GB2203836A - Determination of analyte concentration - Google Patents
Determination of analyte concentration Download PDFInfo
- Publication number
- GB2203836A GB2203836A GB08709814A GB8709814A GB2203836A GB 2203836 A GB2203836 A GB 2203836A GB 08709814 A GB08709814 A GB 08709814A GB 8709814 A GB8709814 A GB 8709814A GB 2203836 A GB2203836 A GB 2203836A
- Authority
- GB
- United Kingdom
- Prior art keywords
- analyte
- antibody
- antigen
- unlabelled
- reactant
- 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.)
- Granted
Links
- 239000012491 analyte Substances 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 33
- 239000000376 reactant Substances 0.000 claims abstract description 23
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 238000011067 equilibration Methods 0.000 claims abstract description 19
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 11
- 239000007790 solid phase Substances 0.000 claims abstract description 10
- 238000000670 ligand binding assay Methods 0.000 claims abstract description 6
- 238000000338 in vitro Methods 0.000 claims abstract description 4
- 102000036639 antigens Human genes 0.000 claims description 30
- 108091007433 antigens Proteins 0.000 claims description 30
- 239000000427 antigen Substances 0.000 claims description 29
- 230000000903 blocking effect Effects 0.000 claims description 9
- 230000027455 binding Effects 0.000 claims description 8
- 230000000694 effects Effects 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000003556 assay Methods 0.000 description 10
- 239000011230 binding agent Substances 0.000 description 5
- 238000012875 competitive assay Methods 0.000 description 5
- 238000010494 dissociation reaction Methods 0.000 description 4
- 230000005593 dissociations Effects 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 3
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 3
- 102000011923 Thyrotropin Human genes 0.000 description 3
- 108010061174 Thyrotropin Proteins 0.000 description 3
- 108010032595 Antibody Binding Sites Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 101000588258 Taenia solium Paramyosin Proteins 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229940034208 thyroxine Drugs 0.000 description 2
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
An in vitro process for determination of analyte concentration in ligand binding assays, the process comprising an equilibration reaction between the analyte to be determined and a reactant immobilised on a solid phase support, wherein active sites on the support together with some immobilised reactant sites are blocked with protein before the equilibration reaction. The process is applicable to the ligand binding assay techniques to enable the sensitivity thereof to be enhanced, by overcoming the hook effect.
Description
DETERMINATION OF ANALYTE CONCENTRATION
This invention relates to a process for the determination of analyte concentration in ligand binding as says for in vitro analysis of for example thyroxine (T4), insulin and thyroid stimulating hormone levels in blood.
It is known to determine analyte concentration by ligand binding assay techniques, which include competitive binding assay and sandwich assay techniques. In one form of competitive binding assay, the reactant to be measured, such as antigen A, is incubated With labelled reactant of known concenrrat)()n and binder, such as antibody against antigen A, of known concentration, so that labelled and unlabelled antigen compete for binding sites on the binder. The bound antigen-antibody complex is separated from free labelled and unlabelled antigen and the signal from the bound complex bears a mathematical relationship to the unlabelled antigen A to give an estimate of the concentration thereof.
Generally, separation is achieved by immobilising the antibody on a solid phase such as plastics tube, beads, or inert particles, or by precipitation of the complexes.
In the sandwich assay technique, the reactant to be measured, such as antigen B, is added sequentially or simultaneously with another reactant of known concentration such as a labelled binder, for example labelled anti-B antibody, to excess of unlabelled binder of known concentration, to form antibody-antigen-labelled antibody sandwich complexes.
The complexes and excess antibody are separated from the free antigen and free labelled antibody, generally by immobilising the unlabelled antibody on an inert solid phase prior to the reaction so that the sandwich complexes can be physically separated, for example by decantation. The signal from the sandwich complexes bears a mathematical relationship to the unlabelled antigen B to enable an estimate of the concentration thereof to be calculated.
The competitive binding assay technique may be used for determination of thyroxine (T4) and the sandwich assay technique may be used for determination of thyroid stimulating hormone (TSH) and insulin.
In the known techniques, repeated re-exposure of the reacted antigens with the reacted antibodies is used to speed up the attainment of equilIbrium between the reactants and binders, but this causes analyte exhaustion to occur. In the sandwich assay technique, an additional disadvantage is that the bound antigen tends to dissociate, with resultant blocking of the binding of the labelled antibody to the bound antigen to form the sandwich compound. Where sequential addition of reactants is practised, dissociation of the bound antigen-labelled antibody moieties from the immobilised sandwich complexes also occurs. As the concentration of antigen increases, the degree of blocking or dissociation also increases, thus rendering the assay inaccurate. The blocking or dissociation effect is known as the "hook effect".
It is an object of the present invention to increase the sensitivity of the known ligand binding assay techniques.
According to the invention, we propose an in vitro process for determination of analyte concentration in ligand binding assays, the process comprising an equilibration reaction between the analyte to be determined and a reactant immobilised on a solid phase support, wherein active sites on the support together with some immobilised reactant sites are blocked with protein before the equilibration reaction.
As applied to the competitive binding assay technique, the process according to the invention comprises blocking active sites on the support, together with some of the binding sites of the antibody, before the reaction with labelled and unlabelled antigen, and unbound labelled and unlabelled antigen is discarded after the equilibration reaction.
As applied to the sandwich assay technique, the process according to the invention comprises blocking active sites on the support, together with some of the binding sites of the unlabelled antibody, before the reaction with labelled antibody and unlabelled antigen, and unbound labelled antibody and unlabelled ' antigen is discarded after the equilibration reaction.
The advantage of the process according to the invention compared with the prior art is that, whereas in the prior art repeated re-equilibration causes analyte exhaustion to occur, according to the invention re-equilibration with fresh reactants may be carried out and the sensitivity of the resulting assay is enhanced with each re-equilibration by a factor of at least 3. In particular, the process according to the invention maintains the analyte concentration and, in the sandwich assay technique, decreases dissociation of the bound antigen (where sequential addition of reactants is employed) or the antigen-labelled antibody moieties (where simultaneous addition of reactants is employed) from the immobilised sandwich complexes and thus prevents manifestation of the hook effect.
According to a preferred feature of the invention, therefore, fresh analyte to be measured is re-equilibrated with the reactant and analyte on the solid phase support as a result of the initial equilibration reaction, some of the protein blocking the reactant sites being displaced by analyte to be measured, whereby the signal from the bound complexes is more accurately related to the concentration of the analyte to be determined.
In the process according to the invention, separation of the bound complexes is carried out in the usual way and the signals from the labelled reactants measured to give an estimate of the unlabelled antigen or other reactant concentration after comparison with signals from the reactants of known concentration assayed in a similar manner.
Embodiments of the invention will now be described by way of example with reference to the accompanying drawings, in which Figures l(a) and l(b) illustrate diagramatically the process according to the invention are applied to the competitive binding assay technique and
Figures 2(a) and 2(b) illustrate diagrammatically the process according to the invention as applied to the sandwich assay technique.
In the drawings, the symbols have the following meaning:
Fresh unlabelled antigen
Fresh labelled antigen
Protein
Unlabelled antibody
Labelled antibody solid Solid phase support.
Referring firstly to Figure l(a), the initial equilibration reaction is shown, in which fresh labelled and unlabelled antigen is reacted with antibody immobilised on a solid phase support, the remaining active sites on the support together with some of the antibody binding sites having been blocked with protein. The labelled and unlabelled antigen compete for the un-blocked binding sites and free (unbound) labelled and unlabelled antigen is discarded.
In the re-equilibration reaction shown in Figure l(b), fresh labelled and unlabelled antigen is further reacted with the immobilised antibody, some of the protein molecules being displaced by antigen so that the total amount of bound labelled and unlabelled antigen is more accurately in proportion to the amounts in the reactant mixture. Free labelled and unlabelled antigen is discarded, together with displaced protein.
Referring to Figure 2(a), which shows the initial sandwich assay equilibration reaction, fresh labelled antibody and unlabelled antigen are reacted with unlabelled antibody immobilised on a solid phase support, the remaining active sites on the support together with some of the antibody binding sites having been blocked with protein. Unlabelled antibody-antigen-labelled antibody sandwich complexes are formed at the unblocked unlabelled antibody sites and free (unbound) labelled antibody and unlabelled antigen are discarded. In the re-equilibration reaction shown in Figure 2(b), fresh labelled antibody and unlabelled antigen are further reactd with the immobilised antibody, some of the protein molecules being displaced to allow the formation of additional sandwich complexes. Free labelled antibody and unlabelled antigen are discarded, together with displaced protein.
Claims (5)
1. An in vitro process for determination of analyte concentration in ligand binding assays, the process comprising an equilibration reaction between the analyte to be determined and a reactant immobilised on a solid phase support, wherein active sites on the support together with some immobilised reactant sites are blocked with protein before the equilibration reaction.
2. A process according to Claim 1 comprising blocking active sites on the support, together with some of the binding sites of the antibody, before the reaction with labelled and unlabelled antigen, and discarding unbound labellled and unlabelled antigen after the equilibration reaction.
3. A process according to Claim 1, comprising blocking active sites on the support, together with some of the binding sites of the unlabelled antibody, before the reaction with labelled antibody and unlabelled antigen, and discarding unbound labelled antibody and unlabelled antigen after the equilibration reaction.
4. A process according to any preceding claim, in which fresh analyte to be measured is re-equilibrated with the reactant and analyte on the solid phase support as a result of the initial equilibration reaction, some of the protein blocking the reactant sites being displaced by analyte to be measured, whereby the signal from the bound complexes is more accurately related to the concentration of the analyte to be determined.
5. A process as hereinbefore described with reference to and as illustrated in the accompanying drawings.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8709814A GB2203836B (en) | 1987-04-24 | 1987-04-24 | Determination of analyte concentration by re-equilibration |
| MYPI87000672A MY101223A (en) | 1987-04-24 | 1987-05-18 | Determination of analyte concentration |
| SG969/91A SG96991G (en) | 1987-04-24 | 1991-11-18 | Determination of analyte concentration by re-equilibration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8709814A GB2203836B (en) | 1987-04-24 | 1987-04-24 | Determination of analyte concentration by re-equilibration |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB8709814D0 GB8709814D0 (en) | 1987-05-28 |
| GB2203836A true GB2203836A (en) | 1988-10-26 |
| GB2203836B GB2203836B (en) | 1991-01-23 |
Family
ID=10616341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB8709814A Expired - Lifetime GB2203836B (en) | 1987-04-24 | 1987-04-24 | Determination of analyte concentration by re-equilibration |
Country Status (3)
| Country | Link |
|---|---|
| GB (1) | GB2203836B (en) |
| MY (1) | MY101223A (en) |
| SG (1) | SG96991G (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0366092A3 (en) * | 1988-10-25 | 1991-04-03 | Roche Diagnostics GmbH | Method for the detection of antigen and class specific antibodies and reagent therefor |
| US5198340A (en) * | 1991-01-17 | 1993-03-30 | Genentech, Inc. | Assay for free igf-i, igf-ii, and gh levels in body fluids |
| JP2005521032A (en) * | 2001-09-10 | 2005-07-14 | メソ スケイル テクノロジーズ，エルエルシー | Method and apparatus for performing multiple measurements on one sample |
| WO2018095314A1 (en) * | 2016-11-22 | 2018-05-31 | 北京科美生物技术有限公司 | Method, system, reagent kit and system for verifying hd-hook effect sample and for performing immunoassay |
| CN108132344A (en) * | 2016-11-22 | 2018-06-08 | 博阳生物科技（上海）有限公司 | Immunoassay methods, systems and kits for characterizing immunoassays |
| CN108152505A (en) * | 2016-11-22 | 2018-06-12 | 博阳生物科技（上海）有限公司 | Immunoassay methods, systems and kits for characterizing immunoassays |
| CN108802378A (en) * | 2018-05-31 | 2018-11-13 | 重庆中元汇吉生物技术有限公司 | A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution |
-
1987
- 1987-04-24 GB GB8709814A patent/GB2203836B/en not_active Expired - Lifetime
- 1987-05-18 MY MYPI87000672A patent/MY101223A/en unknown
-
1991
- 1991-11-18 SG SG969/91A patent/SG96991G/en unknown
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0366092A3 (en) * | 1988-10-25 | 1991-04-03 | Roche Diagnostics GmbH | Method for the detection of antigen and class specific antibodies and reagent therefor |
| US5376557A (en) * | 1988-10-25 | 1994-12-27 | Boehringer Mannheim Gmbh | Process for the determination of antibodies which are class-specific for an antigen and a reagent for carrying out the process |
| US5198340A (en) * | 1991-01-17 | 1993-03-30 | Genentech, Inc. | Assay for free igf-i, igf-ii, and gh levels in body fluids |
| JP2005521032A (en) * | 2001-09-10 | 2005-07-14 | メソ スケイル テクノロジーズ，エルエルシー | Method and apparatus for performing multiple measurements on one sample |
| CN108152505A (en) * | 2016-11-22 | 2018-06-12 | 博阳生物科技（上海）有限公司 | Immunoassay methods, systems and kits for characterizing immunoassays |
| CN108132344A (en) * | 2016-11-22 | 2018-06-08 | 博阳生物科技（上海）有限公司 | Immunoassay methods, systems and kits for characterizing immunoassays |
| WO2018095314A1 (en) * | 2016-11-22 | 2018-05-31 | 北京科美生物技术有限公司 | Method, system, reagent kit and system for verifying hd-hook effect sample and for performing immunoassay |
| CN109470861A (en) * | 2016-11-22 | 2019-03-15 | 博阳生物科技（上海）有限公司 | Method of immunity, the system for identifying immunoassays and kit |
| CN109470862A (en) * | 2016-11-22 | 2019-03-15 | 博阳生物科技（上海）有限公司 | Method of immunity, the system for identifying immunoassays and kit |
| CN109470860A (en) * | 2016-11-22 | 2019-03-15 | 博阳生物科技（上海）有限公司 | Method of immunity, the system for identifying immunoassays and kit |
| CN109470862B (en) * | 2016-11-22 | 2021-12-03 | 科美博阳诊断技术(上海)有限公司 | Immunoassay method, system and kit for identifying immunoassay |
| CN109470860B (en) * | 2016-11-22 | 2021-12-10 | 科美博阳诊断技术(上海)有限公司 | Immunoassay method, system and kit for identifying immunoassay |
| US12196751B2 (en) | 2016-11-22 | 2025-01-14 | Chemclin Diagnostics Co., Ltd. | Method, system, reagent kit, and device for determining HD-hook-effect sample and immunoassay |
| CN108802378A (en) * | 2018-05-31 | 2018-11-13 | 重庆中元汇吉生物技术有限公司 | A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution |
Also Published As
| Publication number | Publication date |
|---|---|
| GB8709814D0 (en) | 1987-05-28 |
| MY101223A (en) | 1991-08-17 |
| SG96991G (en) | 1992-02-14 |
| GB2203836B (en) | 1991-01-23 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19970424 |