CN106018805B - The HIV antibody immunologic detection method and kit of a kind of non-diagnostic purpose - Google Patents

The HIV antibody immunologic detection method and kit of a kind of non-diagnostic purpose Download PDF

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CN106018805B
CN106018805B CN201610562684.3A CN201610562684A CN106018805B CN 106018805 B CN106018805 B CN 106018805B CN 201610562684 A CN201610562684 A CN 201610562684A CN 106018805 B CN106018805 B CN 106018805B
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biotin
hiv
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CN106018805A (en
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聂新民
桂嵘
董彩霞
黄蓉
贺思涵
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Third Xiangya Hospital of Central South University
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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
    • 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
    • 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

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Abstract

The present invention relates to a kind of HIV antibody immunologic detection method of non-diagnostic purpose and kit, this method to include：With the making HIV antigentic specificities HIV antibody in Acquisition Detection sample, then adds biotin labeling secondary antibody, and incubation forms antigen-antibody biotin labeling secondary antibody compound；Under the conditions of existing for Streptavidin, biotinylated primer Biotin I are added, incubation makes the compound be combined with Biotin I, then adds biotinylated hair fastener chain Biotin H1 and hair clip chain H2 and carries out hybridization reaction；The horseradish peroxidase of Avidin mark is added, catalysis substrate solution colour developing, the quantitative detection of HIV antibody is carried out by determining absorbance.HCR is introduced ELISA by the present invention, is carried out signal amplification using HCR, is realized the super sensitivity detection of HIV antibody, Monitoring lower-cut as little as 9.5pg/mL, 2 orders of magnitude are about improved than traditional ELISA.

Description

The HIV antibody immunologic detection method and kit of a kind of non-diagnostic purpose
Technical field
The invention belongs to biomedical engineering field, more particularly to a kind of method of the detection HIV antibody of non-diagnostic purpose And kit.
Background technology
Human immunodeficiency virus (Human immunodeficiency virus, HIV) is to cause acquired immunity to lack Fall into the pathogen of syndrome (Acquired Immune Deficiency Syndrome, AIDS).HIV antibody is that inhibition of HIV enters Enter after human body as produced by human immunity responsing reaction, this antibody is substantially invalid to inhibition of HIV, but can react to a certain extent HIV Infection Status, it is one physical signs of human body.
HIV antibody in detection blood is the laboratory method of detection AIDS viral infection the most frequently used at present, typically To pass through two steps：Primary Screening Test is done first, if the positive, then does validation test, and the positive ability of validation test is diagnosable to be AIDS viral infection.
The detection method for being presently used for HIV antibody Screening tests mainly has EUSA (enzyme- Linked immunoadsordent assay, ELISA), chemiluminescence immunoassay technology (chemiluminescence Immunoassay, CLIA), Gelatin particle agglutination test (gelatin particles agglutinate experiment, GPAT), spot immune colloidal gold quickly experiment etc..The methods of GPAT and spot immune colloidal gold are quickly tested is judged by naked eyes As a result it is more subjective and expensive, result is not easy to maintain.CLIA shortcomings are to mark catalyzing enzyme or the hair of chemiluminescent molecule Light reaction is unstable, for interruption, glitter light, and easily fission during the course of the reaction, cause reaction result unstable It is fixed.In addition, need to be separated during detection to combination phase, free phase, operating procedure is more, and testing cost is high.ELISA is still clinically The method of the most frequently used detection HIV antibody, it has the advantages that sensitive, special, quick, but ELISA Monitoring lower-cut still has There is limitation, it is impossible to meet the horizontal detection of the HIV antibody of infection early stage low concentration.
Cross chain reaction (hybridization chain reaction, HCR) is a kind of new and effective nucleic acid amplification Method, HCR can realize DNA amplification under conditions of without enzyme.Chinese invention patent application CN103940989A discloses one Kind detects TNF-α based on the immunofluorescence technique of HCR and monomolecular counting.Existing HIV antibody screening method still suffers from many ask Topic is, it is necessary to establish detection sensitive height, easily detection method.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of HIV antibody immunologic detection method and kit, to reduce The Monitoring lower-cut concentration of HIV antibody, improve detection sensitivity.
In order to solve the above-mentioned technical problem, the present invention introduces ELISA by HCR systems and carries out signal amplification to realize HIV The detection of antibody.This method is introduced using the HIV antibody in indirect ELISA capture serum by biotin-avidin system HCR systems, HCR systems are expanded at normal temperatures, form DNA double chain polymer.Polymeric marker biotin, Ke Yihe The horseradish peroxidase of Avidin mark combines, so as to carry out color developing detection.
To achieve the above object, the present invention adopts the following technical scheme that：A kind of HIV antibody immune detection of non-diagnostic purpose Method, including：
With the making HIV antigentic specificities HIV antibody in Acquisition Detection sample, biotin labeling secondary antibody is then added, be incubated Form Ag-Ab-biotin labeling secondary antibody compound；
Under the conditions of existing for Streptavidin, add biotinylated primer Biotin-I, incubation make the compound with Biotin-I is combined, and is then added biotinylated hair fastener chain Biotin-H1 and hair clip chain H2 and is carried out hybridization reaction；
The horseradish peroxidase of Avidin mark is added, catalysis substrate solution colour developing, is resisted by determining absorbance progress HIV The quantitative detection of body；
The Biotin-I base sequences are 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT T-biotin-3’；
The Biotin-H1 base sequences are 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT CAA GGA CCA CCG CAT-biotin-3’；
The base sequence of the H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC TTG AGA ACT TTG-3’。
In a detailed embodiment, HIV antigen coats after being handled with confining liquid, will be contained into HIV on ELISA Plate The detection sample of antibody adds microwell plate, 36~38 DEG C of incubations, captures HIV antibody with making HIV antigentic specificities；Add biotin Change goat anti-human igg in 36~38 DEG C of incubations, formation Ag-Ab-biotin labeling secondary antibody compound on microwell plate.
The biotin labeling secondary antibody can be biotinylated goat anti-human igg or biotinylated mouse anti-human igg.
In a detailed embodiment, Biotin-H1 and H2 is first through following pretreatments：Through 95~98 DEG C of water-baths 1~ After 3min, ice bath immediately.
In a detailed embodiment, after immune response, Streptavidin is added into microwell plate at 36~38 DEG C Incubate, get rid of in hole and patted dry after liquid, add Biotin-I and incubated at 36~38 DEG C, the compound is tied with Biotin-I Close.
In a detailed embodiment, add Biotin-H1 and H2, at room temperature react 1~3h, make Biotin-H1 and H2 opens hairpin structure, alternately hybridized, and forms double-stranded DNA polymer jaggy.
In a detailed embodiment, the horseradish peroxidase for adding Avidin mark falls after 36~38 DEG C of incubations Go out liquid, add substrate buffer solution and substrate solution and incubated at 36~38 DEG C, add terminate liquid, colorimetric after mixing.
The present invention also provides a kind of HIV antibody immunity detection reagent, including：
HIV antigens, biotin labeling secondary antibody, Streptavidin, biotinylated primer Biotin-I, biotinylated hair fastener Chain Biotin-H1, hair clip chain H2 and Avidin mark horseradish peroxidase；
The Biotin-I base sequences are 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT T-biotin-3’；
The Biotin-H1 base sequences are 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT CAA GGA CCA CCG CAT-biotin-3’；
The base sequence of the H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC TTG AGA ACT TTG-3’。
In a detailed embodiment, the HIV antigen concentrations are 80~120ng/mL；
The biotin labeling secondary antibody concentration is 1.0~2.0 μ g/mL；
The Streptavidin concentration is 0.8~1.2 μ g/mL；
The Biotin-I concentration is 4~6nM, adds SPSC buffer solutions during configuration；
Biotin-H1 the and H2 concentration is 80~120nM, adds SPSC buffer solutions during configuration；
The horseradish peroxidase concentration of the Avidin mark is 18~22 μ g/mL.
In a detailed embodiment, in addition to coating buffer, confining liquid, washing lotion, substrate solution, substrate buffer solution and termination Liquid；Coating buffer is 8~12mM PBSs of the HIV antigens containing 80~120ng/mL；Confining liquid be containing salmon sperm dna 40~ 60 μ g/mL 0.8~1.2%BSA solution.
The present invention has the following advantages that compared with prior art：
(1) HCR is to realize the chain reaction of hybridization using one group of complementary, hairpin probe that dynamics is limited, it be Isothermal, without what is carried out under conditions of enzyme, it is stable and cheap.HCR is introduced ELISA by the present invention, and antibody is with enzyme in the present invention One-to-many relation, ensureing that detection is specific simultaneously using antigen-antibody reaction, carrying out signal amplification using HCR, realize The super sensitivity detection of HIV antibody, Monitoring lower-cut as little as 9.5pg/mL, 2 orders of magnitude is about improved than traditional ELISA.
(2) this method is easy to operate, without specific apparatus, and with being easy to automation, the spy such as stability is strong, toxicity is low Point.
Brief description of the drawings
Fig. 1 is the principle schematic of HCR-ELISA detections HIV antibody of the present invention；
Fig. 2 is the Comparative result that traditional ELISA and HCR-ELISA quantitatively detect various concentrations HIV antibody；
Fig. 3 is 40 clinical samples testing results.
Embodiment
" room temperature " of the present invention has implication well known in the art, in particular to 20-30 a DEG C, preferably 20-25 DEG C.
Fig. 1 is the principle schematic of HCR-ELISA detections HIV antibody of the present invention.HIV antigen coats are special on microwell plate The HIV antibody in serum is captured different in naturely.After adding biotinylated goat anti-human igg (immunoglobulin G), the shape on microwell plate Into Ag-Ab-biotin goat anti-human igg's compound.Under the conditions of existing for Streptavidin, by biotin-affine Effect between element, the initiation chain I (Biotin-I) that these compounds can be further with biotin labeling are combined.Add hair clip H1 And H2, triggering chain to be catalyzed H1 and H2 and open hairpin structure, H1 and H2 alternately hybridize, and form double-stranded DNA polymer jaggy, SA-HRP is added, substrate for enzymatic activity liquid colour developing, enables signal amplification colour developing.Therefore easy ultraviolet specrophotometer is utilized Quantitative detection can be carried out to the antibody in serum.
In a specific embodiment, a kind of HIV antibody immune detection side based on cross chain reaction of the invention Method, comprise the following steps：
(1) HIV antibody in indirect ELISA Acquisition Detection sample：By HIV antigen coats on microwell plate, refrigerated Night.Add after confining liquid room temperature places 2~4h and discard confining liquid, it is standby to pat dry refrigeration.Will detection sample add microwell plate, 36~ 38 DEG C of 0.5~1h of incubation, HIV antigens can specifically capture the HIV antibody in serum.Add biotinylation goat anti-human igg 36 After~38 DEG C incubate 0.5~1h, Ag-Ab-biotin mouse anti-human igg compound is formed on microwell plate.Often walk reaction PBST washing lotion board-washings are used afterwards, to remove uncombined reagent；
(2) immunoassay technology based on the amplification of HCR signals：After immune response, the strepto- added into microwell plate is close And element, 36~38 DEG C of 20~40min of incubation.Get rid of in hole and patted dry after liquid, addition Biotin-I, 36~38 DEG C of incubations 20~ Board-washing after 40min, Biotin-H1 and H2 is added, mixed, place makes it react 1~3h at room temperature.Board-washing removes uncombined hair Clip chain；
(3) colour developing and signal detection：The horseradish peroxidase of addition Avidin mark, 36~38 DEG C of incubations 20~ 40min, fully pat dry after board-washing, add substrate buffer solution and substrate solution, 36~38 DEG C incubate 20~40 minutes.Add and terminate Liquid, colorimetric after mixing.
Wherein, HIV antigen concentrations are 80~120ng/mL；Biotin labeling secondary antibody concentration is 1.0~2.0 μ g/mL；Strepto- Avidin (SA) concentration is 0.8~1.2 μ g/mL；Biotin-I concentration is 4~6nM, adds SPSC buffer solutions during configuration； Biotin-H1 and H2 concentration is 80~120nM, adds SPSC buffer solutions during configuration；The horseradish peroxidase of Avidin mark (SA-HRP) concentration is 18~22 μ g/mL.
The base sequence of the 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 the 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 ' are as shown in SEQ ID NO.2；
The base sequence of the hair clip chain H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC TTG AGA ACT TTG-3 ' are as shown in SEQ ID NO.3.
Biotin-H1 and H2 need to be pre-processed as follows in step (2)：After 95~98 DEG C of 1~3min of boiling water bath, ice immediately Bath, it is stored refrigerated standby.Boiling can open hairpin structure turns into single stranded DNA.
Technical scheme is described in detail with a specific embodiment below in conjunction with the accompanying drawings.
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) (50mM Na2HPO4/1.0M NaCl)：17.9g Na2HPO4·12H2O, 58.5g NaCl add a little Ultra-pure water dissolves, and adjusts pH to 7.5 with hydrochloric acid, is settled to 100mL.
10mM phosphate buffers (phosphate buffer solution, PBS) (pH=7.4)：8g NaCl、0.2g KCl、1.44g Na2HPO4·12H2O、0.24g KH2PO4It is dissolved into 1L ultra-pure waters, adjusts pH=7.4 with hydrochloric acid, high pressure is gone out 4 DEG C save backup after bacterium.
Biotin-I preparation：The 3.3nmol Biotin-I synthesized by known array are dissolved to 33 μ l ultra-pure waters 100 μM, and it is diluted to 5nM with SPSC buffer solutions.Packing, -20 DEG C of freezen protectives are standby, and multigelation is avoided before use.
Biotin-H1 and H2 preparation：The 2.1nmol H1 synthesized by known array are dissolved with 21 μ l ultra-pure waters, 2.2nmol H2 are dissolved with 21 μ l ultra-pure waters, and 100nM is diluted to SPSC buffer solutions.- 20 DEG C of freezen protectives are standby, kept away before use Exempt from multigelation.
Used washing lotion, substrate buffer solution, substrate solution and terminate liquid, it is ELISA common agents.
The HIV antibody immunologic detection method based on cross chain reaction of the present embodiment, is comprised the following steps that：
(1) HIV antibody in indirect ELISA Acquisition Detection sample：
(1) Biotin-H1 and H2 pretreatments：100nM Biotin-H1 and H2 is after 98 DEG C of boiling water bath 2min, ice immediately Bath, be stored in 4 DEG C it is standby；
(2) 100ng/mL HIV antigenic solutions (the 10mM PBS solutions containing HIV antigen 1s 00ng/mL) are injected into 96 holes In polystyrene micropore plate, per the μ L of hole 200,4 DEG C overnight；Coating buffer is got rid of, the confining liquid (μ containing salmon sperm dna 50 is added per hole G/mL 1%BSA solution) 200 μ L, room temperature place 2h after discard confining liquid, it is standby to pat dry 4 DEG C of refrigerations；
(3) sample that 100 μ L are contained to the HIV antibody of various concentrations adds microwell plate, and 37 DEG C of water baths incubate 1h；
(4) get rid of in hole after liquid, PBST board-washings 1 time, soak 60s, pat dry, add the μ g/mL (1 of 100 μ L 1.5:4000) Biotinylation goat anti-human igg's solution, 37 DEG C of water baths incubate 1h；
(2) immunoassay technology based on the amplification of HCR signals：
(5) get rid of in micropore after liquid, PBST board-washings 1 time, soak 60s, pat dry, addition 100 μ L 1 μ g/mL SA, 37 DEG C water bath incubates 30min；
(6) get rid of in hole and patted dry after liquid, add 100 μ L 5nM Biotin-I, 37 DEG C of incubation 30min；
(7) get rid of in hole after liquid, conventional washing lotion is washed one time, is soaked 60s, is patted dry, add 40nM Biotin-H1 and H2 Each 50 μ L, mix, place makes it react 1h at room temperature；
(3) colour developing and signal detection：
(8) get rid of in hole after liquid, PBST board-washings 1 time, soak 60s, pat dry, add the μ g/mL of 100 μ L 20 SA-HRP, Liquid is poured out after 37 DEG C of incubation 30min；
(9) the PBS washing lotions containing 2% Tween 20 are filled into each hole, washing lotion in hole is discarded after standing 60s, after being repeated 5 times Pat dry；
(10) substrate buffer solution (the citrate-phosphate salt buffer containing urea peroxide) and substrate solution are added (containing the molten of TMB Liquid) each 50 μ L, gently vibration mixes, puts 37 DEG C of water baths and incubate 30 minutes.Add terminate liquid (2mol/L sulfuric acid solutions) 50 μ L, colorimetric after mixing；
(11) absorbance at reaction solution 450nm (OD) is measured using the microplate spectrophotometers of RT 6100. The absorption spectrum in UV-2450 ultravioletvisible absorption spectrophotometer measurement solution 300-700nm wave-length coverages is utilized at room temperature.
Detection performance investigation is carried out to the embodiment institute method for building up, it is as a result as follows：
The HIV antibody sample of various concentrations is prepared, it is respectively 0,10 to make concentration-11、10-10、10-9、10-8G/mL, detection side Method investigates the relation between OD values and HIV antibody concentration at 450nm with embodiment 1.As a result show, when HIV antibody concentration exists 10-11G/mL to 10-8During g/mL scope, the absorbance of HIV antibody concentration and absorbance at 450nm is (attached in pseudo-linear relation Fig. 2).If the three times standard deviation of blank control is defined as into Monitoring lower-cut, the lower limit of this method detection HIV antibody is 9.5pg/ mL.It is respectively 10 to HIV antibody concentration-11g/mL、10-10g/mL、10-9G/m and 10-8G/mL serum carries out 3 parallel surveys Fixed, the relative standard deviation (RSD) of absorbance is respectively 1.1%, 5.3%, 4.3% and 4.7% at 450nm.Therefore, HCR- ELISA method, which quantitatively detects HIV antibody, has higher sensitivity and repeatability.
40 clinical patients serum specimens are detected using the experimental procedure of the embodiment of the present invention 1, while and commercialization HIV antibody ELISA detection kit (Zhuhai Lizhu Reagent Co., Ltd) contrasted, as a result as shown in Figure 3, two Kind method testing result is basically identical, illustrates that present invention detection HIV antibody has preferable accuracy.

Claims (2)

  1. A kind of 1. HIV antibody immunologic detection method of non-diagnostic purpose, it is characterised in that including：
    With the making HIV antigentic specificities HIV antibody in Acquisition Detection sample, biotin labeling secondary antibody is then added, be incubated and formed Ag-Ab-biotin labeling secondary antibody compound；
    Under the conditions of existing for Streptavidin, add biotinylated primer Biotin-I, incubation make the compound with Biotin-I is combined, and is then added biotinylated hair fastener chain Biotin-H1 and hair clip chain H2 and is carried out hybridization reaction；
    The horseradish peroxidase of Avidin mark is added, catalysis substrate solution colour developing, HIV antibody is carried out by determining absorbance Quantitative detection；
    The Biotin-I base sequences are 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT T- biotin-3’；
    The Biotin-H1 base sequences are 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT CAA GGA CCA CCG CAT-biotin-3’；
    The base sequence of the H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC TTG AGA ACT TTG-3’;
    The biotin labeling secondary antibody is biotinylated goat anti-human igg or biotinylated mouse anti-human igg;
    By HIV antigen coats on ELISA Plate, after being handled with confining liquid, the detection sample containing HIV antibody is added into microwell plate, 36 ~ 38 DEG C of incubations, capture HIV antibody with making HIV antigentic specificities；Add biotin labeling secondary antibody to incubate at 36 ~ 38 DEG C, micro- Ag-Ab-biotin labeling secondary antibody compound is formed on orifice plate；
    The Biotin-H1 and H2 are first through following pretreatments：After 95 ~ 98 DEG C of 1 ~ 3min of water-bath, ice bath immediately；
    After immune response, into microwell plate, addition Streptavidin incubates at 36 ~ 38 DEG C, gets rid of in hole and is patted dry after liquid, is added Enter Biotin-I to incubate at 36 ~ 38 DEG C, the compound is combined with Biotin-I；
    Biotin-H1 and H2 is added, reacts 1 ~ 3h at room temperature, Biotin-H1 and H2 opened hairpin structure, alternately hybridized, shape Into double-stranded DNA polymer jaggy；
    The horseradish peroxidase for adding Avidin mark pours out liquid after 36 ~ 38 DEG C of incubations, adds substrate buffer solution and bottom Thing liquid incubates at 36 ~ 38 DEG C, adds terminate liquid, colorimetric after mixing.
  2. A kind of 2. HIV antibody immunity detection reagent, it is characterised in that including：
    HIV antigens, biotin labeling secondary antibody, Streptavidin, biotinylated primer Biotin-I, biotinylated hair fastener chain Biotin-H1, hair clip chain H2 and Avidin mark horseradish peroxidase, coating buffer, confining liquid, washing lotion, substrate solution, substrate Buffer solution and terminate liquid；
    The Biotin-I base sequences are 5 '-TCT CAA GGA CCA CCG CAT CTC TAC TTT TTT TTT T- biotin-3’；
    The Biotin-H1 base sequences are 5 '-GTA GAG ATG CGG TGG TCC TTG AGA CAA AGT TCT CAA GGA CCA CCG CAT-biotin-3’；
    The base sequence of the H2 is 5 '-TCT CAA GGA CCA CCG CAT CTC TAC ATG CGG TGG TCC TTG AGA ACT TTG-3’；
    The HIV antigen concentrations are 80 ~ 120 ng/mL；
    The biotin labeling secondary antibody concentration is 1.0 ~ 2.0 μ g/mL；
    The Streptavidin concentration is 0.8 ~ 1.2 μ g/mL；
    The Biotin-I concentration is 4 ~ 6 nM, adds SPSC buffer solutions during configuration；
    Biotin-H1 the and H2 concentration is 80 ~ 120 nM, adds SPSC buffer solutions during configuration；
    The horseradish peroxidase concentration of the Avidin mark is 18 ~ 22 μ g/mL；
    The coating buffer is 8 ~ 12 mM PBSs containing 80 ~ 120 ng/mL HIV antigens；Confining liquid is containing salmon sperm dna 40 ~ 60 μ g/mL 0.8 ~ 1.2%BSA solution.
CN201610562684.3A 2016-07-15 2016-07-15 The HIV antibody immunologic detection method and kit of a kind of non-diagnostic purpose Expired - Fee Related CN106018805B (en)

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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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