CN106018805A - Non-diagnosis-targeted HIV antibody immunity detecting method and kit - Google Patents
Non-diagnosis-targeted HIV antibody immunity detecting method and kit Download PDFInfo
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- CN106018805A CN106018805A CN201610562684.3A CN201610562684A CN106018805A CN 106018805 A CN106018805 A CN 106018805A CN 201610562684 A CN201610562684 A CN 201610562684A CN 106018805 A CN106018805 A CN 106018805A
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
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- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
- G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
- G01N2333/16—HIV-1, HIV-2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention relates to a non-diagnosis-targeted HIV antibody immunity detecting method and a kit. The method comprises the following steps: specifically capturing and detecting an HIV antibody in a sample by using an HIV antigen, then adding a biotinylated secondary antibody, and incubating to form an antigen-antibody-biotinylated secondary antibody compound; adding a biotinylation primer Biotin-I in the presence of streptavidin, incubating to combine the compound with the Biotin-I, and then adding biotinylated hairpin chain Biotin-H1 and hairpin chain H2 to carry out hybridization reaction; adding avidin marked horseradish peroxidase, catalyzing a substrate solution to develop color, and carrying out quantitative detection on the HIV antibody by determining absorbance. HCR is guided into ELISA, signals are amplified by the HCR, ultra-sensitive detection of the HIV antibody is realized, and lower limit of detection is as low as 9.5 pg/mL, and is higher than that of the traditional ELISA by about 2 orders of magnitude.
Description
Technical field
The invention belongs to biomedical engineering field, particularly relate to method and the reagent of the detection HIV antibody of a kind of non-diagnostic purpose
Box.
Background technology
HIV (human immunodeficiency virus) (Human immunodeficiency virus, HIV) is to cause acquired immunodeficiency comprehensive
The pathogen of disease (Acquired Immune Deficiency Syndrome, AIDS).HIV antibody is that inhibition of HIV enters people
Being produced by human immunity responsing reaction after body, this antibody is the most invalid to inhibition of HIV, but can react HIV to a certain extent
Infection Status, be one physical signs of human body.
HIV antibody in detection blood is the laboratory method of the most the most frequently used detection AIDS viral infection, typically to pass through
Two steps: first do Primary Screening Test, if the positive, then do validation test, and the validation test positive is the most diagnosable for acquired immune deficiency syndrome (AIDS)
Virus infects.
The detection method being presently used for HIV antibody Screening tests mainly has elisa (enzyme-linked
Immunoadsordent assay, ELISA), chemiluminescence immunoassay technology (chemiluminescence immunoassay,
CLIA), Gelatin particle agglutination test (gelatin particles agglutinate experiment, GPAT), spot immune colloidal gold
Fast testing etc..The methods such as GPAT and spot immune colloidal gold fast testing are the most subjective by naked eyes judged result, and price
Expensive, result is difficult to preserve.CLIA shortcoming is that the luminescence-producing reaction of labelling catalyzing enzyme or chemiluminescent molecule is unstable, for being interrupted
, glitter luminous, and easily fission in course of reaction, cause reaction result unstable.Additionally, need to be right during detection
Separating in conjunction with phase, free phase, operating procedure is many, and testing cost is high.ELISA is still the most the most frequently used detection HIV
The method of antibody, it has the advantages such as sensitive, special, quick, but the Monitoring lower-cut of ELISA still has limitation, it is impossible to
Meet the detection infecting the HIV antibody level of low concentration in early days.
Cross chain reaction (hybridization chain reaction, HCR) is a kind of new and effective nucleic acid amplification method, HCR
The amplification of DNA can be realized under conditions of without enzyme.Chinese invention patent application CN103940989A disclose a kind of based on
The immunofluorescence technique detection TNF-α of HCR and monomolecular counting.Existing HIV antibody screening method still suffers from problems, needs
Set up detection sensitive height, easily detection method.
Summary of the invention
The technical problem to be solved is to provide a kind of HIV antibody immunologic detection method and test kit, anti-to reduce HIV
The Monitoring lower-cut concentration of body, improves detection sensitivity.
In order to solve above-mentioned technical problem, the present invention by HCR system introduce ELISA carry out signal amplify realize HIV resist
The detection of body.The method utilizes the HIV antibody in indirect ELISA capture serum, is introduced by biotin-avidin system
HCR system, HCR system expands at normal temperatures, forms DNA double chain polymerization thing.This polymeric marker biotin,
Can combine with the horseradish peroxidase of Avidin labelling, thus carry out color developing detection.
For achieving the above object, the present invention adopts the following technical scheme that the HIV antibody immunologic detection method of a kind of non-diagnostic purpose,
Including:
Make the HIV antibody in HIV antigenic specificity ground Acquisition Detection sample, be subsequently adding biotin labeling two and resist, hatch shape
Become the anti-complex of Ag-Ab-biotin labeling two；
Under conditions of Streptavidin exists, add biotinylated primer Biotin-I, hatch and make described complex and Biotin-I
In conjunction with, it is subsequently adding biotinylated hair fastener chain Biotin-H1 and hair clip chain H2 and carries out hybridization；
Adding the horseradish peroxidase of Avidin labelling, catalytic substrate liquid develops the color, and carries out HIV antibody by measuring absorbance
Detection by quantitative；
Described Biotin-I base sequence is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT
T-biotin-3’；
Described Biotin-H1 base sequence is 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT
CAA GGA CCA CCG CAT-biotin-3’；
The base sequence of described H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC
TTG AGA ACT TTG-3’。
In a detailed description of the invention, by antigen coated for HIV in ELISA Plate, after processing with confining liquid, will resist containing HIV
The detection sample of body adds microwell plate, 36～38 DEG C of incubations, captures HIV antibody with making HIV antigenic specificity；Add biotin
Goat anti-human igg is at 36～38 DEG C of incubations in change, forms the anti-complex of Ag-Ab-biotin labeling two on microwell plate.
Described biotin labeling two is anti-can be biotinylated goat anti-human igg or biotinylated mouse-anti human IgG.
In a detailed description of the invention, Biotin-H1 and H2 is first through following pretreatment: after 95～98 DEG C of water-baths 1～3min,
Ice bath immediately.
In a detailed description of the invention, after immunoreation, in microwell plate, addition Streptavidin is at 36～38 DEG C of incubations,
Getting rid of in hole and pat dry after liquid, Biotin-I is at 36～38 DEG C of incubations in addition, makes described complex be combined with Biotin-I.
In a detailed description of the invention, add Biotin-H1 and H2, react 1～3h under room temperature, make Biotin-H1 and H2
Open hairpin structure, alternately hybridization, form double-stranded DNA polymer jaggy.
In a detailed description of the invention, the horseradish peroxidase adding Avidin labelling pours out liquid after 36～38 DEG C of incubations,
Addition substrate buffer solution and substrate solution, at 36～38 DEG C of incubations, add stop buffer, colorimetric after mixing.
The present invention also provides for a kind of HIV antibody immunity detection reagent, including:
HIV antigen, biotin labeling two are anti-, Streptavidin, biotinylated primer Biotin-I, biotinylated hair fastener chain
Biotin-H1, hair clip chain H2 and the horseradish peroxidase of Avidin labelling；
Described Biotin-I base sequence is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT
T-biotin-3’；
Described Biotin-H1 base sequence is 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT
CAA GGA CCA CCG CAT-biotin-3’；
The base sequence of described H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC
TTG AGA ACT TTG-3’。
In a detailed description of the invention, described HIV antigen concentration is 80～120ng/mL；
The described anti-concentration of biotin labeling two is 1.0～2.0 μ g/mL；
Described Streptavidin concentration is 0.8～1.2 μ g/mL；
Described Biotin-I concentration is 4～6nM, adds SPSC buffer during configuration；
Described Biotin-H1 and H2 concentration is 80～120nM, adds SPSC buffer during configuration；
The horseradish peroxidase concentration of described Avidin labelling is 18～22 μ g/mL.
In a detailed description of the invention, also include being coated liquid, confining liquid, washing liquid, substrate solution, substrate buffer solution and stop buffer；
Being coated liquid is the 8～12mM PBS containing 80～120ng/mL HIV antigens；Confining liquid is containing salmon sperm dna 40～60
0.8～the 1.2%BSA solution of μ g/mL.
The present invention has the advantage that compared to existing technology
(1) HCR is the chain reaction utilizing the hairpin probe that a group complementary, kinetics is limited to realize hybridization, and it is to wait
Temperature, without carry out under conditions of enzyme, stably cheap.HCR is introduced ELISA by the present invention, and in the present invention, antibody with enzyme is
The relation of one-to-many, the while that detection being specific utilizing antigen antibody reaction to ensure, utilizes HCR to carry out signal amplification, it is achieved
The super sensitivity detection of HIV antibody, Monitoring lower-cut as little as 9.5pg/mL, about improves 2 orders of magnitude than traditional E LISA.
(2) this method is easy and simple to handle, it is not necessary to specific apparatus, and has and be prone to the features such as automatization, stability is strong, toxicity is low.
Accompanying drawing explanation
Fig. 1 is the principle schematic of HCR-ELISA of the present invention detection HIV antibody；
Fig. 2 is traditional E LISA and the Comparative result of HCR-ELISA detection by quantitative variable concentrations HIV antibody；
Fig. 3 is 40 example clinical samples testing results.
Detailed description of the invention
" room temperature " of the present invention has implication well known in the art, specifically refers to 20-30 DEG C, preferably 20-25 DEG C.
Fig. 1 is the principle schematic of HCR-ELISA of the present invention detection HIV antibody.HIV is antigen coated on microwell plate, special
Capture the HIV antibody in serum different in naturely.After adding biotinylated goat anti-human igg (immunoglobulin G), at microwell plate
On define Ag-Ab-biotin goat anti-human igg's complex.Under conditions of Streptavidin exists, by biotin-parent
And the effect between element, these complex can combine with biotin labeled initiation chain I (Biotin-I) further.Add hair clip
H1 and H2, causes chain can be catalyzed H1 and H2 and opens hairpin structure, H1 and H2 alternately hybridization, forms double-strand jaggy
DNA polymer, adds SA-HRP, and this substrate for enzymatic activity liquid develops the color, and makes signal be amplified colour developing.Hence with easily
Ultraviolet spectrophotometer can carry out detection by quantitative to the antibody in serum.
In a specific embodiment, a kind of based on cross chain reaction the HIV antibody immunologic detection method of the present invention, bag
Include following steps:
(1) HIV antibody in indirect ELISA Acquisition Detection sample: by antigen coated for HIV on microwell plate, cold preservation
Night.Add after confining liquid room temperature places 2～4h and discard confining liquid, pat dry cold preservation standby.To and detect sample addition microwell plate, 36～38 DEG C
Incubation 0.5～1h, HIV antigen can specifically capture the HIV antibody in serum.Add biotinylation goat anti-human igg 36～38 DEG C
After incubation 0.5～1h, microwell plate forms Ag-Ab-biotin mouse-anti human IgG complex.Often all use PBST after step reaction
Washing liquid washes plate, to remove unconjugated reagent；
(2) immunoassay technology amplified based on HCR signal: after immunoreation, the strepto-added in microwell plate is affine
Element, 36～38 DEG C of incubations 20～40min.Get rid of in hole and pat dry after liquid, add Biotin-I, 36～38 DEG C of incubations 20～40min
After wash plate, add Biotin-H1 and H2, mixing, ambient temperatare is put and is made it react 1～3h.Wash plate and remove unconjugated hair clip chain；
(3) colour developing and signal detection: add the horseradish peroxidase of Avidin labelling, 36～38 DEG C of incubations 20～40min, wash
Fully pat dry after plate, add substrate buffer solution and substrate solution, 36～38 DEG C of incubations 20～40 minutes.Add stop buffer, compare after mixing
Color.
Wherein, HIV antigen concentration is 80～120ng/mL；The anti-concentration of biotin labeling two is 1.0～2.0 μ g/mL；Strepto-is affine
Element (SA) concentration is 0.8～1.2 μ g/mL；Biotin-I concentration is 4～6nM, adds SPSC buffer during configuration；Biotin-H1
It is 80～120nM with H2 concentration, during configuration, adds SPSC buffer；The horseradish peroxidase (SA-HRP) of Avidin labelling
Concentration be 18～22 μ g/mL.
The base sequence of described biotinylated primer (Biotin-I) is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC
TTT TTT TTT T-biotin-3 ', as shown in SEQ ID NO.1；
The base sequence of described biotinylated hair fastener chain H1 (Biotin-H1) is 5 '-GTA GAG ATG CGG TGG TCC
TTG AGA CAA AGT TCT CAA GGA CCA CCG CAT-biotin-3 ' is as shown in SEQ ID NO.2；
The base sequence of described hair clip chain H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG
TCC TTG AGA ACT TTG-3 ' is as shown in SEQ ID NO.3.
In step (2), Biotin-H1 and H2 needs following pretreatment: after 95～98 DEG C of boiling water baths 1～3min, ice bath immediately,
Stored refrigerated is standby.Boil and hairpin structure can be made to open become single stranded DNA.
Technical scheme is described in detail by below in conjunction with the accompanying drawings with one specific embodiment.
Embodiment 1
The various solution formulas used in embodiment are as follows:
Sodium phosphate-sodium chloride buffer solution (sodium phosphate-sodium chloride buffer solution, SPSC) (50
mM Na2HPO4/ 1.0M NaCl): the Na of 17.9g2HPO4·12H2The NaCl of O, 58.5g adds a little ultra-pure water and dissolves, and uses
Hydrochloric acid adjusts pH to 7.5, is settled to 100mL.
10mM phosphate buffer (phosphate buffer solution, PBS) (pH=7.4): 8g NaCl, 0.2g KCl,
1.44g Na2HPO4·12H2O、0.24g KH2PO4It is dissolved in 1L ultra-pure water, regulates pH=7.4, autoclaving with hydrochloric acid
Latter 4 DEG C save backup.
The preparation of Biotin-I: the 3.3nmol Biotin-I synthesized by known array is dissolved to 100 μMs with 33 μ l ultra-pure waters,
And it is diluted to 5nM with SPSC buffer.Subpackage ,-20 DEG C of freezen protective are standby, avoid multigelation before using.
The preparation of Biotin-H1 and H2: the 2.1nmol H1 synthesized by known array is dissolved with 21 μ l ultra-pure waters, 2.2nmol
H2 dissolves with 21 μ l ultra-pure waters, is diluted to 100nM with SPSC buffer.-20 DEG C of freezen protective are standby, avoid anti-before using
Multiple freeze thawing.
Used washing liquid, substrate buffer solution, substrate solution and stop buffer, for the common agents of ELISA.
The HIV antibody immunologic detection method based on cross chain reaction of the present embodiment, specifically comprises the following steps that
(1) HIV antibody in indirect ELISA Acquisition Detection sample:
(1) Biotin-H1 and H2 of Biotin-H1 and H2 pretreatment: 100nM is after 98 DEG C of boiling water bath 2min, immediately
Ice bath, be saved in 4 DEG C standby；
(2) the HIV antigenic solution (containing the 10mM PBS solution of HIV antigen 1 00ng/mL) of 100ng/mL is noted
Entering in 96 hole polystyrene microwell plates, every hole 200 μ L, 4 DEG C overnight；Getting rid of and be coated liquid, every hole adds confining liquid (containing salmon
The 1%BSA solution of essence DNA 50 μ g/mL) 200 μ L, room temperature discards confining liquid after placing 2h, pats dry 4 DEG C of cold preservations standby；
(3) sample of the HIV antibody that 100 μ L contain variable concentrations adds microwell plate, 37 DEG C of water bath incubation 1h；
(4) getting rid of in hole after liquid, PBST washes plate 1 time, soaks 60s, pats dry, add 100 μ L 1.5 μ g/mL (1:4000)
Biotinylation goat anti-human igg's solution, 37 DEG C of water bath incubation 1h；
(2) immunoassay technology amplified based on HCR signal:
(5) getting rid of in micropore after liquid, PBST washes plate 1 time, soaks 60s, pats dry, add the SA of 100 μ L 1 μ g/mL,
37 DEG C of water bath incubation 30min；
(6) get rid of in hole and pat dry after liquid, add the Biotin-I of 100 μ L 5nM, 37 DEG C of incubation 30min；
(7) getting rid of in hole after liquid, conventional washing liquid is washed one time, soaks 60s, pats dry, add 40nM Biotin-H1 and
The each 50 μ L of H2, mixing, ambient temperatare is put and is made it react 1h；
(3) colour developing and signal detection:
(8) getting rid of in hole after liquid, PBST washes plate 1 time, soaks 60s, pats dry, add the SA-HRP of 100 μ L 20 μ g/mL,
Liquid is poured out after 37 DEG C of incubation 30min；
(9) the PBS washing liquid of the Tween 20 containing 2% is filled each hole, discard washing liquid in hole after standing 60s, be repeated 5 times
After pat dry；
(10) substrate buffer solution (the citrate-phosphate salt buffer containing urea peroxide) and substrate solution (molten containing TMB are added
Liquid) each 50 μ L, mixing of vibrating gently, put 37 DEG C of water bath incubations 30 minutes.(2mol/L sulphuric acid is molten to add stop buffer
Liquid) 50 μ L, colorimetric after mixing；
(11) absorbance (OD) at reaction solution 450nm uses RT 6100 microplate spectrophotometer to be measured.
The absorption spectrum in UV-2450 ultravioletvisible absorption spectrophotometer measurement solution 300-700nm wave-length coverage is utilized under room temperature.
Carrying out this embodiment institute method for building up detecting Performance, result is as follows:
The HIV antibody sample of preparation variable concentrations, makes concentration be respectively 0,10-11、10-10、10-9、10-8G/mL, detection side
Method, with embodiment 1, investigates the relation between OD value and HIV antibody concentration at 450nm.Result shows, works as HIV antibody
Concentration is 10-11G/mL to 10-8During the scope of g/mL, at HIV antibody concentration and 450nm, the absorbance of absorbance is plan
Linear relationship (accompanying drawing 2).If the three of blank times of standard deviations to be defined as Monitoring lower-cut, under the method detection HIV antibody
It is limited to 9.5pg/mL.HIV antibody concentration is respectively 10-11g/mL、10-10g/mL、10-9G/m and 10-8The blood of g/mL
Carrying out clearly 3 parallel assays, at 450nm, the relative standard deviation (RSD) of absorbance is respectively 1.1%, 5.3%, 4.3%
With 4.7%.Therefore, HCR-ELISA standard measure detection HIV antibody has higher sensitivity and repeatability.
Using the embodiment of the present invention 1 experimental procedure to detect 40 example clinical patients serum specimens, the HIV with commercialization resists simultaneously
Body ELISA detection kit (Zhuhai Lizhu Reagent Co., Ltd) contrasts, result as shown in Figure 3, two kinds of sides
Method testing result is basically identical, illustrates that the present invention detects HIV antibody and has preferable accuracy.
Claims (10)
1. the HIV antibody immunologic detection method of a non-diagnostic purpose, it is characterised in that including:
Make the HIV antibody in HIV antigenic specificity ground Acquisition Detection sample, be subsequently adding biotin labeling two and resist, hatch shape
Become the anti-complex of Ag-Ab-biotin labeling two；
Under conditions of Streptavidin exists, add biotinylated primer Biotin-I, hatch and make described complex and Biotin-I
In conjunction with, it is subsequently adding biotinylated hair fastener chain Biotin-H1 and hair clip chain H2 and carries out hybridization；
Adding the horseradish peroxidase of Avidin labelling, catalytic substrate liquid develops the color, and carries out HIV antibody by measuring absorbance
Detection by quantitative；
Described Biotin-I base sequence is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT
T-biotin-3’；
Described Biotin-H1 base sequence is 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT
CAA GGA CCA CCG CAT-biotin-3’；
The base sequence of described H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC
TTG AGA ACT TTG-3’。
HIV antibody immunologic detection method the most according to claim 1, it is characterised in that by antigen coated for HIV in ELISA Plate,
After processing with confining liquid, the detection sample containing HIV antibody is added microwell plate, 36～38 DEG C of incubations, makes HIV antigen-specific
Property ground capture HIV antibody；Add biotin labeling two and resist at 36～38 DEG C of incubations, microwell plate is formed Ag-Ab-biology
The element anti-complex of labelling two.
HIV antibody immunologic detection method the most according to claim 1 and 2, it is characterised in that described biotin labeling two resists and is
Biotinylated goat anti-human igg or biotinylated mouse-anti human IgG.
HIV antibody immunologic detection method the most according to claim 1, it is characterised in that Biotin-H1 and H2 is first through following pre-
Process: after 95～98 DEG C of water-baths 1～3min, ice bath immediately.
HIV antibody immunologic detection method the most according to claim 1, it is characterised in that after immunoreation, in microwell plate
Addition Streptavidin, at 36～38 DEG C of incubations, gets rid of in hole and pats dry after liquid, and Biotin-I is at 36～38 DEG C of incubations in addition, makes institute
State complex to be combined with Biotin-I.
6. according to the HIV antibody immunologic detection method described in claim 1,2,4 or 5, it is characterised in that add Biotin-H1 and
H2, reacts 1～3h under room temperature, make Biotin-H1 and H2 open hairpin structure, alternately hybridization, form double-stranded DNA jaggy
Polymer.
HIV antibody immunologic detection method the most according to claim 1, it is characterised in that add the Radix Cochleariae officinalis peroxide of Avidin labelling
Compound enzyme pours out liquid after 36～38 DEG C of incubations, and addition substrate buffer solution and substrate solution, at 36～38 DEG C of incubations, add stop buffer,
Colorimetric after mixing.
8. a HIV antibody immunity detection reagent, it is characterised in that including:
HIV antigen, biotin labeling two are anti-, Streptavidin, biotinylated primer Biotin-I, biotinylated hair fastener chain
Biotin-H1, hair clip chain H2 and the horseradish peroxidase of Avidin labelling；
Described Biotin-I base sequence is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT
T-biotin-3’；
Described Biotin-H1 base sequence is 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT
CAA GGA CCA CCG CAT-biotin-3’；
The base sequence of described H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC
TTG AGA ACT TTG-3’。
HIV antibody immunity detection reagent the most according to claim 8, it is characterised in that:
Described HIV antigen concentration is 80～120ng/mL；
The described anti-concentration of biotin labeling two is 1.0～2.0 μ g/mL；
Described Streptavidin concentration is 0.8～1.2 μ g/mL；
Described Biotin-I concentration is 4～6nM, adds SPSC buffer during configuration；
Described Biotin-H1 and H2 concentration is 80～120nM, adds SPSC buffer during configuration；
The horseradish peroxidase concentration of described Avidin labelling is 18～22 μ g/mL.
HIV antibody immunity detection reagent the most according to claim 8 or claim 9, it is characterised in that also include being coated liquid, closing
Liquid, washing liquid, substrate solution, substrate buffer solution and stop buffer；Being coated liquid is the 8～12mM PBS containing 80～120ng/mL HIV antigens
Buffer；Confining liquid is the 0.8～1.2%BSA solution containing salmon sperm dna 40～60 μ g/mL.
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| CN106546570A (en) * | 2016-11-03 | 2017-03-29 | 天津市宝坻区人民医院 | A kind of detection method of ABCG2 antibody |
| CN106771174A (en) * | 2016-11-09 | 2017-05-31 | 中南大学湘雅三医院 | The HCV antibody mediated immunities detection method and kit of a kind of non-diagnostic purpose |
| CN110441525A (en) * | 2018-05-02 | 2019-11-12 | 南京大学 | Protein chemistry luminescence imaging analysis method based on DNA microarray |
| CN110940809A (en) * | 2019-12-09 | 2020-03-31 | 福州大学 | Technology for detecting alpha-fetoprotein in blood based on combination of laser-induced fluorescence and paper chip |
| CN111819288A (en) * | 2018-01-26 | 2020-10-23 | 北京生命科学研究所 | Immune signal amplification method based on hybrid chain reaction |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987005394A1 (en) * | 1986-02-26 | 1987-09-11 | United States Of America, Represented By The Unite | Competitive elisa for the detection of antibodies |
| CN1030300A (en) * | 1987-06-15 | 1989-01-11 | 科特公司 | Utilize the enzyme-labeled immunity detection method to measure antigen and/or antibody in the human body simultaneously |
| CN1488943A (en) * | 2002-10-09 | 2004-04-14 | 上海市刑事科学技术研究所 | Method for preparing diaghostic reagent of human immunodeficiency virus antibody |
| CN1673749A (en) * | 2005-03-23 | 2005-09-28 | 北京科卫临床诊断试剂有限公司 | HIV viral antibody/antigen diagnostic reagent kit and preparing method thereof and detecting method |
| CN101581720A (en) * | 2008-05-14 | 2009-11-18 | 上海英旻泰生物技术有限公司 | Kit for confirming human immunodeficiency virus antibody |
| CN103940989A (en) * | 2014-04-25 | 2014-07-23 | 山东大学 | Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor |
-
2016
- 2016-07-15 CN CN201610562684.3A patent/CN106018805B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987005394A1 (en) * | 1986-02-26 | 1987-09-11 | United States Of America, Represented By The Unite | Competitive elisa for the detection of antibodies |
| CN1030300A (en) * | 1987-06-15 | 1989-01-11 | 科特公司 | Utilize the enzyme-labeled immunity detection method to measure antigen and/or antibody in the human body simultaneously |
| CN1488943A (en) * | 2002-10-09 | 2004-04-14 | 上海市刑事科学技术研究所 | Method for preparing diaghostic reagent of human immunodeficiency virus antibody |
| CN1673749A (en) * | 2005-03-23 | 2005-09-28 | 北京科卫临床诊断试剂有限公司 | HIV viral antibody/antigen diagnostic reagent kit and preparing method thereof and detecting method |
| CN101581720A (en) * | 2008-05-14 | 2009-11-18 | 上海英旻泰生物技术有限公司 | Kit for confirming human immunodeficiency virus antibody |
| CN103940989A (en) * | 2014-04-25 | 2014-07-23 | 山东大学 | Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor |
Non-Patent Citations (5)
| Title |
|---|
| JINGJIN ZHAO ET AL: "An electrochemical aptasensor based on hybridization chain reaction with enzyme-signal amplification for interferon-gamma detection", 《BIOSENSORS AND BIOELECTRONICS》 * |
| LUTZ GÜRTLER: ""Difficulties and strategies of HIV diagnosis"，Lutz Gürtler，THE LANCET，第348卷，第176-179卷，19960720", 《THE LANCET》 * |
| SHUYAN NIU ET AL: "Fluorescence detection for DNA using hybridization chain reaction with enzyme-amplification", 《CHEM. COMMUN.》 * |
| WEILING SONG ET AL: "Ultrasensitive electrochemical detection for thrombin using hybridization chain reaction with enzyme-amplification", 《ANALYTICA CHIMICA ACTA》 * |
| 薛茵 等: "间接ELISA检测HIV-1/-2抗体", 《天津医科大学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106546570A (en) * | 2016-11-03 | 2017-03-29 | 天津市宝坻区人民医院 | A kind of detection method of ABCG2 antibody |
| CN106771174A (en) * | 2016-11-09 | 2017-05-31 | 中南大学湘雅三医院 | The HCV antibody mediated immunities detection method and kit of a kind of non-diagnostic purpose |
| CN111819288A (en) * | 2018-01-26 | 2020-10-23 | 北京生命科学研究所 | Immune signal amplification method based on hybrid chain reaction |
| CN111819288B (en) * | 2018-01-26 | 2023-07-14 | 北京生命科学研究所 | Immune signal amplification method based on hybridization chain reaction |
| CN110441525A (en) * | 2018-05-02 | 2019-11-12 | 南京大学 | Protein chemistry luminescence imaging analysis method based on DNA microarray |
| CN110940809A (en) * | 2019-12-09 | 2020-03-31 | 福州大学 | Technology for detecting alpha-fetoprotein in blood based on combination of laser-induced fluorescence and paper chip |
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