CN109991428A - A kind of double antigens sandwich indirect method by Seal treatment - Google Patents
A kind of double antigens sandwich indirect method by Seal treatment Download PDFInfo
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- CN109991428A CN109991428A CN201910283168.0A CN201910283168A CN109991428A CN 109991428 A CN109991428 A CN 109991428A CN 201910283168 A CN201910283168 A CN 201910283168A CN 109991428 A CN109991428 A CN 109991428A
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- 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
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- 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
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Abstract
The present invention discloses the double antigens sandwich indirect method by Seal treatment, the following steps are included: 1. the walks: the antibody identification object A being coupled on closing magnetic bead and enzyme target antibody identification object B being added into detection antibody solution, carries out Magneto separate after mixing reaction and removes supernatant；2. the walks: 1. walking the enzyme-linked object of large biological molecule that identification detection antibody is added in isolated closing magnetic bead to the, carry out Magneto separate after mixing reaction and remove supernatant；3. the walks: 2. walking to the and substrate is added in isolated closing magnetic bead carries out the detection that shines.The present invention is associated with antibody by antithesis and identifies that the magnetic bead of object A carries out Seal treatment, both it had solved the problems, such as that simple dual-antigen sandwich method sensitivity was relatively low, weakly positive sample missing inspection, while also having solved the problems, such as that the distribution of bring negative sample range of signal is wide, shake is excessive, the coefficient of variation is excessive or even false positive results occurs after sandwich method is combined with indirect method.Not only it ensure that high detection specificity, but also be able to satisfy high detection sensitivity.
Description
Technical field
The present invention relates to the technical fields of antibody test, more particularly, are related to a kind of dual anti-original by Seal treatment
Sandwich indirect method.
Background technique
Traditional chemoluminescence method detection antibody generallys use dual-antigen sandwich method principle.In order to meet detection as much as possible
The specificity of antibody, while meeting certain detection sensitivity, diagnostic reagent developers would generally use dual-antigen sandwich method
Principle realizes the purpose of detection antibody.Most of producers use this reaction principle and detect syphilis helicoid antibody on the market, but
The phenomenon that single dual-antigen sandwich method detection sensitivity is low, is easy to appear weakly positive sample missing inspection, to clinical diagnosis and treatment
Bring serious puzzlement.
Since there are two solid phase antigens or two tracer labelled antigens to be integrated to the same antibody for dual-antigen sandwich method
And lead to not detected situation, there is false sun or false problem that is negative and leading to testing result inaccuracy.Then occurring will
The method of sandwich method and indirect method combine detection antibody, but this method will appear what positive sample detected signal value was substantially increased
As a result, there is also with this result, the distribution of negative sample range of signal is wide, shake is excessive, the coefficient of variation is excessive or even goes out
The problem of existing false positive results.
Summary of the invention
Occur in method in order to solve sandwich method in the prior art and indirect method combine detection syphilis helicoid antibody
Positive sample detected signal value be substantially increased with negative sample range of signal be distributed it is wide, shake is excessive, the coefficient of variation is excessive very
To there is the problems such as false positive results, the present invention provides a kind of double antigens sandwich indirect methods by Seal treatment.
The present invention provides a kind of double antigens sandwich indirect methods by Seal treatment, comprising the following steps:
1. the walks: the antibody identification object A being coupled on closing magnetic bead being added into detection antibody solution and enzyme target is anti-
Body identifies object B, carries out Magneto separate after mixing reaction and removes supernatant；
2. the walks: 1. walked to the be added in isolated closing magnetic bead identification detection antibody large biological molecule it is enzyme-linked
Object carries out Magneto separate after mixing reaction and removes supernatant；
3. the walks: 2. walking to the and substrate is added in isolated closing magnetic bead carries out the detection that shines.
According to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment, the envelope of the closing magnetic bead
Closing processing is one of serum closing, BSA closing, gelatin closing and skimmed milk power closing.
According to the present invention by Seal treatment double antigens sandwich indirect method one embodiment, the closing magnetic bead with resist
Body identifies that the mode of object A coupling is inhaled for biotin-avidin coupling, biotin-Streptavidin coupling, chemical crosslinking, physics
Echo one of Electrostatic Absorption.
It is described to be coupled at closing magnetic according to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment
Antibody identification object A and enzyme target antibody identification object B on pearl are that identical antibody identifies that object or different antibody identify object.
It is described to be coupled at closing magnetic according to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment
Antibody identification object A and enzyme target antibody identification object B on pearl are a part of antigen, peptide chain or antigen.
According to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment, the identification is to be detected anti-
The large biological molecule of body be mouse anti-human IgG antibodies, goat anti-human igg antibody, sheep anti-mouse igg antibody, the anti-sheep IgG antibody of mouse,
One of Protein A and Protein G.
According to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment, the identification is to be detected anti-
The enzyme-linked object of the large biological molecule of body is by the large biological molecule conjugated alkaline phosphatase or horseradish peroxide for identifying detection antibody
Compound enzyme obtains.
According to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment, the enzyme target antibody is known
Other object B identifies that object B conjugated alkaline phosphatase or horseradish peroxidase obtain by antibody, and the substrate is alkaline phosphatase or peppery
The substrate of root peroxidase.
According to the present invention by one embodiment of the double antigens sandwich indirect method of Seal treatment, the detection antibody is
Syphilis helicoid antibody.
Compared with prior art, the present invention is associated with antibody by antithesis and identifies that the magnetic bead of object 1 carries out Seal treatment, both solves
The problem of simple dual-antigen sandwich method sensitivity is relatively low, weakly positive sample missing inspection, while also solving sandwich method and indirect method
The distribution of bring negative sample range of signal is wide after combination, shake is excessive, the coefficient of variation is excessive or even false positive results occurs
The problem of.Not only it ensure that high detection specificity, but also be able to satisfy high detection sensitivity.
Specific embodiment
All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive
Feature and/or step other than, can combine in any way.
Any feature disclosed in this specification unless specifically stated can be equivalent or with similar purpose by other
Alternative features are replaced.That is, unless specifically stated, each feature is an example in a series of equivalent or similar characteristics
?.
First the double antigens sandwich indirect method of the invention by Seal treatment is specifically described and is illustrated below.
An exemplary embodiment of the present invention, the double antigens sandwich indirect method by Seal treatment includes following step
Suddenly.
1. the walks:
The antibody identification object A being coupled on closing magnetic bead and enzyme target antibody identification object are added into detection antibody solution
B carries out Magneto separate after mixing reaction and removes supernatant.
Wherein, the Seal treatment for closing magnetic bead can be serum closing, BSA closing, gelatin closing and skimmed milk power closing
One of, the mode of closing magnetic bead and antibody identification object A coupling can be biotin-avidin coupling, biotin-strepto- parent
With one of element coupling, chemical crosslinking, physical absorption and Electrostatic Absorption.For chemical crosslinking, can for utilization-COOH with-
NH4, FITC and anti-FITC antibody etc. chemical crosslinking.
Since the testing result of negative sample false positive occurs in unclosed sandwich indirect method, there is false positive in analysis
The reason is that the IgG in human serum can non-specifically be adsorbed to magnetic bead surfaces, the conjugate of magnetic bead and human IgG is formed, when addition enzyme
When the anti-human IgG antibodies of label, immune complex will be formed in conjunction with the human IgG that magnetic bead adsorbs, catalysis substrate, which generates, to shine
There is the testing result of false positive in signal.Then inventor carries out closed strategy in advance to magnetic bead using human serum etc., makes one
IgG in serum is adsorbed to magnetic bead surfaces in advance and occupies the gap and position of magnetic bead surfaces, just not when detecting serum sample
There can be additional IgG absorption again, will not be generated again thus, it is possible to raise basis signal value in advance, in reaction process non-specific
False positive signal value avoids the occurrence of negative sample false positive measurement result.
According to the present invention, the antibody identification object A and enzyme target antibody identification object B being coupled on closing magnetic bead can be identical
Antibody identify object, or different antibody identifies object.Specifically, be coupled at closing magnetic bead on antibody identification object A and
Enzyme target antibody identification object B is preferably a part of antigen, peptide chain or antigen.
Wherein, enzyme target antibody identification object B preferably identifies object B conjugated alkaline phosphatase or horseradish peroxidase by antibody
Enzyme obtains, and specifically, enzyme target enzyme mainly can carry out chemiluminescent two kinds of enzymes, main at present including alkaline phosphatase and peppery
Root peroxidase, substrate used is also the corresponding substrate of both enzymes later.
By the processing of this step, detection antibody can identify object A and enzyme mark with the antibody being coupled on closing magnetic bead
Antibody identification object B combine form double antibodies sandwich structure.
2. the walks:
The enzyme-linked object of large biological molecule that identification detection antibody is added in isolated closing magnetic bead is 1. walked to the, is mixed
Magneto separate is carried out after reaction and removes supernatant.
In the present invention identify detection antibody large biological molecule can for mouse anti-human IgG antibodies, goat anti-human igg antibody,
One of the anti-sheep IgG antibody of sheep anti-mouse igg antibody, mouse, Protein A and Protein G.And identify the life of detection antibody
The enzyme-linked object of object macromolecular is then by the large biological molecule conjugated alkaline phosphatase or horseradish peroxidase of above-mentioned identification detection antibody
Enzyme obtains.
After this step process, the enzyme-linked object of large biological molecule of the identification detection antibody of addition can be allowed to and magnetic bead
On antibody identification object, detection antibody form immune complex, catalysis substrate shines and improves detection sensitivity.
3. the walks:
It is 2. walked to the and substrate is added in isolated closing magnetic bead carries out the detection that shines.
Wherein, the substrate being added in this step is the substrate of alkaline phosphatase or horseradish peroxidase, according to enzyme target
Difference is selected.
In this step, the alkaline phosphorus being coupled in the enzyme-linked object of large biological molecule of the substrate of addition and identification detection antibody
Sour enzyme reaction generates coloured enzymolysis product, obtains the testing result of detection antibody to the detection of luminous quantity by instrument.
Method of the invention is particularly suitable for the detection of syphilis helicoid antibody.
The present invention is further explained in the light of specific embodiments.
Embodiment 1:
Syphilis helicoid antibody (anti-TP) is detected by the double antigens sandwich indirect method of Seal treatment using the present invention.
Anti-TP reagent comes from Chengdu Bo Aoxin scape medical science and technology Co., Ltd.Reagent components include: to be detected
Anti-TP antibody is coupled at using TP antigen, enzyme target TP antigen, the identification anti-to be detected on the closed magnetic bead of human serum
The enzyme-linked object of mouse anti-human IgG antibodies of TP antibody.Wherein, the enzyme in enzyme target TP antigen is alkaline phosphatase, is identified to be detected
Enzyme in the enzyme-linked object of mouse anti-human IgG antibodies of anti-TP antibody is alkaline phosphatase.
Close the Seal treatment of magnetic bead are as follows: 1. take out the magnetic bead solution for being coupled TP antigen that volume is V；2. carrying out magnetic
It is attached, remove supernatant, the human serum sample of V/10 volume is added；3. room temperature mixes closing 30 minutes on horizontal blending instrument；④
After carrying out magnetic suck, remove supernatant, is cleaned three times using the magnetic bead buffer solution of V/10 volume；5. finally using magnetic bead buffer solution
The magnetic bead closed is settled to V volume again.So far, the Seal treatment step of magnetic bead is completed.Close magnetic bead and TP antigen
Coupling mode be the coupling of biotin-Streptavidin.
Specific step is as follows for detection:
1. the walks: being added and be coupled at using the TP on the closed magnetic bead of human serum into anti-TP antibody-solutions to be detected
Antigen and enzyme target TP antigen carry out Magneto separate after mixing reaction and remove supernatant.
2. the walks: the enzyme-linked object of mouse anti-human IgG antibodies for identifying anti-TP antibody to be detected being added into magnetic bead, mixes anti-
Should after carry out Magneto separate and remove supernatant.
3. the walks: the substrate of addition is APS-5, passes through chemical illumination immunity analysis instrument and carries out result detection.
Testing result is as shown in table 1 below.
Comparative example 1:
Syphilis helicoid antibody (anti-TP) is detected using without the double antigens sandwich indirect method of Seal treatment.
Anti-TP reagent comes from Chengdu Bo Aoxin scape medical science and technology Co., Ltd.Reagent components include: to be detected
Anti-TP antibody, the TP antigen being coupled on magnetic bead, enzyme target TP antigen, the mouse of identification anti-TP antibody to be detected are anti-human
The enzyme-linked object of IgG antibody.Other than magnetic bead does not carry out Seal treatment, remaining is same as Example 1.
Specific step is as follows:
1. the walks: the TP antigen and enzyme target TP antigen being coupled on magnetic bead are added into anti-TP antibody-solutions to be measured,
Magneto separate is carried out after mixing reaction and removes supernatant.
2. the walks: the enzyme-linked object of mouse anti-human IgG antibodies for identifying anti-TP antibody to be detected being added into magnetic bead, mixes anti-
Should after carry out Magneto separate and remove supernatant.
3. the walks: the substrate of addition is APS-5, passes through chemical illumination immunity analysis instrument and carries out result detection.
Testing result is as shown in table 1 below.
The positive and negative sample test result of table 1 embodiment 1 and comparative example 1
Such as table 1 as it can be seen that 20 positive samples are detected respectively using the method for embodiment 1 and comparative example 1, with reference result
The coincidence rate that (source: Beijing section U.S. biology) compares is 20/20 (100%), the two test result indifference.In comparative example 1
The test result shake that 20 negative samples are detected in the unclosed situation of magnetic bead is big, the coefficient of variation 235%, coefficient of variation >
100%, the coincidence rate compared with reference result (source: Beijing section U.S. biology) is 19/20 (95%).And magnetic bead in embodiment 1
Identical 20 negative samples are detected after closing in situation, test result shake is small, and the coefficient of variation is only 9%, with reference result
The coincidence rate that (source: Beijing section U.S. biology) compares is 20/20 (100%).
The above test results show that completely unavailable when directly being detected using double antigens sandwich indirect method.It is mainly manifested in:
1. the test result shake of negative sample is big, the bad division of Cutoff value；2. there is false positive issue.This two o'clock shows directly to make
It cannot be used for detection syphilis helicoid antibody with double antigens sandwich indirect method.
The present invention first closes magnetic bead, so that 1. the test result coefficient of variation of negative sample becomes smaller, Cutoff value
It is easy to divide；2. all pattern detection results are consistent with compareing, non-false positive.It therefore, is correct to the Seal treatment of magnetic bead
Use the necessary condition of double antigens sandwich indirect method.
The invention is not limited to specific embodiments above-mentioned.The present invention, which expands to, any in the present specification to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Claims (9)
1. a kind of double antigens sandwich indirect method by Seal treatment, which comprises the following steps:
1. the walks: the antibody identification object A being coupled on closing magnetic bead being added into detection antibody solution and enzyme target antibody is known
Other object B carries out Magneto separate after mixing reaction and removes supernatant；
2. the walks: the enzyme-linked object of large biological molecule that identification detection antibody is added in isolated closing magnetic bead is 1. walked to the,
Magneto separate is carried out after mixing reaction and removes supernatant；
3. the walks: 2. walking to the and substrate is added in isolated closing magnetic bead carries out the detection that shines.
2. passing through the double antigens sandwich indirect method of Seal treatment according to claim 1, which is characterized in that the closing magnetic bead
Seal treatment be serum closing, BSA closing, gelatin closing and skimmed milk power closing one of.
3. the double antigens sandwich indirect method according to claim 1 or claim 2 by Seal treatment, which is characterized in that the closing
Magnetic bead and the mode of antibody identification object A coupling are that biotin-avidin is coupled, biotin-Streptavidin coupling, chemistry are handed over
One of connection, physical absorption and Electrostatic Absorption.
4. the double antigens sandwich indirect method according to claim 1 or claim 2 by Seal treatment, which is characterized in that the coupling
Antibody identification object A and enzyme target antibody identification object B on closing magnetic bead are that identical antibody identifies that object or different antibody are known
Other object.
5. passing through the double antigens sandwich indirect method of Seal treatment according to claim 4, which is characterized in that described to be coupled at envelope
The antibody identification object A and enzyme target antibody closed on magnetic bead identifies that object B is a part of antigen, peptide chain or antigen.
6. the double antigens sandwich indirect method according to claim 1 or claim 2 by Seal treatment, which is characterized in that the identification
The large biological molecule of detection antibody is mouse anti-human IgG antibodies, goat anti-human igg antibody, sheep anti-mouse igg antibody, the anti-sheep IgG of mouse anti-
One of body, Protein A and Protein G.
7. passing through the double antigens sandwich indirect method of Seal treatment according to claim 6, which is characterized in that the identification is to be checked
The enzyme-linked object of large biological molecule of antibody is surveyed by the large biological molecule conjugated alkaline phosphatase or horseradish of the identification detection antibody
Peroxidase obtains.
8. passing through the double antigens sandwich indirect method of Seal treatment according to claim 4, which is characterized in that the enzyme target is anti-
Body identifies that object B identifies that object B conjugated alkaline phosphatase or horseradish peroxidase obtain by antibody, and the substrate is alkaline phosphatase
Or the substrate of horseradish peroxidase.
9. the double antigens sandwich indirect method according to claim 1 or claim 2 by Seal treatment, which is characterized in that described to be checked
Survey antibody is syphilis helicoid antibody.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113155744A (en) * | 2021-04-26 | 2021-07-23 | 广东长光中科生物科技有限公司 | Method for quantitatively detecting target protein in serum by micro-nano optical fiber coupler label-free biosensing |
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