EP0214167A1 - Method for the determination of antibodies or antigens - Google Patents
Method for the determination of antibodies or antigensInfo
- Publication number
- EP0214167A1 EP0214167A1 EP19860901081 EP86901081A EP0214167A1 EP 0214167 A1 EP0214167 A1 EP 0214167A1 EP 19860901081 EP19860901081 EP 19860901081 EP 86901081 A EP86901081 A EP 86901081A EP 0214167 A1 EP0214167 A1 EP 0214167A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- particles
- layer
- fluorescent
- reaction
- 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.)
- Ceased
Links
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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Definitions
- the present invention is concerned with a fluoro etric or phosphorimetric iirununoassay method in which small polymer particles are used as the solid phase.
- the method in accordance with the invention can be used, besides for immunoassays in general, also for blood group determinations.
- the signal concerned may be, e.g., radioactivity (RIA), fluores ⁇ cence signal (FIA) or even enzyme activity (EIA, i.e. Enzyme Immunoassay) .
- RIA radioactivity
- FIA fluores ⁇ cence signal
- EIA enzyme activity
- the separation of the solid phase from the reaction solution always includes washing of the solid phase, which, at present, requires operations whose automation is difficult. Thus, these operations are, as a rule, carried out manually. If small polymer particles are used, the operations include centrifuging or mag ⁇ netic deposition.
- the principal objective of the present inven ⁇ tion is to provide such a method for the determination of antibodies or antigens in which inconvenient operations of separation are avoided and which is also suitable for being used in connection with such antibodies or anti ⁇ gens as are placed on the surface of cells or other particles of organic origin.
- particles treated with a fluorescent (or phosphorescent) tracer are, together with particles treated with a magnetic material, immobilized on an antibody (or antigen) by means of an antigen-antibody bond.
- the cells and the magnetic and fluorescent particles attached to them are pulled by means of a magnetic field from the reaction layer into the separation layer, whereupon the fluorescence is determined from the separation layer or from the reaction layer. Fluorescence is emitted only if the excitation light meets fluorescent particles. Mere magnetic particles alone do not emit fluorescent radiation.
- the antibody does not become labelled with the polymer particles covered by the antigen concerned, only the magnetic particles arrive in the separation layer, and the fluorescent non-magnetic particles and the unlabelled cells remain behind the separation layer.
- the method is suitable for being used both as a direct method and as an indirect method. Thereat, for example, the determination of blood group can be carried out both from the cell side and from the serum side. The determination from the serum side takes place as indirect.
- the method in accordance with the invention is easy to carry out, because the solutions do not have to be removed from the vessel and because no separate washings have to be carried out.
- Figures 1 to 4 illustrate the determination of dissolved antibody.
- Figures 5 to 8 illustrate the de ⁇ termination of an antigen placed on the surface of a cell
- Figures 9 and 10 illustrate simultaneous de ⁇ termination of several antibodies.
- Figures 1 to 4 illustrate the determination of dissolved antibody.
- a reaction layer 1 In the measurement vessel, there are two liquid layers (Fig. 1) : a reaction layer 1 and a so-called separation layer 2.
- the reaction layer 1 In the reaction layer 1, there is the antibody 3 to be determined, in dissolved form.
- the reaction layer 1 there are polymer particles covered with antigen 4 of the antibody 3, of which said polymer particles the particles 5 include a magnetic material and the particles 6 include a fluores ⁇ cent tracer.
- the polymer particles are pearls made of some suitable material, and their size is 0.1 to 10 ⁇ m.
- the separation layer 2 is placed in the vessel below the reaction layer 1. It has been placed into the vessel before the reaction layer 1 , or it has been added afterwards to underneath the reaction layer.
- the separation liquid 2 is preferably denser than the reaction solution 1 , and of such a colour that it prevents the fluorescence in the -reaction solution from being seen in the measurement.
- Suitable reaction solutions are, e.g., saccharose solution (concentration typically 10 to 60 %) and Ficoll-Paque density-gradient centrifuging solution, etc. If the separation solution is not of suitable colour by nature, a suitable colour is obtained by to it adding a colouring agent which ab ⁇ sorbs " light strongly within the excitation or emission wavelength of fluorescence, or within both wavelengths. In particular, black is a suitable colour.
- the conventional incubation stage follows, whereat the immunological reaction takes place (Fig. 2) .
- the antibody 3 to be determined is bound with the antigen 4 placed on the surface both of the magnetic 5 and of the fluorescent 6 particle.
- the antibody 3 can now be both displaced magnetically and measured fluorometri- cally. If there is no antibody 3 in the sample, only the magnetic particles 5 can be displaced magnetically.
- the magnetic particles 5 are pulled by means of a magnetic field 7 through the separation layer 2 onto the bottom of the measurement vessel (Fig. 3) .
- the fluorescent particles 6 adhering to the magnetic particles by the intermediate of the antibody, follow along with them.
- the separation layer 2 also acts as a physico-chemical washing layer.
- a particular magnetic separation device is built in. The more of the antibody 3 to be determined there is present in the sample, the more particles 6 covered with the fluorescent tracer are there now on the bottom of the vessel.
- the measurement takes place so that the exci ⁇ tation radiation 8 is passed to the bottom of the measure ⁇ ment vessel through its wall, and the emission radiation 9 is also collected along the same route for detection (Fig. 4) .
- the coloured separation layer 2 now acts as an optical shield layer against viewing of the fluorescent reaction layer 1. Thereby, no emitted fluorescent radiation has access from the reaction layer 1 into the detector system, and only the fluorescent particles placed on the bottom of the vessel are detected.
- Figures 5 to 8 illustrate a case in which an antigen placed on the surface of a cell is to be de ⁇ termined.
- the measurement vessel there is also a reaction layer 1 at the top and a separation layer 2 underneath.
- the reaction layer 1 there are cells -10 on whose surface there is antigen 11. More ⁇ over, in the reaction layer, there are polymer particles covered with the antibody 12 of the said antigen, some of the said polymer particles being magnetic 5, and some of them being fluorescent 6.
- the dosage stage Fig. 5
- the incubation stage Fig. 6
- the separation stage Fig. 7
- the measurement stage Fig. 8
- This method can be applied, e.g., to b ood group determinations as follows:
- mag- netic and fluorescent particles covered with a known antibody are measured (the same antibody on the surface of each particle type) . It is assumed that in the mea ⁇ surement vessel A there are magnetic and fluorescent particles covered with anti-A antibody, and in the mea- surement vessel B there are magnetic and fluorescent particles covered with anti-B antibody.
- both of the particle types, magnetic and fluorescent adhere to the same .cell.
- none of the particle types adheres to the cell surface, whereby only the magnetic particles respond to the magnetic field.
- the magnetized particles and the labelled cells, if any, are pulled against the bottom of the measurement vessel.
- the determination of the blood group is performed by in con ⁇ nection with the incubation adding a serum sample to be studied on the basis of known control cells (A and B cells) and corresponding magnetic anti-A and anti-B particles. If in the reaction vessel A the adherence of anti-A particles is prevented and the signal is negative, there has been anti-A antibody present in the serum, which has acted on the basis of competition and prevented the adhesion of anti-A particles. ' If the reaction is posi ⁇ tive in respect of the anti-B particles present in the reaction vessel B, the blood group is B from the serum side.
- the blood group is A from the serum side. If the signal is positive both in the reaction vessel A and in the reaction vessel B, the blood group is AB from the serum side. On the other hand, if the signal is negative in both of the reaction vessels, the blood group is 0.
- the blood group can be determined by covering the particles with the corresponding blood cell antigen, which are also available commercially. In the determination of anti- bodies, when particles covered with red-cell antigens are used, no test cells are needed at all. When cells are used, by means of inhibition it is possible to establish, e.g., a person's secretor property.
- the method can also be carried out by means of competition by using magnetic and fluorescent particles covered with the same antigen in the same reaction vessel. If there is the corresponding antibody (IgM class) in the serum, fluorescent pearls are also deposited in con ⁇ nection with the magnetic pearls, being linked by the antibody.
- IgM class antibody
- the Rh-factor can be established by incubating the cells to be studied with anti-D serum. whereby the antibody adheres to the face of the red blood cell.
- the antibody concerned is of the type IgG, which can be detected by means of anti-human-IgG.
- the antibody may be attached to fluorescent particles, or it may be present as its fluorescent conjugate.
- the methods may be the following: After red blood cells have been incubated in anti-D serum, magnetic pearls covered with anti-human-erythrocyte antibody are added to among the sensitized red blood cells, and the red blood cells are pulled down after the incubation time. Now it is possible to suck off any excess serum and unattached pearls out of the measurement vessel while keeping the magnetic field constantly on.
- the cells can be washed in a suitable medium, while finally depositing the cells down by means of the magnetic field.
- fluorescent pearls covered with anti-human-IgG and to underneath the mixture of cells and pearls, it is possible to add " a dense, coloured, suitable separating substance.
- the cells are again pulled down by means of the magnetic field, whereby detection of the fluorescence is possible.
- the coloured dense substance before the fluorescent particles, in which case the risk of fluo ⁇ rescence contamination is reduced.
- One mode of labelling the Rh-positive cells is to sensitize the cells by means of anti-D, whereupon the cells are pulled by means of magnetic anti-human-erythrocyte particles into the first separation-substance layer, wherein there is a very large quantity of fluorescent particles covered with anti-human-IgG or of fluorescent anti-human- IgG conjugate.
- the magnetic pulling is stopped at this layer for the time of the incubation, and upon completion of the incubation the cells are pulled through another coloured layer, of higher density, to the bottom of the measurement vessel, and the fluorescence is detected.
- Fig. 9 illus- trates simultaneous determination of several antibodies in a case in which the antigens of all of the antibodies are attached onto the surfaces of magnetic particles of one sort.
- Fig. 10 illustrates simultaneous determination of several antibodies in a case in which the antigen of each antibody is attached onto the surface of the par ⁇ ticles in its own group of particles.
- the reaction solution there are magnetic particles 5, 5' and 5", one antigen, either 4, 4' or 4", being attached to the surface of each of the said par- ticles.
- fluorometric measurement is carried out at each of the wavelengths concerned in order to determine the anti ⁇ bodies 3, 3' and 3".
- the methods described above can, of course, also be applied by making use of phosphorescence.
- the methods in the solid phase, there may be an antibody equally well as an antigen.
- the separation layer may be placed on top of the reaction layer. In such a case, the measurement becomes less convenient, but thereby the background fluorescence caused by the vessel wall is avoided. If desired, the separation layer may also be formed in the vessel after the incubation.
- the measurement may also be carried out from the reaction layer.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (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, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Dans un procédé de dépistage d'anticorps, des particules fluorescentes (6) et magnétiques (5) sont ajoutées à l'échantillon, et l'antigène de l'anticorps (3) que l'on veut dépister est placé sur la surface des deux types de particules. Pendant une réaction immunologique, les particules fluorescentes adhèrent aux particules magnétiques par l'intermédiaire de l'anticorps. Après la réaction, les particules magnétiques sont attirées par un aimant dans une couche de séparation (2), dont la fluorescence est alors mesurée.In an antibody screening process, fluorescent (6) and magnetic (5) particles are added to the sample, and the antibody (3) antigen to be screened is placed on the surface of the cells. two types of particles. During an immunological reaction, the fluorescent particles adhere to the magnetic particles via the antibody. After the reaction, the magnetic particles are attracted by a magnet in a separation layer (2), the fluorescence of which is then measured.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FI850481A FI850481A0 (en) | 1985-02-06 | 1985-02-06 | FOERFARANDE FOER BESTAEMNING AV MOTMEDEL ELLER ANTIGENER. |
FI850481 | 1985-02-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0214167A1 true EP0214167A1 (en) | 1987-03-18 |
Family
ID=8520326
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19860901081 Ceased EP0214167A1 (en) | 1985-02-06 | 1986-02-05 | Method for the determination of antibodies or antigens |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0214167A1 (en) |
JP (1) | JPS62501647A (en) |
FI (1) | FI850481A0 (en) |
WO (1) | WO1986004684A1 (en) |
Families Citing this family (32)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5071774A (en) * | 1983-04-05 | 1991-12-10 | Syntex (U.S.A.) Inc. | Multiparameter particle analysis |
AU7399487A (en) * | 1986-05-22 | 1987-12-22 | Unilever Plc | Solid phase immunoassay method |
US5238810A (en) * | 1986-09-22 | 1993-08-24 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and apparatus thereof |
EP0287665B1 (en) * | 1986-09-22 | 1996-07-31 | Nippon Telegraph And Telephone Corporation | Laser magnetic immunoassay method and apparatus therefor |
GB8717862D0 (en) * | 1987-07-28 | 1987-09-03 | Acade Diagnostic Systems Sa Nv | Turbidimetric assay |
CN1034617A (en) * | 1987-10-09 | 1989-08-09 | 奈吉尼股份有限公司 | Identify the method and the device of biochemical species |
JPH05504828A (en) * | 1988-05-04 | 1993-07-22 | ケンブリッジ、バイオテック、コーポレーション | Capillary flow device and dual capture assay method |
US5145784A (en) * | 1988-05-04 | 1992-09-08 | Cambridge Biotech Corporation | Double capture assay method employing a capillary flow device |
DE3840462A1 (en) * | 1988-12-01 | 1990-06-07 | Berthold Lab Prof R | METHOD AND DEVICE FOR MEASURING CHEMILUMINESCENCE |
JP2910929B2 (en) * | 1989-03-23 | 1999-06-23 | 浜松ホトニクス株式会社 | Surface modification method for particles composed of magnetic particles |
GB2236852B (en) * | 1989-09-25 | 1994-04-06 | Scotgen Ltd | DNA probe based assays and intermediates useful in the synthesis of cleavable nucleic acids for use in such assays |
US5279936A (en) * | 1989-12-22 | 1994-01-18 | Syntex (U.S.A.) Inc. | Method of separation employing magnetic particles and second medium |
GB2270976A (en) * | 1992-09-18 | 1994-03-30 | Marconi Gec Ltd | Immunoassay/separation process using an auxiliary species on a support |
EP0660935B1 (en) * | 1992-08-03 | 2000-05-24 | Gec-Marconi Limited | Immunological detection using two detectable labels |
GB2270158B (en) * | 1992-08-03 | 1997-03-19 | Marconi Gec Ltd | Detection |
US5374531A (en) * | 1993-03-22 | 1994-12-20 | Zynaxis, Inc. | Immunoassay for determination of cells |
AUPN214095A0 (en) | 1995-04-03 | 1995-04-27 | Australian Water Technologies Pty Ltd | Method for detecting microorganisms using flow cytometry |
EP1428018B1 (en) | 2001-09-06 | 2010-06-09 | Straus Holdings Inc. | Rapid and sensitive detection of molecules |
US7018849B2 (en) | 2002-01-15 | 2006-03-28 | Piasio Roger N | Process for (A) separating biological/ligands from dilute solutions and (B) conducting an immunochromatographic assay thereof employing superparamagnetic particles throughtout |
JP2006255817A (en) * | 2005-03-16 | 2006-09-28 | Sonac Kk | Metal structure and its manufacturing method |
EP1937829A4 (en) | 2005-09-26 | 2011-08-03 | Rapid Micro Biosystems Inc | Cassette containing growth medium |
CN102224260B (en) * | 2008-09-24 | 2015-11-25 | 施特劳斯控股公司 | For test kit and the device of detect analytes |
JP5578241B2 (en) * | 2010-12-21 | 2014-08-27 | 株式会社島津製作所 | Device and method for manipulating a target component in a tube |
HUE036509T2 (en) | 2011-11-07 | 2018-07-30 | Rapid Micro Biosystems Inc | Cassette for sterility testing |
US10407707B2 (en) | 2012-04-16 | 2019-09-10 | Rapid Micro Biosystems, Inc. | Cell culturing device |
US9657290B2 (en) * | 2012-07-03 | 2017-05-23 | The Board Of Trustees Of The Leland Stanford Junior University | Scalable bio-element analysis |
EP3828284A1 (en) | 2013-03-15 | 2021-06-02 | Abbott Molecular Inc. | One-step procedure for the purification of nucleic acids |
WO2015046293A1 (en) * | 2013-09-30 | 2015-04-02 | 凸版印刷株式会社 | Test substance detection system |
US10370653B2 (en) | 2015-02-22 | 2019-08-06 | The Board Of Trustees Of The Leland Stanford Junior University | Micro-screening apparatus, process, and products |
CN115254210A (en) | 2016-11-14 | 2022-11-01 | 浩康生物系统公司 | Method and apparatus for sorting target particles |
CN107192818B (en) * | 2017-05-23 | 2018-06-29 | 重庆天之助生物科技有限公司 | A kind of particulate coloration clustering method and kit |
WO2019204784A1 (en) * | 2018-04-19 | 2019-10-24 | First Light Biosciences, Inc. | Detection of targets |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1566098A (en) * | 1975-11-14 | 1980-04-30 | Nat Res Dev | Separation of solid and liquid components of mixtures |
US4115535A (en) * | 1977-06-22 | 1978-09-19 | General Electric Company | Diagnostic method employing a mixture of normally separable protein-coated particles |
CA1111762A (en) * | 1977-12-28 | 1981-11-03 | David S. Frank | Fluorescent rare earth chelate in polymeric latex particles |
US4434150A (en) * | 1981-10-19 | 1984-02-28 | Ortho Diagnostic Systems, Inc. | Immunological reagents employing polymeric backbone possessing reactive functional groups |
GB8401368D0 (en) * | 1984-01-19 | 1984-02-22 | Amersham Int Plc | Assay method |
FI841023A0 (en) * | 1984-03-14 | 1984-03-14 | Labsystems Oy | FOERFARANDE FOER UTFOERING AV IMMUNOBESTAEMNINGAR |
FI842992A0 (en) * | 1984-07-26 | 1984-07-26 | Labsystems Oy | IMMUNOLOGISKT DEFINITIONSFOERFARANDE. |
-
1985
- 1985-02-06 FI FI850481A patent/FI850481A0/en not_active Application Discontinuation
-
1986
- 1986-02-05 EP EP19860901081 patent/EP0214167A1/en not_active Ceased
- 1986-02-05 JP JP50102486A patent/JPS62501647A/en active Pending
- 1986-02-05 WO PCT/FI1986/000014 patent/WO1986004684A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO8604684A1 * |
Also Published As
Publication number | Publication date |
---|---|
FI850481A0 (en) | 1985-02-06 |
JPS62501647A (en) | 1987-07-02 |
WO1986004684A1 (en) | 1986-08-14 |
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Legal Events
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
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17P | Request for examination filed |
Effective date: 19860903 |
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AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE FR GB IT LI LU NL SE |
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17Q | First examination report despatched |
Effective date: 19881215 |
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STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED |
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18R | Application refused |
Effective date: 19891214 |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: LUOTOLA, JUHANI, E.I. Inventor name: HARJUNMAA, HANNU |