CN104597232A - Capture antibody competition sandwich immunoassay method capable of expanding detection range and biosensor - Google Patents
Capture antibody competition sandwich immunoassay method capable of expanding detection range and biosensor Download PDFInfo
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- 238000003018 immunoassay Methods 0.000 title claims abstract description 18
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- 229910021421 monocrystalline silicon Inorganic materials 0.000 claims description 7
- 239000004698 Polyethylene Substances 0.000 claims description 5
- 239000004793 Polystyrene Substances 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- -1 polyethylene Polymers 0.000 claims description 5
- 229920000573 polyethylene Polymers 0.000 claims description 5
- 229920002223 polystyrene Polymers 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 3
- 229920001477 hydrophilic polymer Polymers 0.000 claims description 3
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- 238000012360 testing method Methods 0.000 abstract description 14
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- 239000000243 solution Substances 0.000 description 10
- 239000007790 solid phase Substances 0.000 description 9
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 7
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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
-
- 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
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Abstract
The invention discloses a capture antibody competition sandwich immunoassay method capable of expanding a detection range and a biosensor. The invention utilizes capture antibody competition sandwich immunoassay to detect the concentration of the antigen to be detected, namely, the antigen in a sample to be detected is originally reacted with a free capture antibody and a labeled antibody and then reacted with a fixed capture antibody, only the labeled antibody-antigen-fixed capture antibody complex in the formed immune complex can be detected, and the concentration of the antigen is determined by measuring a labeled signal on the labeled antibody of the immune complex. Because the free capture antibody-antigen-labeled antibody complex and the free capture antibody-antigen complex can not be detected by a detection instrument, the aim of indirectly diluting the antigen concentration in a sample to be detected is fulfilled, and the detection range of the antigen concentration of the sample to be detected is expanded. Meanwhile, the biosensor test card prepared by the method has the advantages of wide detection range, high sensitivity and short detection time.
Description
Technical field
The present invention relates to technical field of immunoassay, more specifically, relate to a kind of capture antibody competition sandwich immune detecting method and biology sensor of easily extensible sensing range.
Background technology
Immune detection utilizes immunology principle to detect test substance in sample, and its method can be divided into quilitative method and quantivative approach.Immune detection has special, easy, quick, accurate, sensitive advantage, according to the difference of Cleaning Principle, immune detection can be divided into indirect method, sandwich method (comprising dual-antigen sandwich method and double antibody sandwich method), prize law, competition law etc., and often kind of method has respective advantage and limitation.
The principle of traditional double-antibody sandwich immune response (sandwich immunoassay) is detect target with antigen.Chemical treatment is carried out on the surface of solid phase carrier, surface of solid phase carriers is made to cover the material of energy binding antibody, capture antibody is fixed on solid phase carrier, form fixed trapped antibody, then antigen and labelled antibody are contacted with fixed trapped antibody, and then form fixed trapped antibody-antigene-labelled antibody sandwich complex, by the detection to label on labelled antibody, realize the quantitative detection to antigen.This technology has been widely used in the detection of clinical, environment, food, customs etc., but the shortcoming of this technology to be sensing range narrow, usually only can meet and detect the sensitivity of pattern detection lower limit, but the requirement of upper limit of detection cannot be met, there is HD-Hook district in upper limit of detection, numerical value accurately cannot be provided.Therefore, could must to be detected by dilution for many times for high concentration sample, thus the step of the detection of increase, to introduce larger operate miss, the time that the detection of prolongation is used.Such as High-sensitivity C reactive protein, clinically to the requirement of its detection sensitivity generally at a few to tens of ng/ml, and be 10 μ g/mL for the requirement of upper limit of detection, and the normal range of High-sensitivity C reactive protein is less than 1 μ g/mL, pathology scope is 1-10 μ g/mL, if the testing result that a thus traditional step double-antibody sandwich immunologic detection method provides is in HD-Hook district, need to dilute sample, and then detect, and because of the signal intensity of sample to be tested be positioned at HD-Hook district time, because can not determine the general concentration of sample to be tested, so the extension rate of sample can not be indicated.Thus traditional double-antibody sandwich immunologic detection method has the problem that detecting step is loaded down with trivial details, detection time is long.Double antibody sandwich ELISA traditional at present, that capture antibody is fixed on solid phase surface, and sample to be tested is contacted with capture antibody, form the binary complex of capture antibody-antigen, and then the antibody of enzyme labeling is contacted with this binary complex, form the ternary complex of capture antibody-antigen-enzymic-labelled antibody, the concentration of antigen in sample to be tested is determined by the enzyme-to-substrate reaction solution in ternary complex, sensing range is generally in 1-1000ng/ml scope, although euzymelinked immunosorbent assay (ELISA) has obvious expansion to upper limit of detection, but the needs of detection still can not be met for some antigen, need to be developed the color by substrate due to it simultaneously, thus step is added more, increase detection time, when therefore utilizing traditional double-antibody sandwich immunization and euzymelinked immunosorbent assay (ELISA) to carry out clinical detection, there is upper limit of detection low, need to carry out dilution for many times to sample, the problem of the quick directly testing requirement of high concentration sample can not be met.
Immune competition method principle is that antigen in sample and the competition of a certain amount of labelled antigen are combined with fixed trapped antibody.In sample, antigen amount content the more, and the labelled antigen be combined on fixed trapped antibody is fewer, and last detection signal is more weak.Being incorporated into tested antigen concentration in labelled antigen amount on fixed trapped antibody and sample is negative correlation, and the method upper limit of detection is high, but cannot realize the sensitivity of Monitoring lower-cut, and can not simplify the operation step.
Therefore need to provide a kind of highly sensitive, sensing range is wide, easy and simple to handle, detection time is short, require low detection method to checkout equipment.。
Summary of the invention
The object of the invention is to set up a kind of sensing range wide, detection time is short, easily operation and the few method of step.Following technical proposals is the invention provides: capture antibody competition double-antibody sandwich immunization, comprises free capture antibody, labelled antibody and fixed trapped antibody in order to realize foregoing invention object.Wherein free capture antibody, unmarked, different from the epi-position that labelled antibody combines on antigen.Fixed trapped antibody is fixed on solid phase carrier.First with free capture antibody with fixed trapped antibody competition in conjunction with determined antigen, the antigen antibody complex formed is combined with labelled antibody, four kinds of compounds can be formed in course of reaction, the i.e. compound of labelled antibody-antigen, the compound of free capture antibody-antigen, labelled antibody-antigen-fixed trapped antibody sandwich compound, free capture antibody-antigen-labelled antibody compound, the sandwich complex wherein only having labelled antibody and antigen-fixed trapped antibody to be formed does further detection by marking, due to the compound of labelled antibody-antigen, the free compound of capture antibody-antigen and the compound of free capture antibody-antigen-labelled antibody can not be detected, be equivalent to decrease the antigen amount of reacting with capture antibody in liquid to be measured, thus expanded sensing range.
Antigen of the present invention refers to have immunogenic material, as proteins and peptides, representational antigen includes, but is not limited to: the albumen etc. of cell factor, tumor markers, metalloprotein, non-tumor disease mark or microbial expression.
The inventive method comprises the steps:
1) labelled antibody is mixed with free capture antibody, the mixed liquor of both formation;
2) by sample to be tested and the mixing of described mixed liquor, the potpourri of three is formed;
3) potpourri of described three and fixed trapped antibody response;
4) reactant liquor is removed;
5) detect the marking signal on the labelled antibody in described fixed trapped antibody-antigene-labelled antibody compound and calculate the concentration of antigen.
Described fixed trapped antibody is fixed on solid phase carrier; The material of described solid phase carrier is monocrystalline silicon, synthetic glass, polystyrene, golden film, nitrocellulose, fluorinated polyethylene, polycation resin, hydrophilic polymer film or porous material, preferably, the material of described solid phase carrier is monocrystalline silicon, resin, glass, nitrocellulose resin, fluorinated polyethylene or polystyrene.
The material surface fixing for fixed trapped antibody needs chemical treatment, and disposal route is silication, amination, aldehyde radical, sulfhydrylation, epoxidation and active esterifying, preferred epoxidation or amination.
Preferably, after fixed trapped antibody is fixing, use PBST solution, the skimmed milk power of 5% or the fish glue from skin of 1% containing 1%BSA to close, more preferably close with the PBST solution of the BSA containing 1%;
Preferably, capture antibody and labelled antibody are incorporated into the different epi-positions of target antigen;
Described free capture antibody and fixed trapped antibody can not form free capture antibody-antigen-fixed trapped antibody complex with determined antigen; Free capture antibody and fixed trapped antibody can be different molecules, and also can be identical molecule, preferably, the two be identical molecule;
Preferably, the mol ratio of free capture antibody and solid-phase capture antibody is 1:1-1:4;
Preferably, the mol ratio of labelled antibody and fixed trapped antibody is 3:1-1:1;
Preferably, reactant liquor incubation time 5-7 minute;
Preferably, the label of labelled antibody is magnetic bead, chemiluminescence, fluorescence, collaurum, collargol, more preferably magnetic bead, chemiluminescence or collaurum;
Preferably, together with free capture antibody is pre-mixed with labelled antibody, storing solution is formed.
The step competition sandwich immunoassay that the present invention still further developed easily extensible sensing range detects biology sensor, and its technical scheme is as follows:
A kind of capture antibody competition sandwich immunoassay of easily extensible sensing range detects biology sensor, it is characterized in that comprising chemical sensor, described chemical sensor comprises cover plate, back up pad and base plate, back up pad is between cover plate and base plate, back up pad is provided with at least one microchannel, described microchannel comprises reagent storage district, immune response district and waste liquid district; Described immune response district comprises fixed trapped antibody, and the cover plate corresponding to position of described reagent storage area is provided with liquid injection hole, and the cover plate corresponding to the position in waste liquid district is provided with outage.Wherein said reagent storage area comprises free capture antibody and labelled antibody.
A kind of capture antibody competition sandwich immunoassay of easily extensible sensing range detects biology sensor, comprise chemical sensor, described chemical sensor comprises cover plate and base plate, microchannel is formed between cover plate and base plate, described microchannel comprises immune response district, described immune response district comprises fixed trapped antibody, and described cover plate has often held one to have note/outage and row/liquid injection hole.
Preferably, base plate size is length is 40-100mm, and width is 25-40mm, and thickness is 1-5mm;
Preferably, the size of cover plate is identical with base plate;
Preferably, the degree of depth of micro channel is 0.1-0.5mm, and length is 6-9cm;
Preferably, micro channel is provided with reagent storage district and waste liquid district;
Preferably, immune response district is used for the fixing of fixed trapped antibody, its material is monocrystalline silicon, glass, polystyrene, golden film, nitrocellulose, fluorinated polyethylene, polycation resin, hydrophilic polymer film, porosint, resin or nitrocellulose resin, be more preferably monocrystalline silicon, resin, glass, nitrocellulose resin, fluorinated polyethylene, polystyrene, nitrocellulose, resin or porosint;
Preferably, the chemical treatment that immune response district is used for the material surface that fixed trapped antibody is fixed is silication, amination, aldehyde radical, sulfhydrylation, epoxidation or active esterifying, is more preferably amination or epoxidation;
Preferably, base plate and cover plate bonding mode are bonding agent bonding, ultra-violet curing, hot pressing or ultrasonic bonding.
Beneficial effect of the present invention is as follows:
This detection method has the advantage that sensing range is wide, detection sensitivity is high, detection time is short, easy and simple to handle; The chemical sensor made by this method sample volume used is little, detection sensitivity is high, sensing range is wide and can adopt Through Several Survey Measure.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 a illustrates chemical sensor 1 front view.
Fig. 1 b illustrates chemical sensor 1 cut-open view.
Fig. 2 a illustrates chemical sensor 2 front view.
Fig. 2 b illustrates chemical sensor 2 cut-open view.
Fig. 3 a illustrates upper limit of detection during traditional dual anti-sandwich immunoassay reaction method detection CRP antigen.
Fig. 3 b illustrates upper limit of detection during capture antibody competition sandwich immunoassay reaction method detection CRP antigen.
Fig. 4 illustrates the CRP antigen concentration-magnetoresistivity variable quantity typical curve in embodiment 2.
100,200 chemical sensors in figure; 130,230 cover plates; 170 back up pads; 140,240 base plates; 150,250 microchannels; 160 reagent storage districts; 120,220 immune response districts; 180 waste liquids; 110 liquid injection holes; 190 outages; 210 rows/liquid injection hole; 260 sensor regions.
Embodiment
In order to be illustrated more clearly in the present invention, below in conjunction with preferred embodiments and drawings, the present invention is described further.Parts similar in accompanying drawing represent with identical Reference numeral.It will be appreciated by those skilled in the art that specifically described content is illustrative and nonrestrictive, should not limit the scope of the invention with this below.
Embodiment 1: the preparation of magnetic particle CRP biosensor test card
1. the preparation of magnetic particle marker antibody: first carry out mark CRP antibody with NHS-Biotin, NHS-Biotin carries out coupling by NHS and antibody and forms Biotin-CRP antibody, then use Avidin-magnetic particle and Biotin-CRP antibody to carry out coupling by the interaction of Avidin and Biotin, obtain the CRP antibody of magnetic particle marker.The CRP antibody preparation process of magnetic particle marker is as follows:
(1) the DMF solution of the NHS-Biotin of 10mM is prepared.
(2) sodium azide in CRP antibody is got rid of in ultrafiltration.
(3) amount being 1:50 according to CRP antibody and NHS-Biotin mol ratio mixes, and 4 DEG C of incubations vibrate 2 hours (or standing preservation is spent the night) to make CRP antibody coupling Biotin, obtain Biotin-CRP antibody.
(4) 4 DEG C with the condition of 9000rmp under the above-mentioned mixed solution of ultrafiltration, remove unreacted NHS-Biotin.
(5) add PBS dilute solution by NHS-Biotin obtained above, wherein PBS dilute solution contains the BSA of 0.1% and the sodium azide of 0.4%, and the concentration of NHS-Biotin is 0.5mg/mL.
(7) by Biotin-CRP antibody and Avidin-magnetic particle in molar ratio 4:1 mix, at 4 DEG C of vibration 2h, then use Magneto separate frame separation and purification magnetic particle, i.e. the CRP antibody of obtained magnetic particle marker.
2. the process in immune response district: utilize oxygen plasma treatment monocrystalline silicon surface 10 minutes, to be immersed in afterwards in the ethanol solution containing 2-10%APTES and to be placed in 37 DEG C of oscillators and reacted 0.5-2h, then absolute ethanol washing is used 5 times, finally dry up with nitrogen, now the surface of monocrystalline silicon is by amination, i.e. obtained amino silicon chip.
3. the connection of immobilization carrier: amino silicon chip is immersed in the PBS solution containing 5% glutaraldehyde, room temperature reaction 1h, then after cleaning up with PBS, then direct by CRP capture antibody (because foregoing Claims in have both in conjunction with different epi-position, so I here underlines is capture antibody.) (50 μ g/mL) be added drop-wise to-amino silicon chip surface, be placed in 4 DEG C and spend the night, then amino silicon chip extracting PBS is rinsed well, utilize the BSA solution of 5% to close amino silicon chip afterwards, made totally enclosed amino silicon chip.
4. biosensor test card assembling: be assembled in test card by complete totally enclosed amino silicon chip, use ultra-sonic welded cover plate and base plate to be engaged, free capture antibody and labelled antibody freeze-drying are kept at bottom sample holes.
Embodiment 2: the preparation of magnetic particle CRP biosensor test card
The capture antibody competition sandwich immunoassay of easily extensible sensing range detects biology sensor, comprise chemical sensor 100, described chemical sensor 100 comprises cover plate 130, back up pad 170 and base plate 140, back up pad 170 is between cover plate 130 and base plate 140, back up pad 170 is provided with at least one microchannel 150, described microchannel comprises reagent storage district 160, immune response district 120 and waste liquid district 180; Described immune response district 120 comprises fixed trapped antibody, and the cover plate corresponding to position of described reagent storage area 160 is provided with liquid injection hole 110, and the cover plate corresponding to the position in waste liquid district 180 is provided with outage 190.Wherein said reagent storage area 160 comprises free capture antibody and labelled antibody.
Embodiment 3: the preparation of magnetic particle CRP biosensor test card
The capture antibody competition sandwich immunoassay of easily extensible sensing range detects biology sensor, comprise chemical sensor 200, described chemical sensor 200 comprises cover plate 230 and base plate 240, microchannel 250 is formed between cover plate 230 and base plate 240, described microchannel 250 comprises immune response district 220 and sensor regions 260, described immune response district 220 comprises fixed trapped antibody, and described cover plate has often held one to have note/outage 210 and row/liquid injection hole 210.
Embodiment 4: the test of sample
1. the matching of typical curve:
CRP psma ligand is made the antigen standard series of 0ng/mL, 5ng/mL, 50ng/mL, 1000ng/mL, 5000ng/mL, 10000ng/mL and 15000ng/mL, measure different standard items respectively, obtain corresponding magnetic signal intensity (i.e. magnetoresistivity changing value), and simulate typical curve according to antigen concentration and magnetic signal intensity.See Fig. 4, CRP antigen concentration-magnetoresistance rate of change formula is: y=0.0001x+0.1963 (in formula: y is magnetoresistivity changing value, x is CRP concentration).
2. the test of sample: sample is injected in the sample holes of the test card of embodiment 3, the capture antibody mixing 2min being stored in the magnetic particle marker antibody bottom sample holes with freeze-drying and dissociating, afterwards mixed liquor is injected into sensor regions, make it react 2min with fixing capture antibody detect the quantity of magnetic particle by magneto-dependent sensor and the CRP in sample carried out quantitatively, wherein magnetoresistivity changing value is 0.2962, and the concentration according to the CRP antigen of CRP antigen concentration-magnetoresistance rate of change formulae discovery acquisition is 999ng/ml.
Embodiment 5: the mensuration of detectability
1. CRP psma ligand is made the antigen standard series of 0ng/mL, 5ng/mL, 50ng/mL, 1000ng/mL, 5000ng/mL, 10000ng/mL and 15000ng/mL, measure different standard items respectively, obtain corresponding magnetic signal intensity (i.e. magnetoresistivity changing value), and simulate typical curve according to antigen concentration and magnetic signal intensity.The formula of CRP antigen concentration-magnetoresistance rate of change is: y=7062x-1366.8 (in formula: y is magnetoresistivity changing value, x is CRP concentration).
2. the determination of capture antibody competition sandwich immunoassay reaction method Monitoring lower-cut, measures blank 20 times, calculates mean value and the standard deviation of skip test value, detects the antigen concentration be limited to corresponding to blank averages+3 × standard deviation.Be 0.06618 by calculating blank mean value, standard deviation is 0.02687, and blank averages+3 × standard deviation equals 0.1468, is equivalent to the test value of 5ng/mL CRP, and therefore the detectability of the method and sensor can reach 5ng/mL.
3. the determination of capture antibody competition sandwich immunoassay reaction method upper limit of detection, CRP psma ligand is made the antigen standard series of 0ng/mL, 5ng/mL, 50ng/mL, 1000ng/mL, 5000ng/mL, 10000ng/mL, 15000ng/mL, 20000ng/mL, 30000ng/mL, 45000ng/mL, measure different standard items respectively, obtain corresponding magnetic signal intensity level, and simulate curve according to antigen concentration-magnetoresistivity variable quantity, see figure (3b).From experimental result as seen when antigen concentration is higher than 15000ng/mL, no longer can fit to straight line, therefore the upper limit of detection of the method is set as 15000ng/mL.
Obviously; the above embodiment of the present invention is only for example of the present invention is clearly described; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot give exhaustive to all embodiments, every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.
Claims (10)
1. the capture antibody competition sandwich immunoassay of easily extensible sensing range detects a biology sensor, comprises chemical sensor, it is characterized in that: chemical sensor comprises cover plate, back up pad and base plate, and back up pad is between cover plate and base plate; Described back up pad is provided with at least one microchannel, and described microchannel comprises reagent storage district, immune response district and waste liquid district, and surface, described immune response district is provided with fixed trapped antibody, and described reagent storage area stores free capture antibody and labelled antibody; The cover plate corresponding to position of described reagent storage area is provided with liquid injection hole, and the cover plate corresponding to position in described waste liquid district is provided with outage.
2. the capture antibody competition sandwich immunoassay of easily extensible sensing range detects a biology sensor, comprises chemical sensor, it is characterized in that: chemical sensor comprises cover plate and base plate; Form microchannel between cover plate and base plate, described microchannel comprises immune response district, and surface, described immune response district is provided with fixed trapped antibody, and described cover plate is provided with liquid injection hole and outage.
3. the capture antibody competition sandwich immunoassay of a kind of easily extensible sensing range according to claim 1 and 2 detects biology sensor, it is characterized in that: the material in the immune response district of the microchannel of described chemical sensor is monocrystalline silicon, glass, polystyrene, golden film, nitrocellulose, fluorinated polyethylene, polycation resin, hydrophilic polymer film, porosint, resin, nitrocellulose resin.
4. the capture antibody competition sandwich immunoassay of a kind of easily extensible sensing range according to claim 1 and 2 detects biology sensor, it is characterized in that: the degree of depth of described chemical sensor micro channel is 0.1-0.5mm, and length is 6-9cm.
5. a capture antibody competition sandwich immune detecting method for non-diagnostic object easily extensible sensing range, is characterized in that, comprise the steps:
1) labelled antibody is mixed with free capture antibody, the mixed liquor of both formation;
2) by sample to be tested and the mixing of described mixed liquor, the potpourri of three is formed;
3) potpourri of described three and fixed trapped antibody response;
4) reactant liquor is removed;
5) detect the marking signal on the labelled antibody in described fixed trapped antibody-antigene-labelled antibody compound and calculate the concentration of antigen.
6. capture antibody competition sandwich immune detecting method according to claim 5, is characterized in that: described in reaction system, the mol ratio of free capture antibody and described fixed trapped antibody is 1:1-1:4.
7. capture antibody competition sandwich immune detecting method according to claim 5, is characterized in that: the mol ratio of described labelled antibody and described fixed trapped antibody is 3:1-1:1.
8. capture antibody according to claim 6 competition sandwich immune detecting method, is characterized in that: described free capture antibody and fixed trapped antibody and antigen can not form free capture antibody-antigen-fixed trapped antibody complex.
9. capture antibody competition sandwich immune detecting method according to claim 6, is characterized in that: described free capture antibody and fixed trapped antibody are identical molecule; Preferably, also can be different molecules.
10. capture antibody competition sandwich immune detecting method according to claim 6, is characterized in that: the label of described labelled antibody is magnetic bead, chemiluminescence, fluorescence, collaurum or collargol.
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