WO1990001557A1 - Enzyme assays - Google Patents

Enzyme assays Download PDF

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Publication number
WO1990001557A1
WO1990001557A1 PCT/GB1989/000900 GB8900900W WO9001557A1 WO 1990001557 A1 WO1990001557 A1 WO 1990001557A1 GB 8900900 W GB8900900 W GB 8900900W WO 9001557 A1 WO9001557 A1 WO 9001557A1
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WO
WIPO (PCT)
Prior art keywords
enzyme
solution
inhibitor
development reagent
assay
Prior art date
Application number
PCT/GB1989/000900
Other languages
French (fr)
Inventor
David Leslie Bates
Warwick Roy Bailey
Original Assignee
Novo Biolabs Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Biolabs Limited filed Critical Novo Biolabs Limited
Publication of WO1990001557A1 publication Critical patent/WO1990001557A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • C12Q2334/40—Triphenylmethane dye chromogens, e.g. fluorescein derivatives

Definitions

  • ENZYME ASSAYS This invention relates to assays for enzymes.
  • the invention has particular application in the field of immunoassays, in which enzymes are used as labels, 5 although the method can, if desired, be used to assay free enzymes in solution.
  • the term "enzyme” as used herein includes not only free enzymes, but also immobilised enzymes and enzyme conjugates. " In general enzyme assays are carried out by causing the enzymes to react with various development reagents, including an enzyme substrate, appropriate buffers, and, if appropriate for the particular system, co-enzymes etc.
  • the development reaction ⁇ results in a measurable change in some physical property, typically the development of a colour which may be measured spectrophotometrically, or, in more recent times, the production of a substance which may be measured electrochemically.
  • enzyme action is catalytic, rather than stoichiometric in nature, it is necessary to stop the enzyme reaction, after is has been allowed to proceed to a predetermined extent. Normally, this "stopping” is carried out by adding a “stopping" solution to the reaction mixture, after a predetermined reaction period has been allowed.
  • EP-a-0202081 describes the addition of an inhibitor at the outset of an enzyme reaction.
  • the term inhibitor is used in this context however to mea n 5 a substance such as ascorbic acid, which delays the production of the signal rather than progressively causes the signal to decrease.
  • US 4038485 is concerned with the use of certain inhibitor systems for stopping an enzyme development 0 reaction.
  • Examples given are heat generating chemical reactions, which thermally denature the enzyme, inhibitor systems of cross-linkable polymers, such that after a pre-determined time, a hard gel or polymer is formed which effectively stops molecular mobility and , thereby stops the reaction, and the use of microcapsules, which rupture upon wetting to release an inhibitor system.
  • All of the systems disclosed ar e complex to put into operation, and this results in a substantial complication and consequent cost in the ⁇ production of commercial systems.
  • US 4219417 discloses an enzyme assay, in which at least two consecutive sets of reaction conditions are required to complete the assay.
  • Microcapsules are employed, which provide delayed release of agents to 5 change the reaction conditions from the first to th e second set of reaction conditions.
  • NaOH-filled microcapsules maybe employed, to stop enzymic catalysis, and allow the detection of a dephosphorylated substrate, in an acid phosphatase dephosphorylation reaction. Again, complex and expensive reagents are required in order to put the method in practice.
  • an enzyme assay which is essentially 5 self-stopping but is also simple and inexpensive to put into effect, by reacting the enzyme with the development reagent, in the presence of a chelating agent which funtions as a progressive enzyme inhibitor such that the ability of the enzyme to react .with the development reagent to produce the determinable change is progressively inhibited as the reaction progresses.
  • the enzyme inhibitor can be chosen such that its presence does not reduce the initial enzyme activity to such an extent that the assay does not have sufficient sensitivity.
  • the assay method in accordance with the invention has the advantage that it is considerably simpler to perform than assays requiring a separate stopping step, and is much less complex and expensive than systems requiring the presence of slow release stopping agents or immobilising polymers.
  • the enzyme inhibitor may desirably be provided in a solution containing the development reagent or reagents, and the solution containing both may be added to the enzyme, in order to carry out the development.
  • a development reagent solution for use in an enzyme assay comprising at least one development reagent for the enzyme, and a chelating agent which is progressive inhibitor for the enzyme.
  • a solution may be incorporated into a test kit, which may incorporate other reagents, for example for carrying out an immunoassay.
  • the progressive enzyme inhibitor is a chelating agent, such as ethylendiaminetetraacetic acid (EDTA).
  • EDTA ethylendiaminetetraacetic acid
  • Such chelating agents can be chosen so as to be effective in binding metal ions in the enzyme used so as effectively to render the enzyme inactive.
  • Alternative chelating agents are, for example, ethylenedioxydiethylenedinitrotetraacetic acid (EGTA) and diaminocyclohexanetetracetic acid (CDTA).
  • the time at which the assay is "stopped” is primarily determined by the concentration of the progressive inhibitor, in the reaction system.
  • the method of the invention is suited particularly for use in enzyme development reactions in which the detected product is measured spectrophotometrically, it is not limited to such processes and can equally well be utilised where measurement is by fluorescence, or when the product is determined electrochemically.
  • Example 1 Standard solutions containing calf intestinal alkaline phosphatase having concentrations in the range 0-100 ng/ml were assayed by the following procedure.
  • a solution was prepared of phenolphthalein monophosphate (di AMPD salt - 15mM), sodium carbonate buffer - pH 10.0 (100- mM), EDTA (disodium salt - 25 mM), and 0.02% sodium azide as a preservative.
  • 10 microlitre portions of the various phosphatase standard solutions were added to respective wells of a 96 well microtitre plate (NUNC).
  • 200 microlitre portions of the inhibited phenolphthalein solution prepared above was then added to each well.
  • the plate was incubated at 20 ⁇ C, and colour development of each well was monitored at 1 minute intervals over a 1 hour period. After 60 minutes, the rate of colour development had slowed to less than a 1% change in optical density per minute.
  • Figure 1 illustrates the relationship between the alkaline phosphatase concentration in the starting standard solution, and the final optical density, after 60 minutes. It can be seen that Figure 1 shows excellent linearity (showing that enzyme inhibition is independent on enzyme concentration), and good
  • Example 2 Self-Stopping enzyme immunoassay for follicle stimulating hormone
  • FSH follicle stimulating hormone
  • microtitre plate was incubated for two hours and the wells washed three times with a washing buffer. 100 microlitre portions of an inhibited development reagent solution as utilised in Example 1 was then added to each of the wells. The colour development at 550 nanometres was monitored for 60 minutes, by measurement of the optical density at 1 minute intervals.
  • Figure 3 shows the calibration curve of optical density after 60 minutes, for the varying FSH concentrations. Figure 3 again illustrated excellent linearity, and adequate sensitivity. The working range of the assay was from
  • An inhibited development reagent solution was prepared, containing ortho-nitrophenyl beta-galactopyranoside (20 mM), EDTA (40 mM), and 0 diethanolamine buffer pH 8.0 (500 mM).
  • 25 illustrates excellent linearity between optical density and concentration, showing that the enzyme inhibition is independent of enzyme concentration.
  • the working range of the assay was (0-20 ng) beta-Galactosidase.
  • the method of the invention may be any method of the invention.
  • the method of the invention may be any method of the invention.
  • the method in accordance with the invention may be used to assay a reagent which takes part in the "preliminary” chemical • reaction.
  • the method in accordance with the invention may be utilised 0 to determine a wide variety of biochemical substances.
  • Test kits for carrying out enzyme assays may be produced in various formats, comprising the development reagent solution in accordance with the invention.
  • a test kit may contain an antibody-coated 5 microplate, and separate containers for (i) analyte standards, (ii) enzyme-labelled conjugate, (iii) the dry components of a washing buffer, and (iv) colour development reagents including the enzyme inhibitor.
  • the reagent solution may be used in 0 conjunction with an automated analyser which will generally use the same reagents as reagents (i) to (iv) noted above, but the microtitre plate is generally replaced by small polymer particles, having antibody absorbed or covalently bound to its surface.
  • J particles may or may not have magnetic properties.
  • the various reagents of a test kit may be dried into an absorbent material, such as paper or the like, in a number of layers, to which a test solution is added.
  • an absorbent material such as paper or the like
  • 0 development reagent and the inhibitor may be present either within the same layer, or within different layers, such that they come together when the test solution is added.
  • test kit formats are also possible, as is well known in the art .
  • the determinable change which is measured may be, for example, an electrochemical change, or a change in fluorescence, rather than a spectrophotometric one.

Abstract

A method of carrying out an assay for an enzyme, for example as part of an enzyme immunoassay, comprises reacting the enzyme with a development reagent in the presence of a chelating agent such as EDTA, which is a progressive enzyme inhibitor, under conditions such that the ability of the enzyme to react with the development reagent to produce the determinable change is progressively inhibited as the reaction progresses.

Description

ENZYME ASSAYS This invention relates to assays for enzymes. The invention has particular application in the field of immunoassays, in which enzymes are used as labels, 5 although the method can, if desired, be used to assay free enzymes in solution. Accordingly, the term "enzyme" as used herein includes not only free enzymes, but also immobilised enzymes and enzyme conjugates. " In general enzyme assays are carried out by causing the enzymes to react with various development reagents, including an enzyme substrate, appropriate buffers, and, if appropriate for the particular system, co-enzymes etc. The development reaction ^ results in a measurable change in some physical property, typically the development of a colour which may be measured spectrophotometrically, or, in more recent times, the production of a substance which may be measured electrochemically. " Because enzyme action is catalytic, rather than stoichiometric in nature, it is necessary to stop the enzyme reaction, after is has been allowed to proceed to a predetermined extent. Normally, this "stopping" is carried out by adding a "stopping" solution to the reaction mixture, after a predetermined reaction period has been allowed.
The addition of materials such as alkali, or chelating agents after completion of an enzyme assay, in order to terminate the enzyme reaction is well 0 known, for example, from US 3799383, EP-a-0199363,
EP-a-0207493. "Analytical Chemistry, volume 51, No: 2 (1979) page 199" discloses the addition of materials which are described as "competitive inhibitors" to enzyme reactions for the purpose of improving reaction linearity, and the measurement of kinetic rates is also known, from US 4218535.
EP-a-0202081 describes the addition of an inhibitor at the outset of an enzyme reaction. The term inhibitor is used in this context however to mea n 5 a substance such as ascorbic acid, which delays the production of the signal rather than progressively causes the signal to decrease.
US 4038485 is concerned with the use of certain inhibitor systems for stopping an enzyme development 0 reaction. Examples given are heat generating chemical reactions, which thermally denature the enzyme, inhibitor systems of cross-linkable polymers, such that after a pre-determined time, a hard gel or polymer is formed which effectively stops molecular mobility and , thereby stops the reaction, and the use of microcapsules, which rupture upon wetting to release an inhibitor system. All of the systems disclosed ar e complex to put into operation, and this results in a substantial complication and consequent cost in the ^ production of commercial systems.
US 4219417 discloses an enzyme assay, in which at least two consecutive sets of reaction conditions are required to complete the assay. Microcapsules are employed, which provide delayed release of agents to 5 change the reaction conditions from the first to th e second set of reaction conditions. For example, NaOH-filled microcapsules maybe employed, to stop enzymic catalysis, and allow the detection of a dephosphorylated substrate, in an acid phosphatase dephosphorylation reaction. Again, complex and expensive reagents are required in order to put the method in practice.
We have now discovered that it is possible to produce an enzyme assay which is essentially 5 self-stopping but is also simple and inexpensive to put into effect, by reacting the enzyme with the development reagent, in the presence of a chelating agent which funtions as a progressive enzyme inhibitor such that the ability of the enzyme to react .with the development reagent to produce the determinable change is progressively inhibited as the reaction progresses. Surprisingly, the enzyme inhibitor can be chosen such that its presence does not reduce the initial enzyme activity to such an extent that the assay does not have sufficient sensitivity.
The assay method in accordance with the invention has the advantage that it is considerably simpler to perform than assays requiring a separate stopping step, and is much less complex and expensive than systems requiring the presence of slow release stopping agents or immobilising polymers. The enzyme inhibitor may desirably be provided in a solution containing the development reagent or reagents, and the solution containing both may be added to the enzyme, in order to carry out the development.
Accordingly, in a further embodiment of the invention, there is provided a development reagent solution for use in an enzyme assay, comprising at least one development reagent for the enzyme, and a chelating agent which is progressive inhibitor for the enzyme. Such a solution may be incorporated into a test kit, which may incorporate other reagents, for example for carrying out an immunoassay.
Not only does the method of invention result in greater simplicity, as compared with the addition of a separate "stopping" solution, it also reduces the scope for operator error, both in the addition of reagent solutions, and because no timing operation is required. The progressive enzyme inhibitor is a chelating agent, such as ethylendiaminetetraacetic acid (EDTA). Such chelating agents can be chosen so as to be effective in binding metal ions in the enzyme used so as effectively to render the enzyme inactive. Alternative chelating agents are, for example, ethylenedioxydiethylenedinitrotetraacetic acid (EGTA) and diaminocyclohexanetetracetic acid (CDTA).
The time at which the assay is "stopped" is primarily determined by the concentration of the progressive inhibitor, in the reaction system.
Although the method of the invention is suited particularly for use in enzyme development reactions in which the detected product is measured spectrophotometrically, it is not limited to such processes and can equally well be utilised where measurement is by fluorescence, or when the product is determined electrochemically.
A preferred embodiment of the invention is described in the following examples.
Example 1 Standard solutions containing calf intestinal alkaline phosphatase having concentrations in the range 0-100 ng/ml were assayed by the following procedure.
A solution was prepared of phenolphthalein monophosphate (di AMPD salt - 15mM), sodium carbonate buffer - pH 10.0 (100- mM), EDTA (disodium salt - 25 mM), and 0.02% sodium azide as a preservative. 10 microlitre portions of the various phosphatase standard solutions were added to respective wells of a 96 well microtitre plate (NUNC). 200 microlitre portions of the inhibited phenolphthalein solution prepared above was then added to each well. The plate was incubated at 20βC, and colour development of each well was monitored at 1 minute intervals over a 1 hour period. After 60 minutes, the rate of colour development had slowed to less than a 1% change in optical density per minute. Figure 1 illustrates the relationship between the alkaline phosphatase concentration in the starting standard solution, and the final optical density, after 60 minutes. It can be seen that Figure 1 shows excellent linearity (showing that enzyme inhibition is independent on enzyme concentration), and good sensitivity.
Example 2 Self-Stopping enzyme immunoassay for follicle stimulating hormone An immunoassay for follicle stimulating hormone (FSH) was carried out as follows: A microtitre plate as used in Example 1 was coated with an anti-FSH antibody, using a conventional method. Separately, a conjugate was prepared of a second anti-FSH antibody with alkaline phosphatase, again using a conventional method.
75 microlitre portions of a solution containing the anti-FSH conjugate were added to each of the wells of the microtitre plate, followed by 25 microlitre portions of solutions of FSH standards, such that final concentrations of FSH were 0, 10, 25, 50 and 100 IU/L FSH.
The microtitre plate was incubated for two hours and the wells washed three times with a washing buffer. 100 microlitre portions of an inhibited development reagent solution as utilised in Example 1 was then added to each of the wells. The colour development at 550 nanometres was monitored for 60 minutes, by measurement of the optical density at 1 minute intervals. Figure 3 shows the calibration curve of optical density after 60 minutes, for the varying FSH concentrations. Figure 3 again illustrated excellent linearity, and adequate sensitivity. The working range of the assay was from
05 0 to 100 IU/L of FSH hormone.
Examϋle 3
An inhibited development reagent solution was prepared, containing ortho-nitrophenyl beta-galactopyranoside (20 mM), EDTA (40 mM), and 0 diethanolamine buffer pH 8.0 (500 mM).
10 microlitre portions of standard solutions containing 170, 150, 100, 70, 50 and 0 ng of beta-Galactosidase were added to respective wells of a microtitre plate as used in Example 1. 200 microlitre 5 portions of the inhibited development reagent solution referred to above were then added to each well. Colour development at 405 nm was monitored for 60 minutes, by determination of the optical density in each well at 1 minute intervals. Figure 5 illustrates ^ the relationship between the optical density after 60 minutes, and the concentration of beta-Galactosidase in the standard solution. After 60 minutes, the rate of colour development had again slowed to less than a 1% change in optical density per minute. Figure 5
25 illustrates excellent linearity between optical density and concentration, showing that the enzyme inhibition is independent of enzyme concentration. The working range of the assay was (0-20 ng) beta-Galactosidase.
3 it will of course, be appreciated that a wide range of other enzyme and developer combinations are possible, in addition to those specifically described herein.
In particular, the method of the invention may be
" used to determine an enzyme which is not the primary product which it is desired to determine but is an intermediate product, or is indeed present in the solution under investigation in a known amount, its activity being under the control of a modulator, which 5 is produced by a preliminary biochemical reaction. By this method, the method in accordance with the invention may be used to assay a reagent which takes part in the "preliminary" chemical • reaction. Thus, the method in accordance with the invention may be utilised 0 to determine a wide variety of biochemical substances.
Test kits for carrying out enzyme assays may be produced in various formats, comprising the development reagent solution in accordance with the invention. For example, a test kit may contain an antibody-coated 5 microplate, and separate containers for (i) analyte standards, (ii) enzyme-labelled conjugate, (iii) the dry components of a washing buffer, and (iv) colour development reagents including the enzyme inhibitor. Alternatively, the reagent solution may be used in 0 conjunction with an automated analyser which will generally use the same reagents as reagents (i) to (iv) noted above, but the microtitre plate is generally replaced by small polymer particles, having antibody absorbed or covalently bound to its surface. The
J particles may or may not have magnetic properties.
In an alternative format, the various reagents of a test kit may be dried into an absorbent material, such as paper or the like, in a number of layers, to which a test solution is added. In such a system, the
0 development reagent and the inhibitor may be present either within the same layer, or within different layers, such that they come together when the test solution is added.
Obviously, a wide range of alternative test kit formats are also possible, as is well known in the art .
In addition, the determinable change which is measured may be, for example, an electrochemical change, or a change in fluorescence, rather than a spectrophotometric one.

Claims

1. A method of carrying out an assay for an enzyme, which method comprises reacting the enzyme with a development reagent to produce a determinable. change characterised in that the reaction is carried out in the presence of a chelating agent which is a progressive enzyme inhibitor, under conditions such that the ability of the enzyme to react with the development reagent to produce the determinable change is progressively inhibited as the reaction progresses.
2. A method as claimed in Claim 1, wherein the chelating agent is ethylendiaminetetraacetic acid.
3. A method as claimed in Claim 1, wherein the amount of enzyme inhibitor present is such as to inhibit the enzyme substantially totally, after a period of approximately 30 minutes.
4. A method as claimed in claim 1, wherein a solution containing the inhibitor and the development reagent is added to the enzyme.
5. method as claimed in claim 1, wherein the enzyme is alkaline phosphatase or beta-galactosidase.
6. A method as claimed in claim 1, wherein the enzyme assay is an immunoassay, in which the enzyme is used as a label.
7. A development reagent solution for use in an enzyme assay, which solution comprises at least one development reagent for the enzyme, characterised in that the solution also comprises a chelating agent which is a progressive inhibitor for the enzyme.
8. A reagent solution as claimed in Claim 7, wherein the chelating agent is ethylendiaminetetraacetic acid.
9. A reagent solution as claimed in Claim 7 wherein the solution includes a colour development reagent.
PCT/GB1989/000900 1988-08-05 1989-08-07 Enzyme assays WO1990001557A1 (en)

Applications Claiming Priority (2)

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GB8818658.0 1988-08-05
GB888818658A GB8818658D0 (en) 1988-08-05 1988-08-05 Enzyme assays

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3799843A (en) * 1972-07-10 1974-03-26 Dow Chemical Co Determination of acid phosphatase
EP0207493A2 (en) * 1985-07-02 1987-01-07 Oriental Yeast Co., Ltd. Method of terminating isocitrate dehydrogenase reaction

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3799843A (en) * 1972-07-10 1974-03-26 Dow Chemical Co Determination of acid phosphatase
EP0207493A2 (en) * 1985-07-02 1987-01-07 Oriental Yeast Co., Ltd. Method of terminating isocitrate dehydrogenase reaction

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Biological Abstracts, vol. 76, no. 7, 1983, (Philadelphia, PA, US) J. Mallol et al.: "Modification of 5'-nucleotidase (EC 3.1.3.5) activity by divalent cations and nucleotides", see page 5574 *

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GB8818658D0 (en) 1988-09-07

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