distinct from, and additional to that by which methanol operates.

The temperature of the liquid medium during the performance of the method of the present invention will normally be between 1 OOC and 450C and will conveniently be 370C.

It will be apparent to those skilled in the art that time must be allowed (the incubation period) to enable the appropriate immunological reactions to occur, i.e. the reaction of the antibody or binding protein specific to the substance with the conjugate and with the substance in the medium, when present, and also to enable the enzyme to reach its appropriate activated state.

The precise stages at which it is necessary to carry out incubations will depend on the particular circumstances of the assay, for example the order of addition of the reagents, but incubation will always occur before addition of the substrate. A man skilled in the art will be able to judge when a particular incubation stage is appropriate.

The substance, conjugate, enzyme and antibodies will normally be soluble in the medium in which the method is carried out. The substrate may or may not be soluble in the medium but conveniently is soluble in the medium. In certain circumstances it may be desirable to provide a synthetic substrate which is not soluble or employ an insoluble natural substrate.

Conveniently an excess of normal immunoglobulin, for example normal sheep immunoglobulin is included in the assay of the present invention to neutralise the effect of antibodies accidentally present in the medium which may interact with the antibodies capable of activating the substantially inactive enzyme.

Mutant forms of p-galactosidase are known to contain a thiol group essential to their activity.

When such enzymes are used in the assays of the present invention, dithiolthreitol or a similar reagent will normally be included to prevent oxidation of the thiol group occurring. Suitable preservatives, such as sodium azide, can conveniently and advantageously be included in the assay of the present invention.

In the method of the present invention there is competition between the substance to be quantified, when present, and the conjugate consisting of the substance and an antibody capable of activating the substantially inactive enzyme for binding to the specific antibody or binding protein. As the level of enzyme activation is dependent upon the exact degree of binding between the conjugate and the specific antibody or binding protein, then the enzyme activity of the sample is dependent on the amount of substance in the sample.

The manner of assaying the enzyme activity may be varied according to requirements.

Conveniently spectrophotometric or fluorometric measurements can be made to determine the level of the enzyme activity.

The conjugates consisting of the substance to be determined linked to the antibody capable of activating the substantially inactive enzyme are novel and form a further important aspect of the present invention.

The present invention also provides a test kit comprising Reagent 1; antibody specific to the substance to be analysed mixed with substantially inactive enzyme.

Reagent 2; conjugate.

Reagent 3; substrate for enzyme.

Reagent 4; stopping reagent Suitably Reagent 1 includes an antibody capable of activating the substantially inactive enzyme. Suitably the substantially inactive enzyme is a mutant form of p-galactosidase in which case Reagent 1 normally includes dithiothreitol or similar reagent for prevent oxidation of the thiol group contained in the pgalactosidase. Conveniently Reagent 1 also includes normal immunoglobulin and preferably contains normal sheep immunoglobulin. Suitably Reagent 1 and 2 contain a polyol preferably Darabitol.

Suitably Reagent 2 contains a normal immunoglobulin, and preferably contains normal sheep immunoglobulin.

Preferably Reagent 3 contains an alkanol, such as methanol. The stopping agent is a mixture of a high pH buffer, a chelating agent for magnesium and a lipid solubilising agent. Suitably the pH buffer comprises sodium hydroxide in glycine the chelating agent is EDTA and the lipid solubilising agent is sodium cholate. The preparation of the components of the present test method and the operation of the test method are illustrated by the following: Enzyme purification Strains of E. colt which produced activatable mutant p-galactosidase as described by Melchers and Messer (Eur. J. Biochem. 17(1970), 267) were grown in a mineral salts medium supplemented with sodium succinate and thiamine (Fowler, J. Bacterial, 112 (1972), 856).

A further strain A324-5 as described by Fowler (see above) was grown to provide enzymically active wild type p-galactosidase.

The bacteria were harvested by centrifugation and stored frozen until required. After thawing and sonication, p-galactosidase and its various antibody activable mutant forms were purified by classical chromatographic techniques as described by Craven et al. (J. Biol. Chem. 240 (1965), 2468) and Tenu et al. (Eur. J. Biochem 20 (1981), 363). Affinity chromatography could also be used but has the disadvantages of expensive syntheses of column media and low capacities. In a typical purification, 30 litres of bacterial growth medium were harvested, the bacteria were then sonicated and the suspension centrifuged.About 1 litre of supernatant, containing all the Bgalactosidase protein from the bacteria was obtained and the enzyme was partially purified and concentrated by collecting the material precipitating between 1 5 and 45% saturation with ammonium sulphate. After dialysis or gel filtration the enzyme was applied to a column of DEAE-cellulose and eluted with a gradient.

Fractions from the DEAE column combining the enzyme were pooled and applied directly to a column of hydroxylapatite, being eluted with a gradient of phosphate. Again the enzyme containing fractions were pooled and then applied directly to a column of DEAE-Sepharose CL4B.

Elution with a gradient yielded 500-1000 mg of homogeneous /3-galactosidase. The hydroxylapatite column could be replaced with no loss of efficiency by a column of Sephacryl S300.

The purified enzymes may be stored for many months with no loss of actual or potential catalytic activity by suspending them in a buffered solution of 50% saturated ammonium sulphate containing mercaptoethanol or other sulphydryl protecting agent.

Production of antibody Mice were immunised with purified wild type p-galactosidase, and following methods originated by Kohler and Milstein (Eur, J.

Immunol; 6, (1975), 511) their spleens were fused with a myeloma cell line. The fused cells were cultured in vitro in multiwell plates and the supernatant solutions examined for anti pgalactosidase antibodies which activated the otherwise inactive mutant enzymes. Cells from those wells which produced the desired antibodies were cloned repeatedly by the method of limiting dilution. Monoclonal antibody cell lines were thus established and grown up as required either in vitro using tissue culture or in vivo in the peritoneal cavities of mice to give ascitic fluid.

Antibodies were purified from the culture media or ascitic fluid by chromatography on ionexchange cellulose.

Four cell lines producing antibody were established and numbered BG-18, BG-19, BG-79 and BG-81 ; the immunoglobulin produced by these lines when mixed with inactive purified mutant enzyme gave p-galactosidase activity.

Antibody from line BG-79 showed a special property; when mixed with antibody from any of the other three lines and added to inactive mutant enzyme it was found that the activity of the enzyme was synergistically increased giving an activity greater than that expected from the summation of any of the antibodies used singly.

The activity may be increased by the use of methanol, ethanediol, propan-1,2-diol or other compounds containing the hydroxyl functionality; and additionally by a different mechanism, by sugar alcohols, particularly sorbitol and mannitol but especially by D-arabitol.

The observed synergism and the effect of added methanol is shown in Table 1.

Table 1 Activation of 295-type enzyme Antibody No methanol 10% Methanol By: BG-79 7.6+0.16 4.6+0.4 BG-81 0.86+0.04 0.2+0.004 BG-18 12.5+0.03 32+0.25 BG-19 1.0+0.01 0.88+0.003 BG-79+BG-81 28+1.2 46+1.8 BG-79+BG-19 10.9+0.2 16.9+0.1 BG-79+BG-18 42.5+0.5 93.5+3.5 BG-18+BG-19 18 The activity of the enzyme in the presence of excess antibody is expressed in ,moles of onitrophenol/n mole enzyme/min and is determined using o-nitrophenyl-P-D-galactoside as substrate.

Monoclonal antibodies numbers BG-l 8, BG19, BG-79 and BG-81 are available from Wellcome Diagnostics, Dartford, England.

Antibody preparation Monoclonal antibody, preserved as a freezedried powder was rehydrated, then fractionated by precipitation with 50% saturated ammonium sulphate. After resuspension and gel filtration of the collected precipitate the monoclonal antibody was applied to a column of DEAE-cellulose or Procion-3B substituted Bio-Gel DE at pH 7.0 in 10 mM phosphate bluffer. The eluted antibody was virtually homogeneous in a yield of up to 45 mg of antibody per 10 ml of resuspended freeze-dried powder.

Purified monoclonal antibody was substituted by treatment with an N-hydroxysuccinimide ester or other suitable active derivative of the ligand in question. For cortisol an optimum concentration of 10 molecules of N(cortisol-21hemisuccinyl)succinimide was allowed to react with each molecule of purified antibody for 1620 hours at OOC in buffer containing 10% dimethylformamide, phosphate and saline at pH 7.4. The substituted antibody was precipitated at 70% saturated ammonium sulphate and, after washing by resuspension in 70% saturated ammonium sulphate and centrifugation at least twice, all traces of cortisol-21 -hemisuccinate other than protein-bound were removed by gel filtration. The product was diluted appropriately and stored frozen, although freeze drying could be substituted.

For diphenyl hydantoin 20 molecules of the Nhydroxysuccinimide ester of 3-(N-carboxymethyl)diphenyl hydantoin was allowed to react with each molecule of purified antibody for 1 6 hours at room temperature (200C) in buffer containing phosphate 0.01 M and saline at pH 7.4.

Substituted antibody was separated from free 3 (N-ca rboxymethyl)-diphenyl hydantoin by gel filtration using a column of Biogel P10, 200- 400 Mesh using a 6 ml bed volume with a 1 ml sample and with the column equilibrated with Saline 0.15 M. For fibrinogen and fibrinogen degradation product D the purified protein or D fragment was reacted with S-acetyl mercapto succinic anhydride to introduce protected thiol groups. The monoclonal antibody was reacted with succinimidyl 4-(N maleimidomethyl)cyclohexane- 1 -carboxylate to introduce maleimide residues. For coupling the protected thiol groups in the fibrinogen or D fragment were exposed by treatment with hydroxylamine 25 mM at pH 7 for 1 hour at room temperature; the thiol protein or fragment D was then mixed with the maleimide-monoclonal antibody.After coupling overnight the conjugate was treated with mercaptoethanol to block excess maleimide residues then N-ethyl maleimide to block excess thiol groups; the conjugate was then purified by chromatography on Sepharose CL-4B for fibrinogen-antibody or Sephacryl S-300 for fragment D-antibody, the first eluted proteins being useful.

For the antiviral agent acyclovir, chemical name 9-(2-hydroxy-ethoxymethyl)guanine, ten molecules of the N-hydroxysuccinimide ester of succinyl acyclovir was allowed to react with each molecule of purified antibody for 16 hours at room temperature (200C) in buffer containing phosphate 0.01 M and saline at pH 7.4.

Substituted antibody was separated from free small molecules by gel filtration using a column of Biogel P10,200 9100 mesh using a 6 ml bed volume with a 1 ml sample and with the column equilibrated with saline 0.15 M.

Conduct of a typical homogeneous enzyme assay Example 1 Assay for cortisol 50,us of a sample of serum or urine believed to contain 0--1000 picomoles of cortisol was mixed with 1 picomol of cortisol-monoclonai BG-79 antibody conjugate, 60 picomol of purified IgG from an antiserum directed against cortisol in 20 yl and 50 Ml of 2 mM sodium 8-anilino-1 naphthylene sulphonate (pH 7.6).After incubating for 30 minutes at 37 C, about 1 picomol of activable i3-galactosidase enzyme already mixed with 1 30 nanomol of dithiothreitol and 20 picomol of a second unsubstituted activating antibody BG-81 was added along with 200 yl of o-nitrophenyl-P-D-galactoside substrate 7 mM in 100 mM phosphate buffer pH 7.40 containing 10% methanol. The incubation was continued for 30 more minutes then the optical density read at 420 nm and noted, the reaction being stopped with 1 ml of 0.4 M sodium carbonate containing 10% dimethyl-sulphoxide or other organic solvent or 1% sodium deoxycholate.Standards were prepared over the appropriate range, and also blanks (no anti-cortisol and no substituted antibody) for each sample. The results are shown in Table 2 below: Table 2 Cortisol standard curve Cortisol present (pmol) O.D.420 0 0.68 5 0.95 10 1.07 20 1.12 40 1.18 60 1.21 100 1.25 1000 1.36 Example 2 Assay for diphenyl hydantoin 25 yl of a sample of serum believed to contain 0.5 to 2.5 nmoles of diphenyl hydantoin was mixed with 4 picomol of diphenyl hydantoinmonoclonal BG-79 antibody conjugate in 25 yl and 900 picomol of purified IgG in 25 yI from an antiserum directed against diphenyl hydantoin.

The tubes were put to incubate at 37 0C and 6 picomol of activable B-galactosidase enzyme was added already mixed with 500 nanomol of dithiothreitol and 20 picomol of a second unsubstituted activating antibody BG-81. 200,ul of o-nitrophenyl-P-D-galactoside substrate 7 mM in 100 mM phosphate buffer pH 7.4 was then added to give a total incubation volume of 325 yI.

The incubation was continued at 370C for 15 minutes then the reaction stopped with 1 ml of 0.2 M sodium carbonate containing 1% sodium deoxycholate and the optical density read at 420 nm. Standards of diphenyl hydantoin were prepared over the appropriate range of 5-20 yg/ml serum, and also blanks (no anti phenytoin and no diphenyl hydantoin conjugated antibody) for each sample.The results are shown in Table 3 below: Table 3 Diphenyl hydantoin standard curve Diphenyl hydan toin (yg/ml) 0.D.420 0 0.175 5 0.225 10 0.241 20 0.247 Control: no antibody 0.328 Control: no conjugate 0.074 Example 3 Assay for fibrinogen degradation product D 10 yl of a sample of serum believed to contain 0.6 to 24 picomoles of fragment D was with mixed 20 yI Reagent 1 at room temperature and allowed to react for 5 minutes. 20 yI Reagent 2 was then added and incubation commenced at 370C for 10 minutes. 500 I substrate was then added and incubation continued at 37 OC for 5 minutes; the reaction was then stopped by addition of 100,us stopping reagent and the optical density of the solution read at 420 nm.

Standards of fragment D were prepared over the appropriate range 5-200 yg/ml serum. The reagents used and the results obtained are shown in Table 4 and 5 respectively.

Table 4 Reagents for assay for fibrinogen degradation product D.

Reagent 1 Sheep anti fragment D (purified immunoglobulin) 15 mg/ml 520,us Dithiothreitol 250 mM in water 400 e41 Monoclonal 81 (purified immunoglobulin) 1.9 mg/ml 400,ul D(+) Arabitol 300 mg/ml, Normal sheep immunoglobulin 1 mg/ml in buffer 2680ul Enzyme: 12 yl of 10 mg suspension in ammonium sulphate in 12 mM dithiothreitol, 1 mg/ml normal sheep immunoglobulin in buffer. 4000 yl Reagent 2 Conjugate of monoclonal IgG-D fragment: 800 yI. Arabitol 300 mg/ml, 1 mg/ml normal sheep immunoglobulin: 7,200,ul in buffer.

Buffer used for Reagents 1 and 2 and substrate: 50 mM Tris.

25 mM Sodium Chloride.

10 mM Magnesium chloride.

1 mM EDTA.

at pH 7.5.

Substrate o-Nitrophenyl-p-D-galactoside 2.5 mg/ml in buffer containing 2.5 molal methanol.

Stopping 2 M glycine.

Reagent 200 mM EDTA 0.2% sodium cholate pH adjusted to 10.1 with sodium hydroxide Table 5 Fibrinogen degradation product D standard curve Fragment D Fg/ml D420 0 0.404 15 0.409 25 0.412 40 0.426 65 0.464 100 0.548 200 0.566 Example 4 Assay for fibrinogen 20 jut of a sample of serum believed to contain 0-3 mg/ml fibrinogen was mixed with 20 jul, 260 Hg/ml of a purified IgG from an antiserum directed against fibrinogen. Conjugated fibrinogen -1 gG (79 (20 l, OD28 +0.22 was then added followed by enzyme mixture; 20 l (enzyme ammonium sulphate suspension 6 jut, 10 mg/ml with dithiothreitol 100 l, 250 mM with tris buffer 1 ml with flask grown monoclonal antibody BG-81 1 ml).The mixed solutions were incubated for 5 minutes at 370C then 200 jut of o nitrophenyl-P-D-galactoside 7 mM in tris buffer containing 10% methanol added. The incubation was continued for 20 minutes at 37 C then the reaction was stopped with 500 jut of 0.4 M sodium carbonate containing 1% sodium deoxycholate. Standards were prepared over the appropriate range and also a blank with no substituted monoclonal antibody for each sample.

The results are shown in Table 5 below: Table 6 Fibrinogen standard curve Fibrinogen {mg/ml) OD420 0 0.518 0.031 0.626 0.063 0.667 0.125 0.769 0.25 0.892 0.50 0.953 1.0 1.102 Example 5 Assay for acyclovir Sera believed to contain 10 to 100 picomoles of acyclovir were diluted four fold with buffer, then a 10 jut sample was mixed with 20 l of Reagent A at room temperature and allowed to react for 5 minutes, 20 jul of Reagent B was then added an incubation continued at room temperature for 10 minutes. 500 jul substrate prewarmed at 37 OC was then added and incubation started in a water bath at 37 C for 5 minutes; the reaction was then stopped by addition of 100 jut stopping reagent and the optical density of the solution read at 420 nm.

Standards of acyclovir were prepared over the appropriate range 1 to 10 yM in serum. The reagents used and the results obtained are shown in Tables 7 and 8 respectively.

Table 7 Reagents for assay of acyclovir Reagent A Ascitic fluid containing monoclonal anti acyclovir antibodies AC/AINo 2 200 l Dithiothreitol 250 mM in water 800 jut Monoclonal 81 (purified immunoglobulin), 1.9 mg/ml 200 l D(+) Arabitol enzyme, 1.2 g Enzyme, 10 mg/ml suspension in ammonium sulphate in 12 mM dithiothreitol 12 jut Buffer 3000 flu Reagent B Conjugate of monoclonal IgG-acyclovir at 20 Mg/ml in buffer with 0.1 mg/ml normal sheep immunoglobulin.

Buffer used for Reagent A and B and substrate: 50 mM Tris.

25 mM Sodium chloride.

10 mM Magnesium chloride.

1 mM EDTA.

adjusted to pH 7.5 with hydrochloric acid.

Substrate o-nitrophenyl-p-D-galactoside 2 mg/ml in buffer containing 2.5 molol methanol.

Stopping reagent 2 M glycine.

200 mM EDTA.

0.2% sodium cholate.

adjusted to pH 10.1 with sodium hydroxide.

Table 8 Acyclovir standard curve Acyclovir OD420 yM (duplicate assays) 0 0.329 0.345 2.5 0.391 0.395 5 0.569 0.575 7.5 0.645 0.629 10 0.675 0.665

Claims

1. A method for determining the presence of, or quantifying, a substance in a medium suspected of containing the substance which comprises: a) mixing together the medium with a conjugate consisting of the substance linked to an antibody capable of activating a substantially inactive enzyme, with an antibody or binding protein specific to the substance and with excess of the substantially inactive enzyme, the constituents being added in such an order that the enzyme is not admixed with the conjugate until any substance in the medium has had the opportunity to react with the antibody or binding protein specific to the substance, and b) adding substrate for the enzyme and determining the activity of the enzyme.

2. A method according to claim 1 which comprises: a) adding the medium to a mixture of an antibody or binding protein specific to the substance and substantially inactive enzyme, b) adding a conjugate of the substance linked to an antibody capable of activating the substantially inactive enzyme, and c) adding substrate for the enzyme and determining the activity of the enzyme.

3. A method according to claim 1 which comprises: a) mixing together the medium with a conjugate consisting of the substance linked to an antibody capable of activating a substantially inactive enzyme, and with antibody or binding protein specific to the substance, b) adding excess of the substantially inactive enzyme, and c) adding substrate for the enzyme and determining the activity of the enzyme.

4. A method according to any one of claims 1 to 3, wherein the antibody capable of activating the substantially inactive enzyme is a monoclonal antibody.

5. A method according to any one of claims 1 to 4, wherein a further antibody capable of activating the substantially inactive enzyme is mixed with the enzyme before this is added to any of the other ingredients.

6. A method according to any one of claims 1 to 5, wherein the substantially inactive enzyme is a mutant form of p-galactosidose which has less than 1% of the activity of p-galactosidose when activated.

7. A method according to claim 6, wherein the mutant form of ,3-galactosidase is prepared by the cultivation of a mutant strain of E. coli.

8. A method according to any one of claims 1 to 7, wherein the substance being tested for is an anti-epileptic drug, an antibiotic, an antiviral agent, a hormone or a protein.

9. A method according to any one of claims 1 to 8, wherein the assay is carried out at about pH 7.4 in the presence of an alkanol or polyol at between 100C and 450C.

10. A method according to any one of claims 1 to 9 carried out in the presence of D-arabital.

11. A method according to any one of claims 1 to 10 carried out in the presence of methanol.

12. A method according to any one of claims 1 to 11, wherein the assay is stopped by varying the pH to about pH 10 and by adding a chelating agent for magnesium and a lipid solubilising agent.

13. A conjugate consisting of a substance to be determined, as hereinbefore defined, linked to an antibody capable of activating a substantially inactive enzyme.

14. A test kit comprising: Reagent antibody specific to the substance to be analysed mixed with substantially inactive enzyme, Reagent 2; a conjugate consisting of the substance linked to an antibody capable of activating the substantially inactive enzyme, Reagent 3; substrate for the substantially inactive enzyme, Reagent 4; stopping agent.