EP0398920A1 - Analyses biochimiques a marquage enzymatique visant a determiner la presence de deux analytes - Google Patents

Analyses biochimiques a marquage enzymatique visant a determiner la presence de deux analytes

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Publication number
EP0398920A1
EP0398920A1 EP19890901723 EP89901723A EP0398920A1 EP 0398920 A1 EP0398920 A1 EP 0398920A1 EP 19890901723 EP19890901723 EP 19890901723 EP 89901723 A EP89901723 A EP 89901723A EP 0398920 A1 EP0398920 A1 EP 0398920A1
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EP
European Patent Office
Prior art keywords
enzyme
reaction
amount
development
beta
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19890901723
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German (de)
English (en)
Inventor
David Leslie Bates
Warwick Roy Bailey
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oxoid Ely Ltd
Original Assignee
Novo Biolabs Ltd
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Filing date
Publication date
Application filed by Novo Biolabs Ltd filed Critical Novo Biolabs Ltd
Publication of EP0398920A1 publication Critical patent/EP0398920A1/fr
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • This invention relates to biochemical assays, and has particular application in the field of immunoassays, although it also has application in other biochemical assays, for example DNA probes and the like.
  • Biochemical assays are available to test for a wide range of biological substances, e.g. antigens, hormones, cancer markers etc.
  • substantially all commercial assays of this kind have been capable of estimating only a single analyte. It is a very attractive proposition to be able to determine simultaneously more than a single analyte.
  • Radiochemical assays exist for measurement of two analytes, in which the two analytes are distinguished by employing as labels two different radioisotopes which have different characteristic emissions.
  • Radioimmunoassays are however difficult to carry out and require complex detection apparatus, and detailed safety precautions.
  • beta-galactosidase and alkaline phosphatase are incompatible as regards the pH required for the enzyme development reaction, alkaline phosphatase requiring a much higher pH (9 to 11.5) than does beta-galactosidase (typically around 7.1).
  • the assays disclosed are so-called “stopped” assays, in which the enzyme development is carried out for a predetermined period of time, and the development of colour after that period of time is compared with a standard. It is a consequence of the method of Blake et al, because development reactions are not carried out simultaneously, that at least one of the development reactions must be a "stopped” reaction. Stopped assays are inherently inferior to so-called “kinetic” assays, in which the rate of signal development is observed over a period of time. In particular, kinetic determinations are in general much more accurate, have a substantially greater dynamic range and are less prone to certain types of measurement errors than stopped assays.
  • Blake et al realise the desirability of being able to carry out assays in which the observed development reaction for the two products (i.e. the colour development reactions) are simultaneous, but because of the incompatibility of the two enzymes which they employ, acknowledge that this was not possible in practice.
  • the problem encountered by Blake et al is not unique to the combination of enzymes beta-galactosidase and alkaline phosphatase. In any combination of enzyme markers, it is likely that the conditions required for development of one will effectively prohibit the development of the second, for example by reason of preferred pH, temperature, ionic strength, the requirement for certain substances which would inhibit the reaction of the other enzyme or the requirement by one enzyme reaction of effectors (for example metal ions) which can in other circumstances act as enzyme inhibitors.
  • EP-A-0238353 indicates that it is possible to obtain simultaneous colour developments using alkaline phosphatase and beta-galactosidase labels, by carrying out the development reactions in the presence of high concentrations (e.g. 0.25 to 1M) of diethanolamine (DEA). This is said to lower the pH optimum of alkaline phosphatase activity from 9.5 to around 8.6, at which pH the beta-galactosidase is said still to have substantial activity.
  • DEA diethanolamine
  • a development reaction for a beta-galactosidase enzyme label in a biochemical assay can be carried out at substantially higher pH that hitherto, increasing the compatibility of the enzyme reaction with alkaline phosphatase, by carrying out the develpment reaction in the presence of ethanediol.
  • a method of carrying out a biochemi ⁇ al assay for first and second analytes in a sample comprises:- carrying out an assay so as to localise a first enzyme which is alkaline phosphatase in an amount which is dependent upon the amount of the first analyte present in the sample, and a second enzyme which is beta-gala ⁇ tosidase in an amount whi ⁇ h is dependent upon the amount of the second analyte present in the sample, causing the alkaline phosphatase and beta-galactosidase to take part in respective development reactions to produce, for each development reaction, a determinable change, and observing the determinable change for each of the two development reactions, to determine the presence in the sample of the two analytes, characterised in that at least the development reaction of the beta-galactosidase is carried out in the presence of ethanediol in an amount sufficient to
  • a method of carrying out a biochemical assay for two analytes in a sample comprises:- carrying out an assay so as to localise an amount of a first enzyme dependent upon the amount of the first analyte present in the sample, and an amount of a second enzyme dependent upon the amount of the second analyte present in the sample, causing the first enzyme to take part in a chemical reaction to produce an intermediate substance, in the substantial absence of reaction of the second enzyme, such that the amount of the intermediate substance produced is dependent upon the amount of the first enzyme in the sample, thereafter causing the second enzyme and the intermediate substance to take part simultaneously in development reactions to produce, for each reaction, a determinable change, and observing the determinable change for each of the two development reactions, to determine the presence in the sample of the two analytes, wherein at least one of the said determinations is carried out kinetically.
  • the determination of both of the development reactions is carried out kinetically, since by this method substantially increased dynamic range and accuracy is achieved for the determination of both of the analytes.
  • the amount of ethanediol employed is preferably such as to raise the pH optimum of the beta-galactosidase development reaction by at least 0.5, and is typically such as to shift the pH optimum by about 0.8 for example from 7.1 to 7.9.
  • the pH optimum of the overall colour development reaction may be substantially different from that of the enzyme reaction alone.
  • the feature of interest is the pH at which the overall development reaction of beta-galactosidase takes place, to produce something which is measurable (typically a colour development reaction, although in certain embodiments, a product capable of electrochemical determination).
  • the determinable change produced by each of the development reactions may in one embodiment be a colour change, in which the two development reactions produce substances which give rise to light absorption at differing wavelengths.
  • the determinable change may be an electrical change, for example a current change in which either or both of the enzymes produce a substance which may be determined amperometrically, for example as disclosed in PCT/W086/03837.
  • diaphorase may be employed as a component of an enzyme system as disclosed in WO86/03837, and utilised to reduce ferricyanide to ferrocyanide which can be determined amperometrically.
  • phenyl phosphate may be employed as a substrate for alkaline phosphatase, (Wehemeyer, Clin Chem, 31/9, 1546-1549 [1985]).
  • Electrochemical determination of beta-galactosidase may be carried out, for example, by utilising the galactosidase to cleave an electrochemically inactive substance such as phenyl beta-galactoside or a derivative thereof and produce an electrochemically active one.
  • the development reaction of the second enzyme may be carried out under conditions which inhibit normal development reactions for the first enzyme.
  • the reaction of the first enzyme to produce the intermediate substance may be carried out at different conditions of temperature, pH, ionic strength, or in the presence or absence of substances, which inhibit the development reaction of the second enzyme.
  • the method of the invention is particularly suited to application in the field of immunoassays, and in particular to immunoassays in which the two enzyme labels are bound to a surface, in amounts dependent upon the amounts of, respectively, the two analytes present in the sample.
  • immunoassay techniques are very well known, and. will not be described in detail. Conventional sandwich and/or competition methods may be utilised.
  • Typical pairs of analytes which may be determined by the method of the present invention are the following:- T4 & TSH T3 & T4 FSH & LH hCG & prolactin Hepatitis & AIDS (antigens) Chlamydia & Herpes B12 & Folate CEA & AFP PSA & PAP beta-microglobulin & ferritin oestrad iol & progesterone phenytoin & phenobarbital insulin & glucagon insulin & proinsulin C-peptide CMV & Rubella viable and non-viable pathogens (eg moulds and fungi) pathogens and their toxins (eg Botulinus).
  • pathogens eg moulds and fungi
  • native and glycated proteins e.g. native and glycated haemoglobin, albumin or IgG.
  • the present method is particularly suitable for such pairs, because the result very often needs to be expressed only as % glycation. The method can thus be independent of sample volume.
  • the intermediate substance produced by the first enzyme may, in one embodiment of the invention, be a substance which is quantitively converted in a development reaction, in order to produce a determinable change.
  • the intermediate substance produced in the reaction of the first enzyme is a trigger substance capable of taking part in, or of producing a substance capable of taking part in, a cyclic chemical reaction. thereby "amplifying" the signal produced by the first enzyme, for example as disclosed in European Patent Specification Nos. 19606, 37036, 58635 and 60123.
  • the intermediate substance is nicotinamide adenine dinucleotide (NAD), or a derivative thereof (for example dihydro nicotinamide adenine dinucleotide (NADH)).
  • the first enzyme is alkaline phosphatase
  • the second enzyme is beta-galactosidase
  • the intermediate substance is NAD.
  • the method in accordance with the second aspect of the invention is of utility when the development reactions for the two enzymes used as labels are incompatible in a number of ways, for example when they are incompatible in pH, temperature, ionic strength, or in substrates and co-factors required.
  • the substrate for the first reaction system reacts with the substrate for the second
  • the substrate for the first reaction system competes with the substrate for the second
  • the substrate for the first reaction system inhibits the second enzyme.
  • a prime example of this kind of incompatability is peroxidase, which uses hydrogen peroxide as a substrate, which will react with other enzymes or substrates.
  • Example 1 Mixtures of the two enzymes alkaline phosphatase and beta-galactosidase are prepared as follows.
  • a 5 ⁇ 5 matrix is prepared of solutions containing four concentrations (1.6 pg, 3.2 pg, 4.8 pg, and 6.8 pg, each per 10 microlitres) of alkaline phosphatase and a blank (zero alkaline phosphatase), and four concentrations (10 ng, 20 ng , 30 ng, and 40 ng, each per 10 microlitres) of beta-galactosidase, and a blank.
  • the solutions of the matrix (except those derived from one or both of the blanks) thus contain both enzymes in varying concentrations. Each sample has a total volume of 10 microl itres .
  • the mixtures are pipetted in triplicate into the wells of a 96 well NUNC microplate.
  • Replicate controls are also provided on the plate, containing no enzyme, alkaline phosphatase alone (6.4 pg in 10 microlitres) and beta-galactosidase alone (40 ng in 10 microlites).
  • a first reagent solution is prepared containing the following components: nicotinamide adenine 0.1mM dinucleotide (NADP) diethanolamine buffer 50mM (pH 9.5) magnesium chloride 1mM ethanol 4% sodium azide 0.02%
  • a second development reagent solution is prepared containing the following components. diaphorase/ alcohol dehydrogenase(ADH) 1.5 u/ml (1) sodium phosphate 30mM (pH 7.2) ethanediol 8% iodonitrotetrazolium-violet 1mM (INT-violet) o-nitrophenylgalactoside 1mg/ml sodium azide 0.02%.
  • Reagent 1 100ml is pipetted into each well of the mi ⁇ roplate, and the plate is incubated at 20oC for ten minutes.
  • the second development reagent is then added to each of the wells of the microplate 200ml, and the plate maintained at 20o C, whilst absorbance is measured spectrophotometrically, for ten minutes at two wavelengths (414 and 492 nm). The linear rate of increase is determined for each well at both wavelengths. The results are shown in Table 1. Data Analysis Because of the spectral overlap of the two dyes ( o-nitrophenol and INT-forma zan ) produced in this system , both enzymes contribute to the absorbance measured at each of the wavelengths indicated . It is however possible to resolve the two contributions as follows:-
  • V 1 V 1a + V 1b
  • V 2 V 2a + V 2b
  • V 1 and V 2 are the measured absorbance increase rates at wavelengths 1 and 2 respectively
  • V 1a , V 1b , V 2a , and V 2b represent respectively the contributions of the two enzymes a and b to the absorbance at those two wavelengths.
  • the signal from enzyme a is mostly at wavelength 1, and from enzyme b most at wavelength 2.
  • the overlap of each enzyme at the second wavelength can be defined as a fraction of the signal at the other.
  • K a and K b can be determined from control wells containing only one enzyme.
  • the measured velocities can therefore be rewritten as :
  • V 1 V 1a + K b ⁇ V 2b
  • V 2 K a ⁇ V 1a + V 2b
  • Figures 1 and 2 are respectively a graphical representation of the raw data and resolved rates for alkaline phosphatase, and Figures 3 and 4 show similar representations for beta-galactosidase.
  • the data sets for each enzyme produce good linear calibration curves (r greater than 0.99995).
  • the enzymes alkaline phosphatase and beta-galactosidase are each determined in the presence of the other by a kinetic colour development monitored simultaneously at different characteristic wavelengths.
  • Example 2 Eight sample solutions are prepared having varying pH's in the range 6.5 to 9.5, and each containing 10 mM phosphate, 5 mM aminomethylpropanediol buffer, and 1mM of o-nitrophenylgalactoside (o-NPG) substrate. Two samples are prepared at each pH, one of which contains, and the other of which does not contain 6% ethanediol.
  • o-NPG o-nitrophenylgalactoside
  • Glycated haemoglobin is measured by a variety of analyical techniques (HPLC, electrophoresis etc) as an index of mean blood glucose concentration and control in diabetics over the 4-8 weeks preceeding the sample measurement. Values are usually expressed as the % HbAlc of total haemoglobin.
  • the immunoassay described below uses the method described in Examples 1 and 2 to determine both haemoglobin and glycated haemoglobin HbAlc.
  • IgG subclass antibodies to haemoglobin and HbAlc are raised in mice and conjugated to alkaline phosphtase and beta-glactosidase enzymes respectively using the method of Ishikawa et al (Journal of
  • the conjugates are purified by HPLC and then both diluted to working concentration into a single buffer solution containing: imidazole buffer 100mM, pH 7.5 ammonium sulphate 100mM magnesium chloride 1mM zinc chloride 0.1mM bovine serum albumin 2.5% sodium azide 0.02% This is referred to below as the "Dual Conjugate Solution”.
  • Each individual conjugate is also diluted to working concentration in the absence of the other as controls.
  • HbAlc Three calibrator haemolysates of HbAlc (3.9%, 7.4%, 10.5% HbAlc) are prepared by reconstituting freeze dried haemolysate and diluting 50 microlitre of this solution into 1ml of 3.3mM potassium ferricyanide solution and standing at room temperature for 10 minutes. 500 microlitre of diluted haemolysate is then added to 5.5ml of a buffer solution of 100mM sodium citrate pH 4.5. 100 microlitre of this haemolysate/citrate solution is then added to the wells of a microtitre plate and incubated at 25oC for 15 minutes.
  • haemoglobin and HbAlc is passively adsorbed to the plastic surface of the microtitre well.
  • the microtitre wells are washed 3 times with 250 microlitre of a simple washing buffer and then 100 microlitre of the dual conjugate solution is added to the wells.
  • Individual conjugate controls are also added to some wells without the addition of dual conjugate solution. These control wells allow calculation of spectral overlap factors as required by the data analysis in Example 1 .
  • Conj ugates are incubated in the wells for 2 hours at 25 o C and then washed 3 times with 250 microl itre of a simple washing buffer .
  • phosphate buffer as phosphatase inhibitor
  • o-nitrophenylgalactoside as galactosidase substrate
  • INT-violet as d iaphorase substrate : sodium dihydrogen phosphate 20mM pH 7.4 magnesium chloride 1mM ethanediol 12% o-NPG substrate 5mM INT violet 1mM sodium azide 0.02% diaphorase/ADH 1.5u/ml as defined by
  • Example 4 Dual Enzyme Assay using Electrochemical Detection An analogous system to that described in Example 3 is employed, utilising electrochemical rather than spectropKometric detection.
  • Electrochemical detection of alkaline phosophatase is carried out via an NAD( H) intermed iate as described by Cardosi et al (Journal of Immunological Methods , 112 ( 1988 ) pp 153-161) and detection of beta-galactosidase is carried out using the general method of Wehemeyer et al ( Clinical Chemistry 31/9 ( 1985) pp 1546-1549 ) .
  • the spectrophometric diaphorase substrate , INT of Example 1 is replaced with potassium ferricyanide such that the reduced product , ferrocyanide , may be detected electrochemically.
  • the spectrophotometric galactosidase substrate is replaced with a beta-galactoside (for example , amino-phenyl galactoside or naphthyl galactoside) , which is electrochemically inactive at low potentials but on hydrolysis releases an electrochemically active product (e .g . amino-phenol or naphthol) which shows irreversible electrochemistry at low potentials .
  • the assay may be a sandwich assay or a competition assay, in which the two enzymes are conjugated with different monoclonal antibodies, each of which binds specifically with different antigens which it is desired to determine simultaneously, for. example T4 and TSH, T3 and T4, or one of the other antigen pairs listed above.
  • the assay may be carried out by any of the many known procedures.
  • the assay is carried out so as to bind to a solid support, such as the wall of a microplate, amounts of two antigens to be determined, dependent upon the amounts of the antigens present in the sample under test.
  • Kinetic determination of the two enzymes is then carried out utilising the method just described.

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Abstract

Un procédé de mise en oeuvre d'une analyse biochimique en vue de déterminer la présence d'un premier et d'un second analytes dans un échantillon, consiste à procéder à l'analyse à l'aide de phosphatase alcaline à titre de première marque d'enzyme et de bêta-galactosidase à titre de seconde marque d'enzyme. On procéde à la réaction de développement d'au moins le bêta-galactosidase en présence d'éthanediol dans une quantité suffisante pour élever le pH auquel la réaction de développement de bêta-galactosidase se produit, à environ 7,9, ce qui le rend compatible avec une réaction de développement de phosphatase alcaline, utilisant un cycle NAD/NADH. La détection peut être électrochimique ou spectrophotométrique.
EP19890901723 1988-01-20 1989-01-19 Analyses biochimiques a marquage enzymatique visant a determiner la presence de deux analytes Withdrawn EP0398920A1 (fr)

Applications Claiming Priority (2)

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GB8801199 1988-01-20
GB888801199A GB8801199D0 (en) 1988-01-20 1988-01-20 Dual enzyme assay

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EP0398920A1 true EP0398920A1 (fr) 1990-11-28

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EP (1) EP0398920A1 (fr)
AU (1) AU2948789A (fr)
DK (1) DK173690A (fr)
GB (1) GB8801199D0 (fr)
WO (1) WO1989006802A1 (fr)

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JPH09257794A (ja) * 1996-03-26 1997-10-03 Kikkoman Corp 多重測定法
US6294281B1 (en) 1998-06-17 2001-09-25 Therasense, Inc. Biological fuel cell and method
US7005273B2 (en) 2001-05-16 2006-02-28 Therasense, Inc. Method for the determination of glycated hemoglobin
US7368190B2 (en) 2002-05-02 2008-05-06 Abbott Diabetes Care Inc. Miniature biological fuel cell that is operational under physiological conditions, and associated devices and methods
US20100213057A1 (en) 2009-02-26 2010-08-26 Benjamin Feldman Self-Powered Analyte Sensor

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US4690890A (en) * 1984-04-04 1987-09-01 Cetus Corporation Process for simultaneously detecting multiple antigens using dual sandwich immunometric assay
GB8607101D0 (en) * 1986-03-21 1986-04-30 Serono Diagnostics Ltd Immunoassay
GB8614084D0 (en) * 1986-06-10 1986-07-16 Serono Diagnostics Ltd Immunoassay

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DK173690D0 (da) 1990-07-20
GB8801199D0 (en) 1988-02-17
AU2948789A (en) 1989-08-11
DK173690A (da) 1990-07-20
WO1989006802A1 (fr) 1989-07-27

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