CN110456073A - The method and kit of double antigens sandwich detection antibody - Google Patents

The method and kit of double antigens sandwich detection antibody Download PDF

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Publication number
CN110456073A
CN110456073A CN201910772077.3A CN201910772077A CN110456073A CN 110456073 A CN110456073 A CN 110456073A CN 201910772077 A CN201910772077 A CN 201910772077A CN 110456073 A CN110456073 A CN 110456073A
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marker
antigen
antibody
bacillus
biotin
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CN110456073B (en
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周峻
黄记有
潘少丽
程珍珠
池朗山
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Guangdong Peng Peng Biological Co Ltd
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Guangdong Peng Peng Biological Co Ltd
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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
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to field of biotechnology, in particular to the method and kit of a kind of double antigens sandwich detection antibody.The method is improved on the basis of existing " two step combined techniques of a step " double antigens sandwich, and by forming the anti-marker complex of marker-with amplified signal, this method can improve hook problem and low concentration sample missing inspection problem simultaneously.

Description

The method and kit of double antigens sandwich detection antibody
Technical field
The present invention relates to field of biotechnology, in particular to the method and examination of a kind of double antigens sandwich detection antibody Agent box.
Background technique
Dual-antigen sandwich method is the label secondary antibody replaced in indirect method using labelled antigen, and indirect method is solved from methodology Defect, have the characteristics that highly sensitive, high specific and window phase further shorten.The conventional detection mould of dual-antigen sandwich method Formula can be divided into " one-step method ", " two step method " and " three-step approach ".Because of easy, time-consuming short feature, " one-step method " kit by To the favor in clinical detection market.But " one-step method " there are a drawbacks: " hook effect (hook effect) " works as antibody concentration False negative result is easy to appear when excessively high.
In traditional one-step method, examined samples and labelled antigen are to be added simultaneously, i.e., trip is existed simultaneously in reaction system From labelled antigen and by inspection antibody, by inspection antibody moiety and envelope antigen formed " envelope antigen-antibody " compound, partially with Labelled antigen forms " labelled antigen-antibody " compound.It is anti-by inspection especially when the content in reaction system by inspection antibody is very high " labelled antigen-antibody " compound that body and free labelled antigen are formed has seriously affected that " envelope antigen-antibody-marker is anti- The formation of original " interlayer structure to form hook effect, or even causes the detection leakage phenomenon of false yin.
In order to solve hook effect, applicant has invented a kind of " two step combined techniques of a step " (CN109444434A) before this, I.e. the first step contacts envelope antigen, labelled antigen with object to be checked under conditions of being enough to occur antibody/antigen association reaction, shape At immune complex；Wash away unbonded detection antibody；Labelled antigen is added again and it is immunized again with described for second step Remaining antigen binding site in object to be closed to combine, this method can effectively improve HOOK problem, but when antibody concentration to be checked is too low When still have missing inspection problem appearance.
In view of this, the present invention is specifically proposed.
Summary of the invention
The present invention relates to a kind of methods of double antigens sandwich detection antibody, and the method is to form the first antigen A g1It is to be checked Survey the-the second antigen A of antibody g2Form complete detection, wherein Ag1Coupling has solid support, and the second antigen A g2Include Two kinds of forms, the described method comprises the following steps:
A) by Ag1, the first form Ag2It is contacted under conditions of being enough to occur antibody/antigen association reaction with object to be checked, shape At immune complex；
Wherein in terms of molal quantity, Ag1Content be more than the first form Ag2, and the first form Ag2There is label for coupling The Ag of object2；
B) unbonded detection antibody is washed away；
C) the second form Ag is added2And make it in conjunction with antigen binding site remaining in the immune complex；It is described The second form Ag2To be coupled the Ag for having signal designation object2；
D) the anti-marker that coupling has signal designation object is added, the anti-marker can be formed specifically with the marker The anti-marker complex of marker-and carry out signal amplification；
Step c) and d) without sequencing；
E) marking agent is detected, to indicate the presence and/or content of the detection antibody.
The above method can improve hook problem and low concentration sample missing inspection problem simultaneously.
The invention further relates to the kits for realizing the above method.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the schematic illustration (signal designation object is by taking AE as an example) of one embodiment of the present invention.
Specific embodiment
The present invention relates to a kind of methods of double antigens sandwich detection antibody, and the method is to form the first antigen A g1It is to be checked Survey the-the second antigen A of antibody g2Form complete detection, wherein Ag1Coupling has solid support, and the second antigen A g2Include Two kinds of forms, the described method comprises the following steps:
A) by Ag1, the first form Ag2It is contacted under conditions of being enough to occur antibody/antigen association reaction with object to be checked, shape At immune complex；
Wherein in terms of molal quantity, Ag1Content be more than the first form Ag2, and the first form Ag2There is label for coupling The Ag of object2；
B) unbonded detection antibody is washed away；
C) the second form Ag is added2And make it in conjunction with antigen binding site remaining in the immune complex；It is described The second form Ag2To be coupled the Ag for having signal designation object2；
D) the anti-marker that coupling has signal designation object is added, the anti-marker can be formed specifically with the marker The anti-marker complex of marker-and carry out signal amplification；
Step c) and d) without sequencing；
E) marking agent is detected, to indicate the presence and/or content of the detection antibody.
In the present invention, the first antigen A g1With the second antigen A g2Antigen may be the same or different.
In some embodiments, in step a), with molar ratio computing, Ag1:Ag2=6:2~6:5；Also it can choose 3:2 Or 2:1.
Suitable Ag1With Ag2Adding proportion can avoid hook effect under the premise of guaranteeing sensitivity.According to specific Antigen-antibody characteristic is different, which may have some difference, but verify by inventor, and most antigens are all satisfied above-mentioned Ratio.
In the present invention, the active ratio of " molar ratio " or antigen has the antigen table in conjunction with detection antibody One protein of position may be considered an active unit.
In some embodiments, with molar ratio computing, the middle Ag being added of step a)2With the Ag being added in step c)2Ratio For 6:2~6:4.
In some embodiments, with molar ratio computing, the middle Ag being added of step a)2With the Ag being added in step c)2Ratio For 2:1.
Ag in step c)2Addition can effectively reduce the residual of marker in appropriate proportions, to reduce background.
In the present invention, (anti-marker/marker) combination means the two kinds of compounds that can be combined in a specific way.
In some embodiments, the combination of marker/anti-marker is selected from the anti-marker complex of the marker- Biotin or derivatives thereof/Streptavidin (streptavidin), biotin or derivatives thereof/Avidin (avidin) are raw Object element or derivatives thereof/neutravidin (NeutrAvidin), biotin or derivatives thereof/antibiotin or its spread out Biological antibody, haptens/antibody, antigen/antibody, peptide/antibody, receptor/ligand, digoxin/digoxigenin, carbon hydrate Object/agglutinin and polynucleotides/complementation polynucleotides.
These different combinations and other combination be it is known, be well known to those skilled in the art.
The preferred biotin of the present invention/biotin set protein family.
The protein family that biotin combines includes above-mentioned Streptavidin (streptavidin), Avidin (avidin) With NeutrAvidin albumen, each albumen can be with four biotin molecules of the affinity of height and specific binding.Wherein Most-often used is Streptavidin, it is without glycosylating and with very low non-specific binding level.Avidin is then one The glycoprotein of kind height cationization, 10.5 or so, its positive charge residue and oligosaccharide composition can mediate non-isoelectric point Specific binding, thus the problem for causing background excessively high in some applications.NeutrAvidin albumen is handled by deglycosylation With reduce equipotential points, to reduce its background coloration.
In addition, biotin well known to those skilled in the art can also be substituted with its congenerous derivative, such as D-Biotin, Activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin or desthiobiotin.
In some embodiments, the second form Ag2In, marking agent is coupled to Ag2It is to be realized indirectly by bridge material 's.
In some embodiments, the bridge material is selected from one of albumen, albumen composition or bi-functional cross-linking agent Or it is a variety of.
In some embodiments, the bridge material is selected from the anti-marker complex of marker-, the anti-mark of marker- Remember that object compound is defined in content as above.
In some embodiments, contain bovine serum albumin(BSA), ovalbumin, keyhole in the albumen or albumen composition At least one of hemocyanin, immunoglobulin, thyroglobulin, poly-D-lysine.
In preferred technical solution, bridge material is selected from bovine serum albumin(BSA).Bovine serum albumin(BSA) is not only this field institute It is common, and itself have the function of certain reduction non-specific signals, the signal-to-noise ratio of experiment can be increased.
In some embodiments, the bi-functional cross-linking agent is selected from:
1)MPBH(4-[4-N-maleimidophenyl]butyric acid hydrazide hydrochloride)、 MPEG2A(1-[2-[2-(2-Aminoethoxy)ethoxy]ethyl]maleimide hydrochloride)、BMPH(N- [β-maleimidopropionic acid]hydrazide,trifluoroacetic acid salt)、EMCH(N-[ε- Maleimidocaproic acid)hydrazide,trifluoroacetic acid salt)、KMUH(N-[κ- Any one of maleimidoundecanoic acid] hydrazide, trifluoroacetic acid salt)；
Or 1) 2) any one of the derivative containing maleimide base group and amino or hydrazides group of substance in；
3) or by 1), 2) in material composition group.
In the present invention, bridge material generally also has the function of amplified signal, for example, bridge material can be coupled more letters Number indicant is with amplified signal.
In the present invention, antigen A g1It is usually fixed on solid support in advance, term " fixation " means creation altogether Valence or non-covalent bond.In some embodiments, the solid support is magnetic bead, slide, membrane type substrate or is provided with sample-adding The plate in hole.
In some embodiments, the magnetic bead is γ Fe2O3Or Fe3O4Magnetic nano-particle or they and organic high score The complex of sub- material.
In the present invention, signal designation object refers to provide the substance of detected signal, in some embodiments, In Step c) and d) in, the signal designation object it is independent be selected from chromophore, digoxin labelled probe, electron dense substances, colloid Gold or enzyme are any one or more of.These labels are listed in non-limiting part below:
The enzyme of detectable signal is generated, is such as detected by colorimetric method, fluorescence and shining, such as horseradish peroxidase, alkali Acid phosphatase, beta galactosidase and glucose-6-phosphate dehydrogenase (G6PD).
Chromophore, such as fluorescence, quantum dot, fluorescent microsphere, luminophor and dyestuff.
With can be by electron microscope or by its electrical characteristics, such as conductibility, amperometry, voltage measurement and resistance inspection The group of the electron density of survey.
Detectable group, as its molecular size is enough to induce the detectable modification on its physically and/or chemically characteristic；This Kind of detection can optically (such as diffraction, surface plasmon resonance, surface variations and contact variation angle) or physical method (such as atomic force spectroscopy and tunnel-effect) is realized.
Electron dense substances, (such as such as radioactive molecule32P,35S or125I)。
In some embodiments, the signal designation object is acridinium ester (AE).
The present invention also provides kits comprising anti-marker, the first antigen A g as defined above1And second antigen Ag2。
In some embodiments, the kit further includes one in sample pretreatment liquid, buffer and color developing agent Kind is a variety of.
Pretreatment fluid may include the purification reagent of lysate (cracking tissue or cell), albumen especially antibody, albumen Enzyme inhibitor etc. can discharge, purify, protecting the reagent of antibody.The reagent that pretreatment fluid will not preferably cause antibody to be denaturalized.
In some embodiments, the first antigen A g1And the second antigen A g2By that can be produced with disease pathogen The antigen that raw antibody combines；
In some embodiments, the disease pathogen includes one of virus, bacterium, fungi or helminth or more Kind；
In some embodiments, the virus includes: Adenoviridae (adenoviridae), Arenaviridae (arenaviridae), Astroviridae (astroviridae), this refined Viraceae (bunyaviridae), Caliciviridae (caliciviridae), flaviviridae (flaviviridae), hepatitis virus section (hepeviridae), Order Mononegavirales RNA Virales (mononegavirales), net nest virales (nidovirales), Picornaviridae (picornaviridae), Orthomyxoviridae family (orthomyxoviridae), Papillomaviridae (papillomaviridae), Parvoviridae (parvoviridae), polyomavirus section (polyomaviridae), Poxviridae (poxviridae), Reoviridae (reoviridae), one of Retroviridae (retroviridae) and Togaviridae (togaviridae) or It is a variety of；
In some embodiments, the bacterium includes: staphylococcus, streptococcus, Lee's formula Bacillus, Erysipelothrix Category, Renibacterium category, bacillus, fusobacterium, mycobacterium, actinomyces, slave block Pseudomonas, corynebacterium, Rhodococcus sp One of category is a variety of, and/or, bacillus anthracis, erysipelas bacillus, clostridium tetani, Li bacillus, bacillus chauvoei tuberculosis bar Bacterium, Escherichia coli are outer, proteus, shigella dysenteriae, pneumobacillus, Brucella, produce gas folder film bacillus, haemophilus influenzae, Haemophilus parainfluenzae, catarrh Moraxella, acinetobacter, yersinia's genus, legionella pneumophilia, Bordetella pertussis, pair One of Bordetella pertussis, Shigella, Pasteurella, comma bacillus and parahemolyticas bacillus are a variety of；
In some embodiments, the fungi includes: posadasis spheriforme, Pu Saidesi ball armful daughter bacteria, capsule tissue born of the same parents Starch bacterium, Du Shi histoplasma capsulatum, blastomyces loboi, Paracoccidioides brasiliensis, Blastomyces dermatitidis, sporotrichum schenckii, Ma Er Buddhist nun's phenanthrene Penicillium notatum, Candida albicans, Candida glabrata, Candida tropicalis, Candida lusitaniae, Aspergillus, Exophiala jeanselmei, Fonsecaea pedrosoi, Fonsecaea compacta, excipuliform coloring is mould, dermatitis coloring is mould, geotrichum candidum, Podbielniak foot swollen bacterium, Cryptococcus neoformans, silk spore Saccharomycete, Rhizopus oryzae, India Mucor, absidia corymbifera, Syncephalastrum racemosum, frog excrement be mould, Conidiobolus coronatus, Conidiobolus incongruus, the primary nose in west One of pityrosporion ovale, transparent wire spore are mould and dark-coloured silk spore is mould or a variety of；
In some embodiments, the helminth include: alimentary canal entozoa, liver entozoa, intrapulmonary helminth, Brain tissue helminth, blood vessel entozoa, lymphatic vessel entozoa, musculature helminth, cytozoon, bone tissue are posted One of infested and intraocular helminth is a variety of.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.
Embodiment 1
1. magnetic bead HCV antigen coat (MP-HCVAg)
With EDC and NHS activated carboxyl magnetic bead, the carboxyl magnetic bead that HCV antigen and activation is then added is incubated for coupling, magnetic point altogether From supernatant is removed, the magnetic bead of HCV antigen coat is stored in magnetic bead and is saved in liquid.
2.HCV antigen biotin labeling (HCVAg-bio)
HCV antigen is reacted with the mixing of the biotin (Bio-NHS) of activation.It is dialyzed overnight with bag filter, recycling is saturating Residue is the biotin labeling HCV antigen purified in analysis bag.
3. streptavidin AE marks (SA-AE)
Streptavidin is reacted with the mixing of the acridinium ester (AE-NHS) of activation.It is dialyzed overnight, is recycled with bag filter Residue is the streptavidin AE marker (SA-AE) purified in bag filter.
4. bovine serum albumin(BSA) AE marks (BSA-AE)
BSA solid is weighed, buffer solution is added.The acridinium ester (AE-NHS) of activation is added into BSA solution.Room temperature condition Lower reaction.Sample is dialyzed overnight with bag filter.Residue obtains bovine serum albumin(BSA) AE marker (BSA- in recycling bag filter AE)。
5. maleimide activation ester activated b SA-AE
The activation of maleimide activation ester is added into BSA-AE sample.Sample is transferred to bag filter dialysis.Recycling dialysis Residue obtains the BSA-AE of maleimide activation in bag.
The coupling of 6.HCVAg-BSA-AE
By the BSA-AE sample of maleimide activation and HCVAg sample hybrid reaction.Sample is transferred to bag filter dialysis, Residue obtains HCV antigen A E cross-linking agent (HCVAg-BSA-AE) in recycling bag filter.
7, prepared by HCVAg-MPBH-AE
MPBH and AE Acibenzolar hybrid reaction.BSA reaction is added and removes unreacted AE Acibenzolar.MPBH-AE is coupled Object and HCVAg sample hybrid reaction.Sample is transferred to bag filter dialysis, residue in bag filter is recycled and obtains HCV antigen A E Marker (HCVAg-MPBH-AE).
Embodiment 2
In the present embodiment, unless otherwise specified, each group is based on two step combined techniques of a step, and the technology about this method is thin Section can refer to the prior art: Chinese patent CN109444434A, and publication date on 03 08th, 2019.
By taking the B-mode in (1) as an example:
Its testing process are as follows:
1) it takes sample to be tested (antibody containing HCV) 50uL that the magnetic microsphere 50uL of coating HCV antigen is added, while biology is added Element mark antigen 50ul, 37 DEG C of incubation 15min；
2) magnetic field is added and carries out Magneto separate, remove supernatant；
3) it washs 4 times, each 250uL washing lotion, repeats step 2 operation；
4) SA-AE (Ag-BSA-AE) mix reagent 100uL, 37 DEG C of incubation 10min is added；
5) magnetic field is added and carries out Magneto separate, remove supernatant；
6) it washs 4 times, each 250uL washing lotion, repeats step 4 operation；
7) light excitation A liquid is first added (containing H in every hole2O2) 100uL, light exciting liquid B liquid (containing NaOH) 100uL is added, is added dropwise While the relative light units (RLU) in each hole, 1 second/hole of time of measuring are measured with flash of light photon counter.
8) using 4 times of negative control RLU average value as threshold value (cutoff value), the RLU value and threshold value comparison in each hole, sample Product measured value is more than or equal to threshold value, then is judged as positive, is otherwise feminine gender.
(1) hook Resolving probiems Contrast on effect
3 times test result is as follows:
Note: the first file is the positive quality control blood of different dilutions.
Mode A: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms sandwich；Second step is anti- A certain amount of SA-AE should be added, for providing signal (RLU value).
B-mode: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms sandwich；Second step is anti- A certain amount of SA-AE and Ag-BSA-AE mixture should be added, for providing signal (RLU value).
C mode: envelope antigen, serum and a certain amount of Ag-BSA-AE is added in first step reaction, preliminarily forms sandwich；Second A certain amount of Ag-BSA-AE is added in step reaction, and two steps provide signal (RLU value) jointly.
Wherein, Ag-Bio, Ag-BSA-AE are the amounts for being enough sufficiently to react with detection antibody, and the amount of SA-AE is also enough It is sufficiently reacted with Ag-Bio, i.e., its amount being added is excessive.Other following experiments are similarly.
As a result: A, B and C detection pattern data comparison, when positive sample concentration increases to certain value, mode A signal value Decline, B and C mode concentration still keep being positively correlated with signal value.The result shows that " a step two-step method " is a kind of effective The method for solving hook phenomenon.
(note: envelope antigen: it is coated with the magnetic bead of HCV antigen；Ab: antibody；Ag-Bio: biotinylation HCV antigen；SA- AE: streptavidin AE marker；Ag-BSA-AE:HCV antigen bovine serum albumin(BSA) AE marker；Ag-MPBH-AE:HCV is anti- Original-MPBH-AE marker；Similarly hereinafter)
(2) low value Resolving probiems Contrast on effect
3 times test result is as follows:
Note: " 1/24K " indicates dilution 2.4 × 104Positive quality control blood again.
B-mode: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms sandwich；Second step is anti- A certain amount of SA-AE and Ag-BSA-AE mixture should be added, for providing signal (RLU value).
C mode: envelope antigen, serum and a certain amount of Ag-BSA-AE is added in first step reaction, preliminarily forms sandwich；Second A certain amount of Ag-BSA-AE mixture is added in step reaction, and two steps provide signal jointly.
As a result: P/N Value Data shows detection sensitivity B-mode > C mode.The result shows that on the basis of " a step two-step method " On, using two kinds of different bridging label system combinations, especially HCVAg-bio- marker and HCVAg-BSA- marker is combined, Hook problem and low concentration sample missing inspection problem can be improved simultaneously, than a kind of bridging label system (such as Ag-BSA-AE) is applied alone Detection sensitivity it is high.
(3) bridging system
Note: " 1/24K " indicates dilution 2.4 × 104Positive quality control blood again.
A combination: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms sandwich；Second step is anti- A certain amount of SA-AE and Ag-BSA-AE mixture should be added, for providing signal (RLU value).
B combination: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms sandwich；Second step is anti- A certain amount of SA-AE and Ag-MPBH-AE mixture should be added, for providing signal.
C in combination: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms sandwich；Second step is anti- A certain amount of SA-AE and Ag-Bio/SA-AE mixture should be added, for providing signal.
(4) antigen A E mark mode effect compares in composition
Note: " 1/24K " indicates dilution 2.4 × 104Positive quality control blood again.
2nd column data testing procedure: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms It is sandwich；A certain amount of SA-AE and Ag-BSA-AE mixture is added in second step reaction, for providing signal (RLU value).
3rd column data testing procedure: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms It is sandwich；A certain amount of SA-AE and Ag-MPBH-AE mixture is added in second step reaction, for providing signal.
4th column data testing procedure: envelope antigen, serum and a certain amount of Ag-Bio is added in first step reaction, preliminarily forms It is sandwich；A certain amount of SA-AE and Ag-AE mixture is added in second step reaction, for providing signal.
As a result: P/N value size order is Ag-BSA-AE > Ag-MPBH-AE > Ag-AE.The result shows that using two kinds of bridgings When system, with Ag-Bio/SA-AE associated with another bridging mark system, it is better than direct mark mode in a manner of indirect bridging.
(5) high level and low value pattern detection
It chooses 20 negative samples and 20 positive samples while being detected using several reaction patterns, each hole of mean value RLU value is compared with CUTOFF, sample measurements >=20 × feminine gender Quality Control, then is judged as positive, is otherwise feminine gender, with+indicate sun Property ,-indicate negative, as a result as follows:
The above result shows that the method for the present invention not only can solve the hook missing inspection problem of one-step method, but also can be in a step two The false dismissal probability (such as sample P5) that weak positive sample is further decreased on the basis of footwork, improves detection sensitivity.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution The range of art scheme.

Claims (10)

1. a kind of method of double antigens sandwich detection antibody, the method is to form the first antigen A g1Detection antibody-the second is anti- Former Ag2Form complete detection, wherein Ag1Coupling has solid support, and the second antigen A g2It is described comprising two kinds of forms Method the following steps are included:
A) by Ag1, the first form Ag2It is contacted under conditions of being enough to occur antibody/antigen association reaction with object to be checked, formation is exempted from Epidemic disease compound；
Wherein in terms of molal quantity, Ag1Content be more than the first form Ag2, and the first form Ag2There is marker for coupling Ag2；
B) unbonded detection antibody is washed away；
C) the second form Ag is added2And make it in conjunction with antigen binding site remaining in the immune complex；Second shape Formula Ag2To be coupled the Ag for having signal designation object2；
D) the anti-marker that coupling has signal designation object is added, the anti-marker can form special mark with the marker The anti-marker complex of note object-simultaneously carries out signal amplification；
Step c) and d) without sequencing；
E) marking agent is detected, to indicate the presence and/or content of the detection antibody.
2. the method according to claim 1, wherein marker in the anti-marker complex of the marker -/anti- The combination of marker is selected from biotin or derivatives thereof/Streptavidin (streptavidin), biotin or derivatives thereof/parent With plain (avidin), biotin or derivatives thereof/neutravidin (NeutrAvidin), biotin or derivatives thereof/ Antibiotin or derivatives thereof antibody, haptens/antibody, antigen/antibody, peptide/antibody, receptor/ligand, digoxin/digoxin Aglucon, carbohydrate/agglutinin and polynucleotides/complementation polynucleotides；
Wherein the derivative of biotin is D-Biotin, activated biotin, biocytin, ethylenediamine biotin, cadaverine biotin Or any one of desthiobiotin.
3. the method according to claim 1, wherein the second form Ag2In, marking agent is coupled to Ag2It is logical Cross what bridge material was realized indirectly.
4. according to the method described in claim 3, it is characterized in that, the bridge material is selected from albumen, albumen composition or double function One of energy crosslinking agent is a variety of；
Optionally, the bridge material is selected from the anti-marker complex of marker-, and the anti-marker complex of marker-is power Benefit requires defined in 1 or 2.
5. according to the method described in claim 4, containing bovine serum albumin(BSA), albumen egg in the albumen or albumen composition At least one of white, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin and poly-D-lysine.
6. according to the method described in claim 4, it is characterized in that, the bi-functional cross-linking agent is selected from:
1) any one of MPBH, MPEG2A, BMPH, EMCH, KMUH；
Or 1) 2) any one of the derivative containing maleimide base group and amino or hydrazides group of substance in；
3) or by 1), 2) in material composition group.
7. the method for described in any item double antigens sandwich detection antibody according to claim 1~6, which is characterized in that described solid Phase support is magnetic bead, slide, membrane type substrate or the plate for being provided with well；
Optionally, the magnetic bead is γ Fe2O3Or Fe3O4Magnetic nano-particle or the complex of they and high-molecular organic material.
8. the method for described in any item double antigens sandwich detection antibody according to claim 1~6, which is characterized in that in step C) in and d), the signal designation object it is independent selected from chromophore, digoxin labelled probe, electron dense substances, colloidal gold or Enzyme is any one or more of；
Optionally, the signal designation object is acridinium ester.
9. kit comprising anti-marker defined in any one of claim 1~8, the first antigen A g1And second is anti- Former Ag2；
Optionally, the kit further includes one of sample pretreatment liquid, buffer and color developing agent or a variety of.
10. kit according to claim 9, which is characterized in that the first antigen A g1And the second antigen A g2For energy It is enough with disease pathogen caused by antigen in conjunction with antibody；
Optionally, the disease pathogen includes one of virus, bacterium, fungi or helminth or a variety of；
Optionally, the virus include: Adenoviridae (adenoviridae), it is Arenaviridae (arenaviridae), starlike Viraceae (astroviridae), this refined Viraceae (bunyaviridae), Caliciviridae (caliciviridae), jaundice Malicious section (flaviviridae), hepatitis virus section (hepeviridae), Mononegavirales (mononegavirales), net nest virales (nidovirales), Picornaviridae (picornaviridae), positive mucus Viraceae (orthomyxoviridae), Papillomaviridae (papillomaviridae), Parvoviridae (parvoviridae), polyomavirus section (polyomaviridae), Poxviridae (poxviridae), Reoviridae (reoviridae), one of Retroviridae (retroviridae) and Togaviridae (togaviridae) or It is a variety of；
Optionally, the bacterium includes: staphylococcus, streptococcus, Lee's formula Bacillus, erysipelothrix, Renibacterium category, bud Spore Bacillus, fusobacterium, mycobacterium, actinomyces, slave block one of Pseudomonas, corynebacterium, Rhod or more Kind, and/or, outside bacillus anthracis, erysipelas bacillus, clostridium tetani, Li bacillus, bacillus chauvoei tubercle bacillus, Escherichia coli, Proteus, shigella dysenteriae, pneumobacillus, Brucella, produce gas folder film bacillus, haemophilus influenzae, haemophilus parainfluenzae, Catarrh Moraxella, acinetobacter, yersinia's genus, legionella pneumophilia, Bordetella pertussis, parapertussis bacillus, will are congratulated One of Pseudomonas, Pasteurella, comma bacillus and parahemolyticas bacillus are a variety of；
Optionally, the fungi includes: posadasis spheriforme, Pu Saidesi ball embraces daughter bacteria, Histoplasma capsulatum, Du Shi organize born of the same parents Starch bacterium, blastomyces loboi, Paracoccidioides brasiliensis, Blastomyces dermatitidis, sporotrichum schenckii, penicillium Marneffei, white It is candida albicans, Candida glabrata, Candida tropicalis, Candida lusitaniae, Aspergillus, Exophiala jeanselmei, Fonsecaea pedrosoi, close Colour the coloring of mould, excipuliform is mould, dermatitis coloring is mould, geotrichum candidum, Podbielniak enough swollen bacterium, Cryptococcus neoformans, Trichosporon Behrend, Rhizopus oryzae, India Mucor, absidia corymbifera, Syncephalastrum racemosum, frog excrement be mould, Conidiobolus coronatus, Conidiobolus incongruus, Rhinosporidium seeberi, transparent wire spore Mould and dark-coloured silk spore is one of mould or a variety of；
Optionally, the helminth include: alimentary canal entozoa, liver entozoa, intrapulmonary helminth, brain tissue helminth, Blood vessel entozoa, lymphatic vessel entozoa, musculature helminth, cytozoon, bone tissue helminth and intraocular One of helminth is a variety of.
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