CA1208548A - Method for the determination of antigens - Google Patents
Method for the determination of antigensInfo
- Publication number
- CA1208548A CA1208548A CA000425012A CA425012A CA1208548A CA 1208548 A CA1208548 A CA 1208548A CA 000425012 A CA000425012 A CA 000425012A CA 425012 A CA425012 A CA 425012A CA 1208548 A CA1208548 A CA 1208548A
- Authority
- CA
- Canada
- Prior art keywords
- reaction
- antigen
- antibody
- buffer substance
- buffer
- 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.)
- Expired
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
- G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
- Semiconductor Memories (AREA)
- Amplifiers (AREA)
- Junction Field-Effect Transistors (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Abstract In the photometric determination of the antigen content by the antigen-antibody-reaction at least one of the two reaction components is diluted with a physiological salt solution, which contains, beside polyethylenglycol a buffer substance, before the two reaction components are combined.
Description
- ~Z08548 The invention relates to a method for the determination of the antigen content in a sample by the antigen-antibody-reaction. It also relates to a diluent for an antigen and antibody component, respectively, for preforming this method.
The determination of diagnostic relevant parameters with antigens in samples, as serum, plasma or liquor, by the immunological or antigen-antibody-reaction, which is evaluated photometrically for instance by nephelometry or turbidimetry, makes frequently a special preparation of both reaction components necessary. Usually the re-action component which contains the sample which antigen content has to be determined, and the other component which contains the antibody, i.e. which is normally the corresponding antiserum, are diluted with physiological salt solution or with physiological salt solution which contains in addition 4~ polyethylenglycol (average molecular weight 6000).
The content of polyethylenglycol serves particularly to avoid a precipitation of the antigen-antibody-complexes formed during the reaction, i.e. to avoid the formation of a precipitation in the test solution formed by the com-bination of both reaction components during the antigen determination, i.e. during the measurement. This property of polyethylenglycol is known for a certain period of time and is a necessity for these measurements, because otherwlse not all formed antigen-antibody-comple~es are subjected to the measurement.
l'he determination of immune globulins by laser turbidimetry according to the "Arbeitsanleitung fur das Lasermed-Turbidometer (pm 4) zur Bestimmung der Immunglobuline IgA, IgG, IgM mit ATAB-Antiseren (Instructions for the Lasermed-turbidometer (pm 4) for determining the immune globulins '~
208S~8 IgA, IgG, IgM with ATAB-anitsera), AHS Deutschland GmbH, Bereich ~lerz und Dade, Munich, 1982", showed that with different physiological salt solutions with 4% by weight polyethylenglycol different measurement signals were obtained with the laser turbidometer during the assay, although they (the salt solutions) were prepared according to the same formulation and with the same reagents by the same person. Accordingly no sufficiently reproducible results were obtained. The so called kinetic method shows a particular interference when according to said instructions after a prereaction of 10 sec. the rate of the antigen-antibody-reaction is measured and the antigen content is determined. In contrast to the so called "Fix point"-method according to said instructions, i.e. the final poin~ measurement after a reaction period of for instance 30 min, it is not necessary with said kinetic method to wait until the end of the completion of the antigen-antibody-reaction.
According to one aspect of the invention there is provided a method for the determination of the antigen content of a sample by an antigen-antibody reaction involving the mixing of a reaction component containing the antigen and a reaction component containing an antibody, comprising first diluting at least one of the two reaction compon-ents with a physiological saline solution which containspolyethyleneglycol and a buffer substance, thereafter combining both reaction components and determining the antigen-antibody reaction photometrically in the pre.sence of a buffer sub~tance, wherein the buffer substance is selected from the group consisting of a glycin, glycyl-glycin, acetate, citrate, bicine, histidin/HCl, tcis or 5.5-dialkylbarbituric acid buffer~
According to another aspect of the invention there is provided a polyethylene containing, physiological salt ` ~2~)85~18 - 2a -solution which can be used as a diluent of at least one of two reaction components of an antigen-antibody-reaction for the photometrical determination of the antigen content, wherein said solution contains a buffer substance selected from the group consisting of a glycin, glycylglycin, acetate, citrate, bicine, histidin/HCl, tris or 5.5-dialkylbarbituric acid buffer.
The invention solves the object to provide a method and a diluent, respectively, with which the antigen content of sample is exactly and reproducibly determinable even with the kinetic method.
The antibody molecule has a polyvalent and therefore a strongly structured form whereas the polyethyleneglycol is non-polar and therefore not structured. When koth reaction components are combined a fairly homogenous partition, which gives fairly reproducible results, is obtained not before a certain period has lapsed. The method according to the invetion is based on the fact that a fast and homogenous partition of both reaction components is obtained by adding a buffer substance to the polyethyleneglycol containing, physiological salt solution, which eliminates the structured form of the antibody molecule by balancing the charges.
~5 - ~20B54B
It is surprising that theirate of the antigen-antibody-reaction is particularly dependent on the chemical composition of the buffer substance, even far more than the concentration of the buffer substance or the pH-value~ Also important is in connection with the invention that not such buffer substances are selected which provide the fastest reaction rate, but such buffer substances are prefered, with which a constant reaction rate in the beginning is obtained which _ lasts for a certain period of time. This property of the buffer substances used according to the invention stra1ghtens the reaction to a certain degree, so that a linear progress over a relatively long period is obtained. This improves the accuracy of the results of the kinetic method considerably.
If for instance 20 mmol/l 5.5-diethylbarbituric acid, pH 7,5, are added as buffer to the physlological salt solution containing 4 % by weight polyethylenglycol a pre reaction time of for instance 10 sec is followed by a measurement time of for instance 30 sec. The period of 30 sec is a measurement basis for the antigen determination which is sufficient even for very accurate results.
The determination of diagnostic relevant parameters with antigens in samples, as serum, plasma or liquor, by the immunological or antigen-antibody-reaction, which is evaluated photometrically for instance by nephelometry or turbidimetry, makes frequently a special preparation of both reaction components necessary. Usually the re-action component which contains the sample which antigen content has to be determined, and the other component which contains the antibody, i.e. which is normally the corresponding antiserum, are diluted with physiological salt solution or with physiological salt solution which contains in addition 4~ polyethylenglycol (average molecular weight 6000).
The content of polyethylenglycol serves particularly to avoid a precipitation of the antigen-antibody-complexes formed during the reaction, i.e. to avoid the formation of a precipitation in the test solution formed by the com-bination of both reaction components during the antigen determination, i.e. during the measurement. This property of polyethylenglycol is known for a certain period of time and is a necessity for these measurements, because otherwlse not all formed antigen-antibody-comple~es are subjected to the measurement.
l'he determination of immune globulins by laser turbidimetry according to the "Arbeitsanleitung fur das Lasermed-Turbidometer (pm 4) zur Bestimmung der Immunglobuline IgA, IgG, IgM mit ATAB-Antiseren (Instructions for the Lasermed-turbidometer (pm 4) for determining the immune globulins '~
208S~8 IgA, IgG, IgM with ATAB-anitsera), AHS Deutschland GmbH, Bereich ~lerz und Dade, Munich, 1982", showed that with different physiological salt solutions with 4% by weight polyethylenglycol different measurement signals were obtained with the laser turbidometer during the assay, although they (the salt solutions) were prepared according to the same formulation and with the same reagents by the same person. Accordingly no sufficiently reproducible results were obtained. The so called kinetic method shows a particular interference when according to said instructions after a prereaction of 10 sec. the rate of the antigen-antibody-reaction is measured and the antigen content is determined. In contrast to the so called "Fix point"-method according to said instructions, i.e. the final poin~ measurement after a reaction period of for instance 30 min, it is not necessary with said kinetic method to wait until the end of the completion of the antigen-antibody-reaction.
According to one aspect of the invention there is provided a method for the determination of the antigen content of a sample by an antigen-antibody reaction involving the mixing of a reaction component containing the antigen and a reaction component containing an antibody, comprising first diluting at least one of the two reaction compon-ents with a physiological saline solution which containspolyethyleneglycol and a buffer substance, thereafter combining both reaction components and determining the antigen-antibody reaction photometrically in the pre.sence of a buffer sub~tance, wherein the buffer substance is selected from the group consisting of a glycin, glycyl-glycin, acetate, citrate, bicine, histidin/HCl, tcis or 5.5-dialkylbarbituric acid buffer~
According to another aspect of the invention there is provided a polyethylene containing, physiological salt ` ~2~)85~18 - 2a -solution which can be used as a diluent of at least one of two reaction components of an antigen-antibody-reaction for the photometrical determination of the antigen content, wherein said solution contains a buffer substance selected from the group consisting of a glycin, glycylglycin, acetate, citrate, bicine, histidin/HCl, tris or 5.5-dialkylbarbituric acid buffer.
The invention solves the object to provide a method and a diluent, respectively, with which the antigen content of sample is exactly and reproducibly determinable even with the kinetic method.
The antibody molecule has a polyvalent and therefore a strongly structured form whereas the polyethyleneglycol is non-polar and therefore not structured. When koth reaction components are combined a fairly homogenous partition, which gives fairly reproducible results, is obtained not before a certain period has lapsed. The method according to the invetion is based on the fact that a fast and homogenous partition of both reaction components is obtained by adding a buffer substance to the polyethyleneglycol containing, physiological salt solution, which eliminates the structured form of the antibody molecule by balancing the charges.
~5 - ~20B54B
It is surprising that theirate of the antigen-antibody-reaction is particularly dependent on the chemical composition of the buffer substance, even far more than the concentration of the buffer substance or the pH-value~ Also important is in connection with the invention that not such buffer substances are selected which provide the fastest reaction rate, but such buffer substances are prefered, with which a constant reaction rate in the beginning is obtained which _ lasts for a certain period of time. This property of the buffer substances used according to the invention stra1ghtens the reaction to a certain degree, so that a linear progress over a relatively long period is obtained. This improves the accuracy of the results of the kinetic method considerably.
If for instance 20 mmol/l 5.5-diethylbarbituric acid, pH 7,5, are added as buffer to the physlological salt solution containing 4 % by weight polyethylenglycol a pre reaction time of for instance 10 sec is followed by a measurement time of for instance 30 sec. The period of 30 sec is a measurement basis for the antigen determination which is sufficient even for very accurate results.
Claims (7)
1. A method for the determination of the antigen content of a sample by an antigen-antibody reaction involving the mixing of a reaction component containing the antigen and a reaction component containing an antibody, comprising first diluting at least one of the two reaction compon-ents with a physiological saline solution which contains polyethyleneglycol and a buffer substance, thereafter combining both reaction components and determining the antigen-antibody reaction photometeically in the presence of a buffer substance, wherein the buffer substance is selected from the group consisting of a glycin, glycyl-glycin, acetate, citrate, bicine, histidin/HCl, tris or 5.5-dialkylbarbituric acid buffer.
2. A method according to claim 1, wherein the 5.5-dialkyl-barbituric acid is 5.5-diethylbarbituric acid.
3. A method according to claim 1 wherein the concentration of buffer substance in the salt solution is 5 to 200 mmol/l.
4. A method according to any one of claims 1 to 3, wherein the concentration of the polyethyleneglycol in the salt solution is 2 to 6% by weight, and the average molecular weight of the polyethyleneglycol is 4000 to 12000.
5. A method according to any one of claims 1 to 3, wherein the pH-value of the physiological salt solution containing the buffer substance and the polyethylenelgycol is 4,0 to 9,0.
6. A method according to any one of claims 1 to 3, wherein the photometrical determination of the kinetic progression of the antigen-antibody-reaction is performed.
7. A polyethylene containing, physiological salt solution which can be used as a diluent of at least one of two reaction components of an antigen-antibody-reaction for the photometrical determination of the antigen content, wherein said solution contains a buffer substance selected from the group consisting of a glycin, glycylglycin, acetate, citrate, bicine, histidin/HCl, tris or 5.5-dialkylbarbituric acid buffer.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3212658.1 | 1982-04-05 | ||
| DE19823212658 DE3212658C3 (en) | 1982-04-05 | 1982-04-05 | METHOD FOR DETERMINING ANTIGEN |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1208548A true CA1208548A (en) | 1986-07-29 |
Family
ID=6160289
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000425012A Expired CA1208548A (en) | 1982-04-05 | 1983-03-31 | Method for the determination of antigens |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0090966B1 (en) |
| JP (1) | JPH0650313B2 (en) |
| AT (1) | ATE17049T1 (en) |
| CA (1) | CA1208548A (en) |
| DE (1) | DE3212658C3 (en) |
| ES (1) | ES8402425A1 (en) |
| FI (1) | FI76216C (en) |
| IE (1) | IE54400B1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61113271A (en) * | 1984-11-08 | 1986-05-31 | Matsushita Electronics Corp | Semiconductor memory device |
| JPS62159047A (en) * | 1985-12-31 | 1987-07-15 | Chemo Sero Therapeut Res Inst | Reagent for quantitative determination of plasma protein |
| US5371021A (en) * | 1992-07-17 | 1994-12-06 | Beckman Instruments, Inc. | Initial rate photometric method for immunoassay |
| EP3882631A4 (en) * | 2018-11-09 | 2022-08-24 | Sekisui Medical Co., Ltd. | Method for suppressing abnormal detection in immunoassay by automatic analysis device, and immunoassay reagent |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1540098A (en) * | 1975-03-06 | 1979-02-07 | Baxter Travenol Lab | Immunological reagent and nephelometric method of using same |
| JPS605899B2 (en) * | 1975-04-10 | 1985-02-14 | 中外製薬株式会社 | Immunochemical quantitative method |
| GB1590525A (en) * | 1976-12-10 | 1981-06-03 | Technicon Instr | Biological analysis |
| JPS5399319A (en) * | 1977-02-09 | 1978-08-30 | Hidematsu Hirai | Novel qualitative and quantitative detecting method and detecting body for antgenic substance |
| JPS582660A (en) * | 1981-06-30 | 1983-01-08 | Chugai Pharmaceut Co Ltd | Immunological reagent |
| JPS5847256A (en) * | 1981-09-14 | 1983-03-18 | Mitsubishi Chem Ind Ltd | Measuring method for antigen-antibody reaction |
-
1982
- 1982-04-05 DE DE19823212658 patent/DE3212658C3/en not_active Expired - Lifetime
-
1983
- 1983-03-15 EP EP83102566A patent/EP0090966B1/en not_active Expired
- 1983-03-15 AT AT83102566T patent/ATE17049T1/en not_active IP Right Cessation
- 1983-03-25 JP JP58049111A patent/JPH0650313B2/en not_active Expired - Lifetime
- 1983-03-30 FI FI831095A patent/FI76216C/en not_active IP Right Cessation
- 1983-03-30 ES ES521137A patent/ES8402425A1/en not_active Expired
- 1983-03-31 CA CA000425012A patent/CA1208548A/en not_active Expired
- 1983-03-31 IE IE752/83A patent/IE54400B1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IE54400B1 (en) | 1989-09-27 |
| ES521137A0 (en) | 1984-01-16 |
| JPH0650313B2 (en) | 1994-06-29 |
| DE3212658C3 (en) | 1993-08-19 |
| FI76216C (en) | 1988-09-09 |
| DE3212658A1 (en) | 1983-10-06 |
| ES8402425A1 (en) | 1984-01-16 |
| IE830752L (en) | 1983-10-05 |
| JPS58182558A (en) | 1983-10-25 |
| ATE17049T1 (en) | 1986-01-15 |
| EP0090966A1 (en) | 1983-10-12 |
| DE3212658C2 (en) | 1987-11-26 |
| EP0090966B1 (en) | 1985-12-18 |
| FI76216B (en) | 1988-05-31 |
| FI831095A0 (en) | 1983-03-30 |
| FI831095L (en) | 1983-10-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |