CN105181961B - Method for determining antibody titer by using antigen single immunodiffusion - Google Patents
Method for determining antibody titer by using antigen single immunodiffusion Download PDFInfo
- Publication number
- CN105181961B CN105181961B CN201510540046.7A CN201510540046A CN105181961B CN 105181961 B CN105181961 B CN 105181961B CN 201510540046 A CN201510540046 A CN 201510540046A CN 105181961 B CN105181961 B CN 105181961B
- Authority
- CN
- China
- Prior art keywords
- glass tube
- antigen
- antibody titer
- glass
- volume
- 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.)
- Active
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 51
- 102000036639 antigens Human genes 0.000 title claims abstract description 51
- 108091007433 antigens Proteins 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000000951 immunodiffusion Effects 0.000 title claims abstract description 13
- 239000011521 glass Substances 0.000 claims abstract description 68
- 238000001114 immunoprecipitation Methods 0.000 claims abstract description 15
- 229920000936 Agarose Polymers 0.000 claims description 34
- 230000002745 absorbent Effects 0.000 claims description 5
- 239000002250 absorbent Substances 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 230000008023 solidification Effects 0.000 claims description 5
- 238000007711 solidification Methods 0.000 claims description 5
- 238000005259 measurement Methods 0.000 abstract description 8
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000009792 diffusion process Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000010977 jade Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
Abstract
The invention belongs to the technology field of a method for measuring antibody titer, and concretely relates to a method for determining antibody titer by using an antigen single immunodiffusion. The invention mainly solves the technology problems that according to the present determination method of antibody titer, the measurement results have deviations and the measurement process is complicated. According to the invention, the central single radial immunodiffusion used in the present determination method is restrained into a vertical radial single immunodiffusion in a calibrated glass tube, so that the Becker method deviation is eliminated, and the antibody titer is rapidly and accurately measured; a immunoprecipitation zone volume formed by single immunodiffusion can be directly read out from the scale on the calibrated glass tube, and thickness and diameter are not needed to be measured, so the result can be obtained more rapidly and accurately.
Description
Technical field
The invention belongs to measure the method and technology field of antibody titer, and in particular to a kind of to be surveyed using antigen single radial immunodiffusion
The method for determining antibody titer.
Background technology
Antibody protein is the class defense macromolecular of higher mammal wide expression, is capable of identify that pathogen (antigen molecule)
And it is in combination.The assay method of existing antibody titer (potency) is based on antibody and the affine characteristic of antigen.Including ELISA method,
Agarose double-diffusion process, Becker methods and immunoturbidimetry.Nineteen sixty-eight Becker delivered it is a kind of based on antigen on agarose with
Antibody mediated immunity list diffuses to form the principle of immunoprecipitation complex and then the method for determining antibody titer.The method is made known to quality
Antigen on the agarose plate containing unknown titer antibody uni-directional diffusion, make to be formed immune complex precipitation, by determining
Settling zone size carrys out calculating antibody titre.The antibody titer unit that Becker methods are determined is per milliliter of milligram, compared to most
Big extension rate represents that the ELISA of titre (potency) and agarose double diffusion titre are more accurate, and surveys antibody titer with respect to which
His antibody titer assay method is easy to operate, is independent of precision equipment.But becker methods need by measure deposit ring diameter and
Agarose thickness is being calculated antibody titer.And the Ago-Gel of semi-solid property, its thickness measure relative difficulty；
When determining because the dynamic dispersal direction of antigenic solution heterogeneity, diffusion circle diameter measurement also has deviation, leads Becker methods
Cause the precipitation ring area error according to measurement result calculating larger.
The content of the invention
Present invention aim to address the assay method presence of existing antibody titer is easily caused measurement result and there is deviation, survey
The loaded down with trivial details technical problem of amount process, there is provided a kind of method that utilization antigen single radial immunodiffusion determines antibody titer.
For solving above-mentioned technical problem, the technical solution used in the present invention is：
A kind of method that utilization antigen single radial immunodiffusion determines antibody titer, comprises the following steps：
1) one end for having graduated glass tube is sealed with gummed paper；
2) the agarose melt solution containing test antibodies is irrigated in glass tube；
3) after the solidification of agarose melt solution, the gummed paper of glass tube one end sealing is opened, glass tube is upright in
Bottom is placed with the base of absorbent material；
4) 0.01～0.05 milliliter of concentration known antigen is added in the upper end of the glass tube erect；
5) upper end of gummed paper sealed glass tube is used after adding antigen, prevents solution from evaporating from upper end；Then glass tube is put
8 hours are incubated in 37 DEG C of insulating box, so that antigen is diffused into inside Ago-Gel completely；
6), after antigen is spread in the agarose column containing antibody completely, form milky immunoprecipitation area；
7) antibody titer is calculated by following equation：
Wherein：C adds the concentration of the antigen in glass tube, V1 to add the antigen volume in glass tube, V2 glass
Milky immunoprecipitation area volume in pipe, the test antibodies volume added in agarose melt solution body in V3 glass tubes,
V4 glass tubes are contained within the agarose melt solution volume of test antibodies.
The present invention adopts above technical scheme, based on single immunediffusion principle, by the center used in existing assay method
Radial single diffusion, is constrained to single vertical radial direction list diffusion in graduated glass pipe, to eliminate Becker method deviations, more accelerates
Fast accurately measurement antibody titer.The method can directly be read the immunity for singly diffuseing to form and be sunk by the scale on graduated glass pipe
Shallow lake area volume, without the need for passing through thickness and diameter measurement, obtains result more quick and accurate.Additionally, the present invention can also be by
The antigen dissolution of concentration is known in Ago-Gel, the antibody of unknown concentration is spread in gel.Therefore, with background technology phase
Than the present invention has the simple to operate and accurate advantage of measurement.
Specific embodiment
Embodiment 1
The method that a kind of utilization antigen single radial immunodiffusion in the present embodiment determines antibody titer, comprises the following steps：
1) the one end for having graduated glass tube, glass bore 4mm, external diameter 5.5mm are sealed with gummed paper；
2) the agarose melt solution containing test antibodies is irrigated in glass tube, the melting of the agarose containing test antibodies
The volume of solution is 5 milliliters, and the test antibodies volume added in agarose melt solution body is 125 microlitres；
3) after the solidification of agarose melt solution, the gummed paper of glass tube one end sealing is opened, glass tube is upright in
Bottom is placed with the base of absorbent material；
4) 0.01 milliliter of concentration known antigen is added in the upper end of the glass tube erect, and the concentration for adding antigen is 3mg/
ml；
5) upper end of gummed paper sealed glass tube is used after adding antigen, prevents solution from evaporating from upper end；Then glass tube is put
8 hours are incubated in 37 DEG C of insulating box, so that antigen is diffused into inside Ago-Gel completely；
6), after antigen is spread in the agarose column containing antibody completely, milky immunoprecipitation area, milky are formed
The volume in immunoprecipitation area is 0.75ml；
7) antibody titer is calculated by following equation：
Wherein：C adds the concentration of the antigen in glass tube, V1 to add the antigen volume in glass tube, V2 glass
Milky immunoprecipitation area volume in pipe, the test antibodies volume added in agarose melt solution body in V3 glass tubes,
V4 glass tubes are contained within the agarose melt solution volume of test antibodies,
It is 1.6mg/ml that antibody titer is calculated by above-mentioned formula, that is, represent per milliliter of antibody and can neutralize 1.6mg
Corresponding antigens.
Embodiment 2
The method that a kind of utilization antigen single radial immunodiffusion in the present embodiment determines antibody titer, comprises the following steps：
1) the one end for having graduated glass tube, glass bore 4mm, external diameter 5.5mm are sealed with gummed paper；
2) to step 1) glass tube in irrigate the agarose melt solution containing test antibodies, the fine jade containing test antibodies
The volume of lipolysaccharide melt solution is 5 milliliters, and the test antibodies volume added in agarose melt solution body is 300 microlitres；
3) after the solidification of agarose melt solution, the gummed paper of glass tube one end sealing is opened, glass tube is upright in
Bottom is placed with the base of absorbent material；
4) 0.05 milliliter of concentration known antigen is added in the upper end of the glass tube erect, and the concentration for adding antigen is 3mg/
ml；
5) upper end of gummed paper sealed glass tube is used after adding antigen, prevents solution from evaporating from upper end；Then glass tube is put
8 hours are incubated in 37 DEG C of insulating box, so that antigen is diffused into inside Ago-Gel completely；
6), after antigen is spread in the agarose column containing antibody completely, milky immunoprecipitation area, milky are formed
Immunoprecipitation area volume is 1.60ml；
7) antibody titer is calculated by following equation：
Wherein：C adds the concentration of the antigen in glass tube, V1 to add the antigen volume in glass tube, V2 glass
Milky immunoprecipitation area volume in pipe, the test antibodies volume added in agarose melt solution body in V3 glass tubes,
V4 glass tubes are contained within the agarose melt solution volume of test antibodies,
It is 1.56mg/ml that antibody titer is calculated by above-mentioned formula, that is, representing per milliliter of antibody can neutralize
1.56mg corresponding antigens.
Embodiment 3
The method that a kind of utilization antigen single radial immunodiffusion in the present embodiment determines antibody titer, comprises the following steps：
1) the one end for having graduated glass tube, glass bore 4mm, external diameter 5.5mm are sealed with gummed paper；
2) to step 1) glass tube in irrigate the agarose melt solution containing test antibodies, the fine jade containing test antibodies
The volume of lipolysaccharide melt solution is 4.8 milliliters, and the test antibodies volume added in agarose melt solution body is 240 microlitres；
3) after the solidification of agarose melt solution, the gummed paper of glass tube one end sealing is opened, glass tube is upright in
Bottom is placed with the base of absorbent material；
4) in the concentration known antigen of 0.03 milliliter of the upper end addition of the glass tube erect, the concentration for adding antigen is
15mg/ml；
5) upper end of gummed paper sealed glass tube is used after adding antigen, prevents solution from evaporating from upper end；Then glass tube is put
8 hours are incubated in 37 DEG C of insulating box, so that antigen is diffused into inside Ago-Gel completely；
6), after antigen is spread in the agarose column containing antibody completely, milky immunoprecipitation area, milky are formed
Immunoprecipitation area volume is 1.27ml；
7) antibody titer is calculated by following equation：
Wherein：C adds the concentration of the antigen in glass tube, V1 to add the antigen volume in glass tube, V2 glass
Milky immunoprecipitation area volume in pipe, the test antibodies volume added in agarose melt solution body in V3 glass tubes,
V4 glass tubes are contained within the agarose melt solution volume of test antibodies,
It is 7.09mg/ml that antibody titer is calculated by above-mentioned formula, that is, representing per milliliter of antibody can neutralize
7.09mg corresponding antigens.
Claims (1)
1. a kind of method that utilization antigen single radial immunodiffusion determines antibody titer, it is characterised in that：Comprise the following steps：
1) the one end for having graduated glass tube, glass bore 4mm, external diameter 5.5mm are sealed with gummed paper；
2) the agarose melt solution containing test antibodies is irrigated in glass tube；
3) after the solidification of agarose melt solution, the gummed paper of glass tube one end sealing is opened, glass tube is upright in into bottom
It is placed with the base of absorbent material；
4) 0.01～0.05 milliliter of concentration known antigen is added in the upper end of the glass tube erect；
5) upper end of gummed paper sealed glass tube is used after adding antigen, prevents solution from evaporating from upper end；Then glass tube is placed in into 37
DEG C insulating box in be incubated 8 hours so that antigen is diffused into inside Ago-Gel completely；
6), after antigen is spread in the agarose column containing antibody completely, form milky immunoprecipitation area；
7) antibody titer is calculated by following equation：
Wherein：C adds the concentration of the antigen in glass tube, and V1 adds the antigen volume in glass tube, in V2 glass tubes
Milky immunoprecipitation area volume, the test antibodies volume added in agarose melt solution in V3 glass tubes, V4 glass
Pipe is contained within the agarose melt solution volume of test antibodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510540046.7A CN105181961B (en) | 2015-08-28 | 2015-08-28 | Method for determining antibody titer by using antigen single immunodiffusion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510540046.7A CN105181961B (en) | 2015-08-28 | 2015-08-28 | Method for determining antibody titer by using antigen single immunodiffusion |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105181961A CN105181961A (en) | 2015-12-23 |
| CN105181961B true CN105181961B (en) | 2017-03-22 |
Family
ID=54904183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510540046.7A Active CN105181961B (en) | 2015-08-28 | 2015-08-28 | Method for determining antibody titer by using antigen single immunodiffusion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105181961B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4162208A (en) * | 1975-02-03 | 1979-07-24 | Aladjem Frederick J | Quantitative protein analysis by immunodiffusion |
| CN2102721U (en) * | 1991-07-02 | 1992-04-29 | 衡阳市第二医院 | Immunodiffusion rocket electrophoresis test plate |
| WO2003062825A1 (en) * | 2002-01-25 | 2003-07-31 | Forskarpatent I Syd Ab | DIAGNOSTIC TEST METHOD FOR DETECTION OF THE GM ALLOTYPE G2m(n) |
| CN104302318A (en) * | 2012-03-23 | 2015-01-21 | 葛兰素史密丝克莱恩生物有限公司 | Influenza vaccines |
-
2015
- 2015-08-28 CN CN201510540046.7A patent/CN105181961B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4162208A (en) * | 1975-02-03 | 1979-07-24 | Aladjem Frederick J | Quantitative protein analysis by immunodiffusion |
| CN2102721U (en) * | 1991-07-02 | 1992-04-29 | 衡阳市第二医院 | Immunodiffusion rocket electrophoresis test plate |
| WO2003062825A1 (en) * | 2002-01-25 | 2003-07-31 | Forskarpatent I Syd Ab | DIAGNOSTIC TEST METHOD FOR DETECTION OF THE GM ALLOTYPE G2m(n) |
| CN104302318A (en) * | 2012-03-23 | 2015-01-21 | 葛兰素史密丝克莱恩生物有限公司 | Influenza vaccines |
Non-Patent Citations (3)
| Title |
|---|
| Immunoaffinity Isolation and Partial Characterization of the Coccidioides immitis Antigen Detected by the Tube Precipitin and Immunodiffusion-Tube Precipitin Tests;BARBARA L. ZIMMER AND DEMOSTHENES PAPPAGIANIS;《JOURNAL OF CLINICAL MICROBIOLOGY》;19890831;第27卷(第8期);1759-1766 * |
| 改良单向免疫扩散法检测胃液SIgA;王静芝 等;《临床检验杂志》;19920630;第10卷(第2期);92 * |
| 改进的单向免疫扩散法测定正常人及病人尿中4种微量尿蛋白;周永华;《重庆医学》;19970831;第26卷(第4期);208 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105181961A (en) | 2015-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104655751B (en) | A kind of detect the method for organic solvent residual in dapoxetine | |
| CN105092754A (en) | Method for determining sulfonate genotoxic impurity by using HPLC | |
| CN105372337B (en) | A kind of method for detecting vitamin D content in vitamin D drops | |
| CN104502246A (en) | Combined type settlement column and method for researching settlement characteristics by using combined type settlement column | |
| CN105223282A (en) | A kind of Gradient High Performance Liquid Chromatography measures the method for Abiraterone acetate related substance | |
| CN105181961B (en) | Method for determining antibody titer by using antigen single immunodiffusion | |
| WO2023040391A1 (en) | Method for determining content of ethanol in wines on basis of gas chromatographic method | |
| CN104569276A (en) | Method for measuring related substances of Sofosbuvir tablet by using HPLC | |
| CN109212187A (en) | It is a kind of detect polycyclic aromatic hydrocarbon class enzyme linked immunological kit and its application | |
| CN105527353B (en) | A kind of method that utilization tracer headspace gas chromatography determines organic solvent solubility | |
| CN105758972A (en) | Method for determining related substances in paliperidone extended-release tablet | |
| CN104502491B (en) | A kind of headspace sampling measures the method for crude drug oxfendazole dissolvent residual | |
| CN105572240A (en) | Method for detecting content of pharmaceutic adjuvant carmine by using high performance liquid chromatography | |
| CN102636597A (en) | Method for measuring residual solvent in tetracycline hydrochloride bulk drug by utilizing headspace gas chromatography | |
| CN103926365A (en) | Method for detecting ophiopogonin D and ophiopogonin D' in pulse-activating injection | |
| CN106770713A (en) | Residual ethanol, toluene assay method in a kind of Pentoxyverine | |
| CN107238669A (en) | A kind of detection method infused with residual solvent levels in high-barrier outer bag | |
| CN108732281B (en) | Method for measuring content of hydroxymethylfurfural in honey | |
| CN106483205A (en) | A kind of method that employing high performance liquid chromatography detects pharmaceutic adjuvant carmine content | |
| CN107233873B (en) | There is the preparation method of the solid-phase micro-extraction fibre of specificity to sulfa drugs | |
| CN105388222B (en) | A kind of high performance liquid chromatography determines borneol, camphor, the method for isoborneol content simultaneously | |
| CN105954431B (en) | A kind of method of the HPLC separation determination Ao Pei meter Fen bulk pharmaceutical chemicals in relation to substance | |
| CN105067743B (en) | A kind of ethyl chloride method in gas chromatography determination chlortetracycline hydrochloride | |
| CN105842352A (en) | Simple method for determination of content of sodium caprylate in human albumin through GC method | |
| CN105628807B (en) | A kind of quality determining method of the amino piperidines of 1 Boc 4 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180614 Address after: 048100 5, 74, crow rock bottom, Fengshui Town, Fengcheng County, Yangcheng County, Shanxi, Jincheng Patentee after: SHANXI REALLY BIOTECHNOLOGY CO.,LTD. Address before: 048100 No. 74, Yan Yan Di, Fengshui Town, Fengcheng Town, Yangcheng County, Shanxi, Jincheng Patentee before: REALLY TECH CO.,LTD. |
|
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Hongyan Inventor after: Su Shaobo Inventor after: Li Guangfei Inventor before: Wu Hongyan Inventor before: Su Shaobo Inventor before: Li Guangfei |