GB2095400A

GB2095400A - Detection of enzyme using supported enzyme binder

Info

Publication number: GB2095400A
Application number: GB8108928A
Authority: GB
Original assignee: Individual
Current assignee: Individual
Priority date: 1981-03-21
Filing date: 1981-03-21
Publication date: 1982-09-29

Abstract

A method is described for detecting an enzyme in a liquid which method comprises (i) contacting the liquid with a support to which is attached a substance to which the enzyme will bind specifically without eliminating its enzymatic properties so that the enzyme becomes associated with said support, (ii) separating the support with its associated enzyme from the liquid and (iii) using the support with its associated enzyme in an assay for the enzyme in which the enzyme associated with the support produces a modulator for a chemical reaction which causes a detectable change.

Description

SPECIFICATION Separation technique and assay This invention relates to a method by which an enzyme present in a liquid can be detected.

It is frequently desirable to detect enzymes in liquids in which a number of reactive species are present. This can be a particular problem if one is dealing with a biological fluid such as blood, serum or urine which can contain a very large number of reactive species. Clearly it would be desirable to have a method which could be used to separate the enzyme to be detected from other materials by a simple means, followed by a highly sensitive detection of the enzyme activity which could thus take place in the absence of interference by unwanted reactive species. Such a method has now been discovered.

Accordingly the present invention provides a method of detecting an enzyme in a liquid which method comprises (i) contacting the liquid with a support to which is attached a substance to which the enyzme will bind specifically without eliminating its enzymatic properties, so that said enzyme becomes associated with said support, (ii) separating the support with its associated enzyme from the liquid and (iii) using the support with its associated enzyme in an assayforthe enzyme in which assay the enzyme associated with the support produces a modulator for a chemical reaction which causes a detectable change.

Techniques of this kind have not hitherto been worthy of development since in general assay methods for enzymes have not been suffiently sensitive to make such methods easy to apply. The use of enhanced assay methods such as those of my (as yet unpublished) specifications numbers European No.803034784, PCT/G B 80/00153 and US Serial No. 193647 (which is incorporated herein by cross reference) are sufficiently sensitive to enable ready use of the new techniques disclosed herein.

The chemical reaction which causes a detectable change is aptly an enzymatic process which is caused to operate by the introduction of a modulator. Such systems are described in herebefore mentioned patent application (incorporated herein by cross reference).

The chemical reaction which causes a detectable change is aptly a cyclic chemical reaction. Such cyclic chemical reactions is most aptly an oxidation/ reaction and will favourable employ a NAD/NADH or NADP/NADPH interconversion of which the NAD/ NADH interconversion is preferred. This latter interconversion most desirably employs NAD as modulator in which case the enzyme most aptly detected is a phosphatase. Such a phosphatase may be an acid phosphatase or an alkaline phosphatase. Such cyclic reactions modulated by NAD are described in my compending unpublished UK Patent application no.

8104395 (which is incorporated herein by cross reference).

The method of this invention is most suitably adapted to the detection of an enzyme in blood, serum or urine or, in extract, of tissues or microbiological extracts. Since enzymes are often used as markers in the diagnosis of human pathological conditions it follows that the method of this invention is preferably adapted to the detection of an enzyme in human blood, serum or urine. Of course use may be made of this invention in thevetinary field in analogous manner.

The support used in this invention may be in the form of discrete surface such as that of a stick or dish or filter system or in the form of small particules such as a powder or in the form of gel materials.

Aptly the support may be polystyrene, nylon, cross linked dextrans (such as sephadex, sepharose and agarose), polyacrylamide, bromacetyl-cellulose or the like. Such materials are presently known for the immobilization of non-enzymatic biological materials such as proteins, for example antigens, nucleic acids and the like. The skilled worker will appreciate that such supports can bind a wide range of materials so that, in conventional manner, in order to prevent uptake of unwanted enzymes or the like from the liquid the support will be neutralised to prevent this, for example by treatment with innocuous protein after the substance to which the enzyme to be detected is bound to the support.

The substance to be bound to the support will normally and preferably be an antibody to the enzyme with the proviso that the antibody employed will not be one that eliminates the enzymatic properties of the enzyme to be detected. Very desirably the antibody will be a monoclonal antibody.

The binding of the antibody (or other substance) to the support may be physical or chemical binding.

Physical binding includes adsorption onto the surface of the support and chemical binding may be ionic or covalent. Sandwich systems are also envisaged. The antibody may be bound in any manner as long as it is still capable of specifically binding the enzyme in a manner which will not eliminate the enzyme activity.

Alternatively the substance to be bound to the support may be some other substance with specific affinity for the enzyme in question, such as a substrate, substrate-analogue or inhibitor substance for that enzyme. Such approaches are possible with enzymes having more than one active enzymic site per enzyme molecule for - if only one site is taken up in binding to the support through the substrate, substrate analogue, inhibitor - the other site may still be available for catalytic activity.

Generally sufficient binding of the enzyme to the support with its bound substance will take place within 10 seconds to 30 minutes depending on the reaction parameters, concentrations employed and degree of accuracy required. More usually the binding will take place within 30 seconds to 10 minutes and preferably from 1 to 5 minutes. Mild agitation may be employed to facilitate the binding.

At the end of the binding stage the liquid may be separated and the supportwith the enzyme bound thereto washed. Generally it is preferred that this washing is thorough in orderto remove unwanted unbound material. Washing may be carried out with water, saline, buffer or the like of which phosphate buffered saline is favoured. Suitably the washing liquid may include a mild surfactant such as Tween.

At this point the support with the enzyme bound thereto may be promptly used in an assay for the enzyme.

As previously indicated that assay will aptly be one employing an amplification system such as that described in my earlier patent application hereinbefore identified.

Enzymes to be detected in this manner include kinases such as pyruvate kinase (especially type 11) and creatin kinase and their various isoenzymes; phosphatases such as acid phosphatase and alkaline phosphatase and its various isoenzymes; dehydrogenases such as lactice dehydrogenase and its various iso-enzymes; and the like.

Normally and preferably the assay employed will be adpted to produce a visually or spectorphotometrically determinable end point.

The following example illustrates the invention: Detection of alkaline phosphatase.

The alkaline phosphatase used in this example is as obtainable from the Sigma Chemical Co (London) Ltd Type VII from bovine (calf) intestine catalogue number P4502 (1981). Antibody is raised against this material by any of the standard procedures. For example 0.5mg of the alkaline phosphatase suspen sion is taken and made to 1.0my with distilled water and shaken gently. One ml of Freund's complete adjuvant is then added and the mixture homoge nised through a connector between two hypodermic syringes back and forth until a stable water-in-oil emulsion is produced. Approximately 1 ml of the emulsion is then injected intramuscularly into each hindquarter of a rabbit. The injections are repeated two and four weeks later with the same amount of alkaline phosphatase but without the added Freund's adjuvant.Blood is then taken from the immunised animal two weeks after the last injection and the immune-serum taken off after clotting has taken place. An IgG fraction of the serum is then obtained by the standard procedure of DEAE Cellu lose Chromatography set out in Methods in Im munology by D. H. Cambell, J. S. Garvey, N. E.

Cremer and D. H. Sussdorf, 2nd Edition (1970) pages 193-197, published by W. A. Bejamin, Reading Massachusetts. An aliquot of the IgG fraction is then diluted to a protein concentration of 0.5mg/ml in 0.05M ca rbonatg buffer at pH 9.6 and is used to coat the wells of polystyrene micro-haemagglutination trays (Type M29 AR Microtitre, Dynatech Laborator ies) by the addition of 0.3ml of diluted antiserum being added to each well and left either for 6-8 hours at room temperature or 1 hour at 37"C. The solutions are then taken out of the wells by suction and replaced with 0.2% lactalbium and left overnight.

The wells are emptied and then washed three times with phosphate buffered saline containing 0.05% Tween 20 (PBS Tween). The plates are now ready for use.

Starting with a 1:1000 dilution, ten-fold dilutions of the original source of alkaline phosphatasewere made down to 1:107 and o.25ml of each placed separately into the coated wells. The solutions are left for 30 minutes at room temperature (230C) and then removed by aspiration. The wells are again washed three times with PBS-Tween. The wells are then assayed for their alkaline phosphatase activity.

To each well is added 210ul of 0.14Methanola- mine-HC1 buffer at pH 9.3 and containing 5mM MgC12 followed by 10ul 1 0mM thiazolylblue (Sign London Chemical Co), 10ul of 40 mM phenazine ethosulphate (Signma London Chemical Co), 5ul of alcohol dehydrogenase especially free from NAD (Sigma London Chemical Co - catalogue number A3263) and 5ul of 1M ethanol. Reactions are initiated by the addition of 10ul of a 10mMsolution og NADP (Sigma London Chemical Co) to each well, the plate gently agitated and left at room temperature.The solutions in the wells to which a higher concentration of alkaline phosphatase was added show a change of colour from pale yellow to black much faster than did those which had been exposed to lower concentration, indicating that the method not only detects the presence of alkaline phosphatase in the solution added to the wells but is also capable of quantification.

In a simple alternative, suitable (e.g. polystyrene) 1.0my plastic colourimeter cuvettes are coated with the IgG fraction of the antiserum as above. One ml samples of the alkaline phosphatase dilutions are then placed in individual cuvettes and incubated and washed as above. To each cuvette is then added 0.96ml of 0.14M ethanolamine-HC1 buffer at pH 9.3 and containing 5mMMgCi2followed by 10ul 10mM thiazolyl blue, 10u140mM phenazine ethosulphate, 5ul alcohol dehydrogenase (A3263) and 5ul 1M ethanol. Reactions are initiated as before by the addition of 10u11 OmM NADP and the solutions mixed. The colour changes may then be monitored by eye or at 570nm in a colourimeterfor more precise quantification.

The general method is adaptable to the detection of other alkaline phosphatases including those from human origin, however, in each case it is usually advisable to prime the wells with a specific anti serum raised against the particular alkaline phos phatase in question. Furthermore, different alkaline phosphatases from human origin (such as from liver and bone) may be separately determined in a mixture of different types by means of IgG fractions directed specifically against particular types being used to charge specific wells.

CLAIMS (Filed on 2213/82) 1. A method of detecting an enzyme in a liquid which method comprises (i) contacting the liquid with a support to which is attached a substance to which the enzyme will bind specifically without eliminating its enzymatic properties so that the enzyme becomes associated with said support, (ii) separating the support with its associated enzyme from the liquid and (iii) using the support with its associated enzyme in an assay for the enzyme in which the enzyme associated with the support produces a modulator for a chemical reaction which cases a detectable change.

2. A method as claimed in claim 1 wherein the chemical reaction which causes the detectable change is an enzymatic reaction.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **.

liquid may include a mild surfactant such as Tween.

At this point the support with the enzyme bound thereto may be promptly used in an assay for the enzyme.

As previously indicated that assay will aptly be one employing an amplification system such as that described in my earlier patent application hereinbefore identified.

Enzymes to be detected in this manner include kinases such as pyruvate kinase (especially type 11) and creatin kinase and their various isoenzymes; phosphatases such as acid phosphatase and alkaline phosphatase and its various isoenzymes; dehydrogenases such as lactice dehydrogenase and its various iso-enzymes; and the like.

Normally and preferably the assay employed will be adpted to produce a visually or spectorphotometrically determinable end point.

The following example illustrates the invention: Detection of alkaline phosphatase.

The alkaline phosphatase used in this example is as obtainable from the Sigma Chemical Co (London) Ltd Type VII from bovine (calf) intestine catalogue number P4502 (1981). Antibody is raised against this material by any of the standard procedures. For example 0.5mg of the alkaline phosphatase suspen sion is taken and made to 1.0my with distilled water and shaken gently. One ml of Freund's complete adjuvant is then added and the mixture homoge nised through a connector between two hypodermic syringes back and forth until a stable water-in-oil emulsion is produced. Approximately 1 ml of the emulsion is then injected intramuscularly into each hindquarter of a rabbit. The injections are repeated two and four weeks later with the same amount of alkaline phosphatase but without the added Freund's adjuvant.Blood is then taken from the immunised animal two weeks after the last injection and the immune-serum taken off after clotting has taken place. An IgG fraction of the serum is then obtained by the standard procedure of DEAE Cellu lose Chromatography set out in Methods in Im munology by D. H. Cambell, J. S. Garvey, N. E.

Cremer and D. H. Sussdorf, 2nd Edition (1970) pages

193-197, published by W. A. Bejamin, Reading Massachusetts. An aliquot of the IgG fraction is then diluted to a protein concentration of 0.5mg/ml in 0.05M ca rbonatg buffer at pH 9.6 and is used to coat the wells of polystyrene micro-haemagglutination trays (Type M29 AR Microtitre, Dynatech Laborator ies) by the addition of 0.3ml of diluted antiserum being added to each well and left either for 6-8 hours at room temperature or 1 hour at 37"C. The solutions are then taken out of the wells by suction and replaced with 0.2% lactalbium and left overnight.

The wells are emptied and then washed three times with phosphate buffered saline containing 0.05% Tween 20 (PBS Tween). The plates are now ready for use.

Starting with a 1:1000 dilution, ten-fold dilutions of the original source of alkaline phosphatasewere made down to 1:107 and o.25ml of each placed separately into the coated wells. The solutions are left for 30 minutes at room temperature (230C) and then removed by aspiration. The wells are again washed three times with PBS-Tween. The wells are then assayed for their alkaline phosphatase activity.

To each well is added 210ul of 0.14Methanola- mine-HC1 buffer at pH 9.3 and containing 5mM MgC12 followed by 10ul 1 0mM thiazolylblue (Sign London Chemical Co), 10ul of 40 mM phenazine ethosulphate (Signma London Chemical Co), 5ul of alcohol dehydrogenase especially free from NAD (Sigma London Chemical Co - catalogue number A3263) and 5ul of 1M ethanol. Reactions are initiated by the addition of 10ul of a 10mMsolution og NADP (Sigma London Chemical Co) to each well, the plate gently agitated and left at room temperature.The solutions in the wells to which a higher concentration of alkaline phosphatase was added show a change of colour from pale yellow to black much faster than did those which had been exposed to lower concentration, indicating that the method not only detects the presence of alkaline phosphatase in the solution added to the wells but is also capable of quantification.

In a simple alternative, suitable (e.g. polystyrene) 1.0my plastic colourimeter cuvettes are coated with the IgG fraction of the antiserum as above. One ml samples of the alkaline phosphatase dilutions are then placed in individual cuvettes and incubated and washed as above. To each cuvette is then added 0.96ml of 0.14M ethanolamine-HC1 buffer at pH 9.3 and containing 5mMMgCi2followed by 10ul 10mM thiazolyl blue, 10u140mM phenazine ethosulphate, 5ul alcohol dehydrogenase (A3263) and 5ul 1M ethanol. Reactions are initiated as before by the addition of 10u11 OmM NADP and the solutions mixed. The colour changes may then be monitored by eye or at 570nm in a colourimeterfor more precise quantification.

The general method is adaptable to the detection of other alkaline phosphatases including those from human origin, however, in each case it is usually advisable to prime the wells with a specific anti serum raised against the particular alkaline phos phatase in question. Furthermore, different alkaline phosphatases from human origin (such as from liver and bone) may be separately determined in a mixture of different types by means of IgG fractions directed specifically against particular types being used to charge specific wells.

CLAIMS (Filed on 2213/82) 1. A method of detecting an enzyme in a liquid which method comprises (i) contacting the liquid with a support to which is attached a substance to which the enzyme will bind specifically without eliminating its enzymatic properties so that the enzyme becomes associated with said support, (ii) separating the support with its associated enzyme from the liquid and (iii) using the support with its associated enzyme in an assay for the enzyme in which the enzyme associated with the support produces a modulator for a chemical reaction which cases a detectable change.
2. A method as claimed in claim 1 wherein the chemical reaction which causes the detectable change is an enzymatic reaction.
3. A method as claimed in claim 2 wherein the

enzymatic chemical reaction is part of a cyclic reaction.
4. A method as claimed in claim 3 wherein the cyclic reaction is an oxidation-reduction reaction.
5. A method as claimed in claim 4 wherein the oxidation-reduction reaction is a NADP/NADPH interconversion.
6. A method as claimed in claim 4 wherein the oxidation-reduction reaction is a NAD/NADH interconversion.
7. A method as claimed in claim 6 wherein NAD is the modulator.
8. A method as claimed in any claims 1 to 7 carried out on human blood, serum or urine.
9. A method as claimed in any claims 1 to 8 wherein the substance to which the enzyme to be determined becomes attached is an antibody to the enzyme.
10. A method as claimed in claim 9 wherein the antibody is a monoclonal antibody.