EP0223816A1 - Enzyme assay of body fluids - Google Patents
Enzyme assay of body fluidsInfo
- Publication number
- EP0223816A1 EP0223816A1 EP19860903511 EP86903511A EP0223816A1 EP 0223816 A1 EP0223816 A1 EP 0223816A1 EP 19860903511 EP19860903511 EP 19860903511 EP 86903511 A EP86903511 A EP 86903511A EP 0223816 A1 EP0223816 A1 EP 0223816A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- enzyme
- assay
- hrp
- analyte
- sample
- 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- Withdrawn
Links
Classifications
-
- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
Definitions
- Assays for various analytes in body fluids using reagents labelled with radioactive isotopes are well known and widely available commercially. Examples of such assays are radioim uno assays which employ a radioactively labelled version of the analyte; and radioimmunometric assays which employ a radioactively labelled antibody to the analyte. These assays are
- Certain substances in certain patient sera bind to the ⁇ -galactosidase part of the conjugate thereby inhibiting the binding of the macromolecular substrate to the enzyme independently of the binding of antibody.
- the ?-galactosidase part of the conjugate has been coated with albumin and an excess of inactivated fl-galactosidase is added. It is not suggested that the addition of inactivated ⁇ -galactosidase would be useful by itself.
- a homogeneous assay as described depends on inactivation of the label under some circumstances, and it may be difficult to avoid unintentional inactivation when this is not desired.
- the invention provides a method of performing an assay on a sample of a body fluid including a step of incubating a mixture of the sample with a reagent labelled with an enzyme, wherein the sample may contain an inhibitor for the enzyme, which method comprises including in the incubation mixture a correction factor to block the action of any inhibitor present in the sample.
- the assay is one which involves an immune reaction between an analyte and its specific binding partner, in which immune reaction the reagent labelled with enzyme also participates.
- the analyte may be an antigen or hapten and the specific binding partner its associated antibody; or the analyte may be an antibody, with an antigen as its specific binding partner.
- the inhibitor is a substance, usually macromolecular, which binds to the enzyme in such a way as to inhibit participation of the labelled reagent in the immune reaction.
- the inhibitor is an enzyme poison, naturally occurring or as an added preservative, which binds to the enzyme in such a way as to reduce or destroy its enzymic activity.
- the nature of the assay is not critical. It may for example be an uptake assay, or a competition or im unometric assay for total or for free analyte.
- the analyte may be a hormone, an enzyme, a biochemical messenger, a steroid, a drug, a drug metabolite, a polypeptide or protein, a catechol-amine, a vitamin, a tumour antigen, a toxin, an alkaloid, a mono-, di, or poly-saccharide , or a virus or virus particle.
- the analyte may itself be antigenic, or may be a small molecule such as a hapten (which can initiate the production of antibodies only when joined to a larger molecule); or may be an antibody.
- the sample for assay may be taken from any body fluid, such as for example plasma, urine, or serum.
- sample correction factor or SCF for short, which term is occasionally used herein ⁇ after.
- the correction factor or SCF is a material which blocks the action of any inhibitor for the peroxidase enzyme which may be present in the assay sample.
- the correction factor must be .inert to the assay reagents and must not otherwise affect the assay.
- the correction factor should preferably not itself have enzymatic activity in order to maintain assay precision.
- One suitable material is the apo- protein left after gentle removal of the haem group from HRP.
- Two other materials are suitable for use as sample correction factors when an HRP label is used to generate a chemi luminescent signal which is amplified by the known techniques of enhanced luminescence.
- the first is an acidic form of HRP, which is not susceptible to luminescence enhancement and can hence be regarded as enzy ically inactive for the purposes of the assay.
- Sigma Chemical Company Limited sell peroxidase isoenzymes including two acidic isoenzymes (Types VII and VIII), one basic isoenzyme (Type IX), of which only the basic isoenzyme is susceptible to luminescence enhancement. (The terminology is in accordance with the classification of Shannon L.M. et al., J. Biol. Chem., 241, 2166 (1966)).
- the second SCF material for this purpose is a carboxymethylated HRP prepared by the method of Gurd F.R.N., Methods of Enzymology, Vol. 11, 532. Both these materials are suitable for use as sample correction factors: because they are inert to the assay reagents, e.g. analyte, conjugate and antibody; and because they are not susceptible to luminescence enhancement and can hence be regarded as enzymatical ly inactive for the purposes of the assay.
- Aberrant samples may also contain enzyme poisons, which reduce or destroy enzyme activity under all circumstances. These poisons are distinguished from the macromolecular substances which can bind to enzymes and physically block their reaction with macromolecular substrates. Enzyme poisons may be naturally occurring, or deliberately added.
- Serum controls for assay kits often contain sodium azide as a preservative. Sodium azide binds to the haem group of HRP. So serum controls often constitute aberrant samples according to this invention. And apo-HRP enzyme is not satisfactory as a SCF because it lacks the haem group to which sodium azide binds.
- acidic HRP isoenzyme and carboxymethylated HRP are suitable SCF's.
- the amount of correction factor needed to block the action of the inhibitor depends on several factors, such that no overall concentration range can be given. However, the amount of correction factor required to bring an aberrant result obtained from a particular sample back to normality is easily determined by routine experiment. Factors affecting the required amount of correction factor include the following. The quantity and nature of the analyte; more analyte requires more labelled reagent, which in turn requires more correction factor. The volume of the sample; more sample may require more correction factor. The amount of the HRP labelled reagent; more labelled reagent may require more correction factor. The purity of the HRP labelled reagent, and in particular the amount of free HRP present as impurity; use of a pure labelled reagent may require use of more correction factor. Indeed, if enough free HRP is present, there may be no need for any correction factor; but then the precision would be greatly impaired.
- the invention is applicable to various different kinds of assay, of which the following are examples :-
- A. Competition assay for total analyte The sample is incubated with an amount of an analyte-HRP conjugate and an .antibody to the analyte which is insolubi lised either before, during or after the incubation.
- the analyte and analyte-HRP conjugate compete for binding with the antibody.
- the amount of enzyme attached to antibody varies inversely with the amount of analyte in the sample.
- T3 tri-iodothyronine
- TB6 thyroxine binding globulin
- TBPA thyroxin binding pre-albumin
- ALB albumin
- a blocking agent useful with thyroid hormones is 8-ani 1 ino-naphthalene sulphonic acid (ANS).
- ANS 8-ani 1 ino-naphthalene sulphonic acid
- the blocking agent acts on the serum binding proteins and provides all the analyte in the serum in free form for assay; the correction factor is believed to act on some supposed inhibitor and ensures that all the HRP enzyme is available to participate in the assay.
- a one-site im unometric assay for total analyte The sample is incubated with an amount of added immobilised analyte and a limited amount of an HRP conjugate of an antibody to the analyte.
- a blocking agent may also be present if required to ensure that the analyte is all in free form.
- the analyte in the sample and the immobilised analyte compete for reaction with the labelled antibody.
- the amount of insoluble HRP label varies inversely with the amount of analyte in the sample.
- a competition assay for free analyte The procedure here may be as in A or B, with two important differences, both designed to avoid disturbing the free-bound equilibrium of analyte in the sample. The first difference is that the blocking agent must be absent. The second is that the amount of antibody used is insufficient to substantially disturb the free- bound analyte equilibrium; this may require the use of a rather small amount of rather high affinity antibody.
- D. A simultaneous 2-site immunometric assay for total analyte The sample is incubated with an amount of an immobilised antibody to the analyte and an amount of an HRP conjugate of an antibody to the analyte. A blocking agent may also be present if required. This method is only applicable to analytes which have at least two sites which are recognised by antibodies. The amount of immobilised label varies directly with the amount of analyte in the sample.
- E. An analyte uptake assay This is a test to determine the number of available unused analyte binding sites on the natural binding proteins in the sample. For example, T3 and T4 uptake assays are commonly used as adjuncts to total T3 and total T4 assays.
- the sample is incubated with an amount of the unlabelled analyte, an amount of an HRP conjugate of the analyte, and an amount of antibody, which is immobilised either before or after the incubation.
- the amount of insolubi 1 ised HRP label varies directly with the number of vacant binding sites.
- Other assay procedures in which a sample is incubated together with an enzyme-labelled molecule will readily occur to those skil led in the art.
- the inhibitor is usually macromolecular.
- Assays of classes A, C and E above our work suggests that it binds with HRP in a manner which mainly prevents subsequent binding of the analyte/HRP conjugate to antibody; and.that inhibition of the enzymic activity of HRP occurs, if at all, only to a minor degree.
- the inhibitor probably binds with HRP in a manner which mainly prevents subsequent binding of the antibody/HRP conjugate to analyte. Where an enzyme poison such as sodium azide is present, this probably binds to HRP in such a way as to reduce or destroy its enzymic activity.
- this invention can be applied to homogeneous assays, it is better suited to heterogeneous assays, in which bound label is physically separated from not-bound label prior to observation of a signal generated by either the bound or the not-bound label. Separation may readily be achieved by the use of one assay reagent bound to solid particles or to the vial wall, in accordance with well- known techniques.
- the enzyme can be made to generate an observable signal in various ways.
- HRP can be caused to catalyze the the oxidation of luminol with the production of light via chemi luminescence.
- HRP enzyme can be used to generate colour by addition of o-phenylenediamine.
- the nature of the system used to generate an observable signal is not critical to the present invention, and any known and appropriate system can be used.
- Figure 1 is a graph comparing the concentration of T3 in serum samples by two different techniques.
- the X axis shows the concentration, expressed in nanomoles/litre, determined by a commercially available radioimmuno assay.
- the Y axis shows the concentration on a similar basis, determined by a conventional enzyme immunoassay (using enhanced luminescence) without the use of a correction factor.
- Figure 2 represents a graph which is the same as Figure 1 except that the Y axis gives results obtained by an enzyme immunoassay (using enhanced luminescence) including a correction factor according to this invention.
- the X axis represents total T3 concentrations, in nanomoles per litre of serum, determined using the commercially available Amerlex RIA kit, which uses latex beads coated with antibody.
- the Y axis represents total T3 concentrations of the same sera determined using a conventional enzyme immunoassay (enhanced luminescence) test, the protocol of which is set out below.
- the letter H refers to a hypothyroid sample.
- the letter E to a Euthyroid sample, and the letter P to a sample obtained in pregnancy, while the letter T refers to hyperthyroid samples.
- most of the results are quite accurately on a straight line passing through the origin, as is to be expected if the two assay techniques are comparable. But there are four aberrant results indicated by stars. In these, the indicated T3 concentration is too high, which means that the activity of immobilised HRP enzyme was too low. This is consistent with part of the enzyme having been inhibited by some unknown inhibitor in the sample.
- Immobilised HRP enzyme was determined by enhanced luminescence involving a luminol/perborate system.
- the T3 coupled to HRP was replaced with a T3 solution labelled with 125-1. After washing, bound activity in each well was measured for ten minutes in a gamma counter.
- Columns 6 and 7 represent assays performed in accordance with the present invention. Column 6 should be compared to column 5, and also column 1, to show the effect of adding a correction factor to a commercial colorimetric HRP enzyme immunoassay. Column 7 should be compared to column 2, and also to column 1, to show the effect of adding a correction factor to a conventional enhanced luminescent HRP enzyme immunoassay.
- Tests were carried out to determine how much correction factor needed to be added to cope with aberrant samples in a total T3 HRP enzyme immunoassay.
- the assay protocols were as described in Example 1 under 1 and 7. Samples of two sera, one normal and one abnormal, were tested. The results are set out in table 2.
- the 125 I-label was not expected to make any difference to the luminescent results obtained.
- Serum controls for assay kits often contain sodium azide as a preservative. Sodium azide binds to the haem group of HRP. So serum controls often constitute aberrant samples requiring the use of a SCF according to this invention.
- Total T o enhanced luminescence assay as 2 except that carboxymethylated HRP was added at a concentration of 100ng/assay.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Dans une analyse qui fait appel à l'incubation d'un échantillon d'un fluide somatique avec un réactif marqué avec un enzyme, les résultats peuvent être affectés par la présence dans l'échantillon d'un inhibiteur de l'enzyme. Ce problème est résolu en incluant dans le mélange d'incubation un facteur de correction pour arrêter l'action de tout inhibiteur présent dans l'échantillon. Si l'enzyme est une péroxydase de raifort (HRP), le facteur de correction peut être apo-HRP, un isoenzyme HRP acide ou un enzyme HRP carboxyméthylé. La présente invention est particulièrement bien adaptée à des immunoanalyses à luminescence accrue pour haptènes et antigènes.In an assay that involves incubating a sample of a somatic fluid with an enzyme labeled reagent, the results may be affected by the presence in the sample of an enzyme inhibitor. This problem is resolved by including in the incubation mixture a correction factor to stop the action of any inhibitor present in the sample. If the enzyme is horseradish peroxidase (HRP), the correction factor can be apo-HRP, an acidic HRP isoenzyme, or a carboxymethylated HRP enzyme. The present invention is particularly well suited to immunoassays with increased luminescence for haptens and antigens.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB8514288 | 1985-06-06 | ||
GB858514288A GB8514288D0 (en) | 1985-06-06 | 1985-06-06 | Enzyme assay of body fluids |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0223816A1 true EP0223816A1 (en) | 1987-06-03 |
Family
ID=10580275
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19860903511 Withdrawn EP0223816A1 (en) | 1985-06-06 | 1986-06-06 | Enzyme assay of body fluids |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0223816A1 (en) |
JP (1) | JPS62502633A (en) |
GB (1) | GB8514288D0 (en) |
WO (1) | WO1986007462A1 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE4227102C2 (en) * | 1992-08-17 | 2002-11-14 | Dade Behring Marburg Gmbh | Immunochemical method for the detection of an analyte |
AU697785B2 (en) * | 1994-07-19 | 1998-10-15 | Johnson & Johnson Clinical Diagnostics, Inc. | Analytical element,composition and method using modified apo-horseradish peroxidase |
DE19628484A1 (en) * | 1996-07-15 | 1998-01-22 | Boehringer Mannheim Gmbh | Blood substitute suppression by peroxides |
RU2418633C2 (en) | 2004-04-08 | 2011-05-20 | Байоматрика, Инк. | Integration of specimens storage and control in biomedical sciences |
WO2012018638A2 (en) | 2010-07-26 | 2012-02-09 | Biomatrica, Inc. | Compositions for stabilizing dna, rna and proteins in blood and other biological samples during shipping and storage at ambient temperatures |
US9845489B2 (en) | 2010-07-26 | 2017-12-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures |
EP2934572A4 (en) | 2012-12-20 | 2016-11-23 | Biomatrica Inc | Formulations and methods for stabilizing pcr reagents |
EP3154338B1 (en) | 2014-06-10 | 2020-01-29 | Biomatrica, INC. | Stabilization of thrombocytes at ambient temperatures |
EP4242628A3 (en) | 2015-12-08 | 2023-11-08 | Biomatrica, INC. | Reduction of erythrocyte sedimentation rate |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL51668A (en) * | 1977-03-16 | 1981-12-31 | Israel State | Analytical method for the quantitative determination of immunogens and antibodies and a kit therefor |
US4269938A (en) * | 1979-03-08 | 1981-05-26 | Eastman Kodak Company | Assay of peroxidatively active materials |
US4318982A (en) * | 1979-06-04 | 1982-03-09 | Miles Laboratories, Inc. | FMN-Labeled specific binding assay |
US4366143A (en) * | 1979-09-24 | 1982-12-28 | Amersham International Public Limited Company | Assay for the free portion of substances in biological fluids |
WO1983003306A1 (en) * | 1982-03-19 | 1983-09-29 | Roger Philip Ekins | Method and composition for free ligand assays |
DE3463395D1 (en) * | 1983-02-11 | 1987-06-04 | Nat Res Dev | Enhanced luminescent or luminometric assay |
-
1985
- 1985-06-06 GB GB858514288A patent/GB8514288D0/en active Pending
-
1986
- 1986-06-06 EP EP19860903511 patent/EP0223816A1/en not_active Withdrawn
- 1986-06-06 WO PCT/GB1986/000324 patent/WO1986007462A1/en not_active Application Discontinuation
- 1986-06-06 JP JP50326586A patent/JPS62502633A/en active Pending
Non-Patent Citations (1)
Title |
---|
See references of WO8607462A1 * |
Also Published As
Publication number | Publication date |
---|---|
JPS62502633A (en) | 1987-10-08 |
GB8514288D0 (en) | 1985-07-10 |
WO1986007462A1 (en) | 1986-12-18 |
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Inventor name: CHARLES, STEPHEN, ALEXANDERAMERSHAM PLACE Inventor name: MARTIN, JOHN, KINGSLEYAMERSHAM PLACE Inventor name: DENT, ALASTAIR, HOWARDAMERSHAM PLACE |