CN111089976A - ELISA method for detecting specific antibody in blood - Google Patents
ELISA method for detecting specific antibody in blood Download PDFInfo
- Publication number
- CN111089976A CN111089976A CN201911376320.6A CN201911376320A CN111089976A CN 111089976 A CN111089976 A CN 111089976A CN 201911376320 A CN201911376320 A CN 201911376320A CN 111089976 A CN111089976 A CN 111089976A
- Authority
- CN
- China
- Prior art keywords
- antibody
- serum
- antibodies
- elisa
- detection
- 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.)
- Pending
Links
- 238000002965 ELISA Methods 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 12
- 210000004369 blood Anatomy 0.000 title claims abstract description 7
- 239000008280 blood Substances 0.000 title claims abstract description 7
- 239000011248 coating agent Substances 0.000 claims abstract description 11
- 238000000576 coating method Methods 0.000 claims abstract description 11
- 230000027455 binding Effects 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- 238000011161 development Methods 0.000 claims abstract description 4
- 230000000903 blocking effect Effects 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims abstract description 3
- 238000001514 detection method Methods 0.000 abstract description 27
- 210000002966 serum Anatomy 0.000 abstract description 24
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 206010020751 Hypersensitivity Diseases 0.000 abstract description 3
- 238000010790 dilution Methods 0.000 abstract description 3
- 239000012895 dilution Substances 0.000 abstract description 3
- 208000026935 allergic disease Diseases 0.000 abstract description 2
- 230000009610 hypersensitivity Effects 0.000 abstract description 2
- 108060003393 Granulin Proteins 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000003302 anti-idiotype Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
Images
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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
Landscapes
- 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)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an ELISA method for detecting specific antibodies in blood. The ELISA method comprises coating, washing, blocking, sample adding, color development and reaction stopping, and is characterized in that the coating is coated with an anti-antibody 1; and adding an enzyme-labeled anti-antibody 2 after the sample is added and before the color development; the anti-antibody 1 and the anti-antibody 2 bind to different binding domains of the specific antibody. Compared with the dilution requirement of the conventional ELISA on a serum sample by hundreds of times, the hypersensitivity ELISA improves the sensitivity of antibody concentration detection by hundreds of times, and can accurately and accurately detect the antibody with the concentration level of 5 nanograms per milliliter.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to an ELISA method for detecting specific antibodies in blood.
Background
The conventional ELISA method is used for detecting the concentration of the antibody in the serum, and an antigen protein specifically recognized by the antibody or a specific anti-antibody is generally used for coating an enzyme label plate and is specifically combined with the antibody in the serum; monoclonal or polyclonal antibodies against human IgG (anti-huIgG) labeled with HRP were used as detection antibodies. Since a large amount of autologous antibodies in serum will also bind to monoclonal or polyclonal antibodies against human IgG (anti-huIgG) to generate false positive signals (i.e., matrix interference), in order to avoid matrix interference of serum, the sample is usually diluted hundreds of times, which greatly reduces the detection sensitivity. The lowest concentration of antibody that can be detected by conventional ELISA methods is typically in the range of hundreds of nanograms per milliliter to micrograms.
Conventional ELISA methods, typically use an antigen protein specifically recognized by an antibody or a specific anti-antibody to coat an ELISA plate, specifically binding to the antibody in serum; monoclonal or polyclonal antibodies against human IgG (anti-huIgG) labeled with HRP were used as detection antibodies. Samples typically need to be diluted hundreds of times, while the minimum detectable concentration of antibodies typically ranges from hundreds of nanograms per milliliter to micrograms.
Matrix interference is the largest factor affecting the sensitivity of antibody concentration detection in serum.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an ELISA method for detecting specific antibodies in blood in order to overcome the defects of low detection sensitivity and the like of antibody concentration in serum in the prior art.
The difficulty of the invention lies in: it is desirable to screen each antibody of interest for a pair of specific but non-competing anti-antibodies (anti-idiotype antibodies), i.e., two anti-antibodies are required to bind to different binding domains of the antibody of interest. Since the specific sequences of antibodies are very limited (restricted to CDR sequences), fewer sequences can be induced to generate specific anti-antibodies. Screening two anti-antibodies that bind to different binding domains to meet the requirements of this assay is therefore a very challenging requirement.
Two anti-antibodies of the present invention, one of which is suitable for coating and still specifically recognizes the target antibody when bound to a solid surface; another anti-antibody is suitable for detection, i.e., it is required that the HRP-labeled antibody retains a high specific binding. The sandwich ELISA formed by matching the two can effectively eliminate the interference of matrix protein in serum and accurately detect the antibody with ultra-low concentration (ng/mL) in the serum.
The invention solves the technical problems through the following technical scheme: an ELISA method for detecting specific antibodies in blood, the method comprising coating, washing, blocking, loading, developing and terminating a reaction, the coating being coated with anti-antibody 1; and adding an enzyme-labeled anti-antibody 2 after the sample is added and before the color development; the anti-antibody 1 and the anti-antibody 2 bind to different binding domains of the specific antibody.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the hypersensitive ELISA adopts a sandwich structure of bispecific anti-antibodies, the coating antibody and the detection antibody both adopt the specific anti-antibodies, and the dual specificity recognition of the coating antibody and the detection antibody on the antibodies is utilized, so that the matrix interference is effectively avoided, and the interference of the humanized antibodies in serum at least up to 20 percent can be tolerated, thereby greatly reducing the dilution multiple of samples and improving the detection sensitivity. Compared with the dilution requirement of the conventional ELISA on a serum sample by hundreds of times, the hypersensitivity ELISA improves the sensitivity of antibody concentration detection by hundreds of times, and can accurately and accurately detect the antibody with the concentration level of 5 nanograms per milliliter. The interference of matrix protein in serum is eliminated, so that the detection method is suitable for detecting low-concentration antibodies in serum, and the accuracy of a detection result is ensured.
Drawings
FIG. 1 is a serum sample concentration response curve for Ab 1.
Fig. 2 is a serum sample concentration response curve for Ab 2.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1
The detection of two specific monoclonal antibodies, Ab1 and Ab2 is taken as an example to prove the strong interference capability against matrix protein and the ultrahigh sensitivity of the detection method.
Ab1 and Ab2 are respectively coated with anti-Ab1a or anti-Aba2 anti-antibody, and are respectively diluted in PBS containing 1%, 10% and 20% of serum in a gradient manner, PBS containing no serum is used as a control, and then anti-antibody of anti-Ab1b or anti-Ab2b marked by HRP is used as a detection antibody. The absorbance was read on a microplate reader at 450/650nm and the concentration-effect curve was fitted according to a four parameter logistic model.
As shown in fig. 1 and fig. 2, the detection method can tolerate all the detected serum concentrations for two specific monoclonal antibodies, and even in up to 20% of the serum, the highest asymptotic property and the lowest asymptotic property of the sample detection curve are hardly different from those of the sample diluted in PBS: that is, high concentrations of serum matrix protein neither raise the detection background (lowest asymptotic) nor reduce the detection sensitivity of saturating target antibodies (highest asymptotic). Because the detection method effectively avoids the interference of matrix protein in serum, the sensitivity of the detection of the low-concentration antibody in the serum is greatly improved, the minimum effective detection concentration is 1ng/mL, the minimum antibody detection concentration in the serum after conversion can reach 5.0ng/mL, and the detection method is improved by nearly 100 times compared with the conventional detection method.
Claims (1)
1. An ELISA method for detecting specific antibodies in blood, comprising coating, washing, blocking, loading, developing and terminating reactions, characterized in that the coating is a coating of anti-antibody 1; and adding an enzyme-labeled anti-antibody 2 after the sample is added and before the color development; the anti-antibody 1 and the anti-antibody 2 bind to different binding domains of the specific antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911376320.6A CN111089976A (en) | 2019-12-27 | 2019-12-27 | ELISA method for detecting specific antibody in blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911376320.6A CN111089976A (en) | 2019-12-27 | 2019-12-27 | ELISA method for detecting specific antibody in blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111089976A true CN111089976A (en) | 2020-05-01 |
Family
ID=70398353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911376320.6A Pending CN111089976A (en) | 2019-12-27 | 2019-12-27 | ELISA method for detecting specific antibody in blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111089976A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923093A (en) * | 2010-07-13 | 2010-12-22 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN103197077A (en) * | 2013-03-20 | 2013-07-10 | 郑州伊美诺生物技术有限公司 | Assay kit for detecting trace bovine immunoglobulin G |
| CN104034896A (en) * | 2014-06-19 | 2014-09-10 | 华北制药集团新药研究开发有限责任公司 | Double-antibody sandwiched ELISA (enzyme-linked immunosorbent assay) detection method |
-
2019
- 2019-12-27 CN CN201911376320.6A patent/CN111089976A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923093A (en) * | 2010-07-13 | 2010-12-22 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN103197077A (en) * | 2013-03-20 | 2013-07-10 | 郑州伊美诺生物技术有限公司 | Assay kit for detecting trace bovine immunoglobulin G |
| CN104034896A (en) * | 2014-06-19 | 2014-09-10 | 华北制药集团新药研究开发有限责任公司 | Double-antibody sandwiched ELISA (enzyme-linked immunosorbent assay) detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hayrapetyan et al. | Enzyme-linked immunosorbent assay: types and applications | |
| US20160161480A1 (en) | Method for the detection of the prozone effect of photometric assays | |
| Zhang et al. | Development of a new broad-specific monoclonal antibody with uniform affinity for aflatoxins and magnetic beads-based enzymatic immunoassay | |
| CA2794721A1 (en) | Assay utilizing immunochromatography, immunochromatographic test strip, and assay reagent kit for immunochromatography | |
| O'Kennedy et al. | Don't blame it all on antibodies–The need for exhaustive characterisation, appropriate handling, and addressing the issues that affect specificity | |
| US8334103B2 (en) | Composition related to rapid ELISA process | |
| JP2022043219A (en) | Monoclonal antibody to react with glycopeptide and use therefor | |
| JP7141405B2 (en) | Analyte detection immunoassay | |
| US9081016B2 (en) | Assay for measurement of antibodies binding to a therapeutic monoclonal antibody | |
| US20220308067A1 (en) | Applications of serum s100a8/a9 complex level in prognosis assessment of acute myocardial infarction | |
| JP2020506395A5 (en) | ||
| JP5380303B2 (en) | High molecular adiponectin assay | |
| CN111868527B (en) | Kit, assay kit and assay method | |
| CN111089976A (en) | ELISA method for detecting specific antibody in blood | |
| EP4286850A1 (en) | Immunological assay method | |
| WO2020067233A1 (en) | Test piece for immunochromatography | |
| Hall et al. | An extended range generic immunoassay for total human therapeutic antibodies in preclinical pharmacokinetic studies | |
| US10479827B2 (en) | Monoclonal antibody reacting with glycopeptide, and use thereof | |
| US11320427B2 (en) | Tandemly repeated antibody-binding protein and its applications | |
| Son et al. | Strategies for the optimization of bead-immunoassays for the effective detection of target biomolecules | |
| US20180164316A1 (en) | Test object detection method, and immunoassay instrument and monoclonal antibody for same | |
| AU2020101515A4 (en) | A kit for preparation of a reagent for diagnosis of pancreatic ductal adenocarcinoma | |
| JP2020051910A (en) | Immunochromatography test piece | |
| WO2024185720A1 (en) | Antibody or antigen-binding fragment of same | |
| Shrivastav et al. | Development of enzyme-linked immunosorbent assay for estimation of urinary albumin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200501 |