CN105527426A

CN105527426A - A liquid-chip HIV-antigen antibody combined detection kit and a detecting method

Info

Publication number: CN105527426A
Application number: CN201511014153.2A
Authority: CN
Inventors: 袁玉华; 孔伟伟; 李艳
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2015-12-31
Filing date: 2015-12-31
Publication date: 2016-04-27

Abstract

The invention discloses a liquid-chip HIV-antigen antibody combined detection kit and a detecting method and belongs to the field of immunoassay. The kit comprises microspheres respectively covered with HIV gp41, p17, p24, p31, p66 and gp120 antigens, a biotinylated anti-human secondary antibody, an SA-PE solution, microspheres covered with a p24 monoclonal antibody, and a monoclonal antibody labeled with fluorescein and matching the p24 monoclonal antibody. Combined detection for six HIV antibodies and a P24 antigen can be performed simultaneously through a small amount of a sample. Compared with an immunoblotting method, the kit and the detecting method are high in sensitivity and specificity. Through the kit and the detecting method, the HIV antibodies can be detected timely in the earlier period of infection, thus achieving early detection and early intervention of HIV infection, reducing HIV transmission and ensuring safety of blood transfusion. The kit and the detecting method have a wide application prospect.

Description

Luminex HIV antigen-antibody combined detection kit and detection method

Technical field

The invention belongs to immunoassay field, specifically a kind of Luminex HIV antigen-antibody combined detection kit and detection method.

Background technology

HIV detects and comprises the methods such as P24 antigen, HIV antibody, HIV virus load, cd4 cell counting.The method that the clinical HIV of carrying out blood screening is the most frequently used is at present enzyme linked immunosorbent assay (ELISA).Up to the present HIVELISA reagent developed into for the 4th generation.The main flow reagent that the current world and domestic HIV detect and method be the 3rd generation reagent, dual-antigen sandwich method detects the antibody in sample, enzyme marker is specific HIV antigen, the antibody subtype HIV-1/2/OIgG/M/A that HIV is different can be detected, its detect sensitivity and specificity higher than the 1st, 2 generation reagent, improve the reactivity of O group's sample, window phase foreshortens to 22 days.

There are 3 kinds of HIV-1 to confirm experimental technique at present in the world, comprise immunoblot experiment, immunofluorescence experiment and band immunity test.Specifying that the unique confirmation method of China's HIV is immunoblot experiment (WB) in " acquired immune deficiency syndrome (AIDS) inspection specifications " that the Ministry of Public Health promulgates, is also special, the most responsive method confirming HIV.WB is through SDS-PAGE by HIV totivirus antigen, protein bands different for molecular size range separately, and then the albumen electricity of these different molecular weight sizes is forwarded on nitrocellulose filter, then this film is cut into ribbon and to make on every bar nitrocellulose filter all containing through the isolated HIV antigen of SDS-PAGE.Add the serum specimen through damping fluid dilution, if containing HIV antibody in serum specimen, macroscopic band will be shown in the relevant position of nitrocellulose filter after chromogenic reaction.The criterion of HIV immunoblot experiment is: form at least two Env and be with (gp41, gp120/gp160) or an Env band to add that p24 is with result to be judged to be the positive, occur that banding pattern can not meet Positive judgement standards result and be judged to be suspicious, form result without any HIV band and be judged to be feminine gender, the false positive results that primary dcreening operation is tested can be differentiated; The early stage of the HIV course of disease or late period p31 band usually lack, can be used as a prediction index of the HIV course of disease.But HIV immunoblot experiment has the false positive rate of 2%, be more common in greatly vaccine inoculation history and the patient suffering from autoimmune disease.If enzyme linked immunological experiment and immunoblot experiment are combined detection HIV antibody, greatly can improve the recall rate (about 99%) of HIV antibody, false positive rate is reduced to 0.0006.Although WB has higher specificity based on the separation of the different antigen component of HIV, concentrated and purifying, but WB with ELISA and other detection methods are compared, and testing cost is high, experimental technique requires strictly, influence factor is more, the false positive rate of erroneous judgement banding pattern and misoperation is more than 2%, the testing result of the sample WB of the 4%-20% of the first reinspection HIV positive is " antibody is uncertain ", the a part uncertain sample of HIV antibody and p24 are with relation, and this may be relevant with non-specific antibody.Uncertain sample can be determined after need following up a case by regular visits to for 3,6 months, makes Voluntary Blood Donors individual, family, work and life bear huge pressure, also brings inconvenience to staff.

The liquid-phase chip technology being described as genome times afterwards comprehensively chip technology grows up the mid-90 in 20th century, liquid-phase chip technology is also known as Suspension array technique, multifunctional suspending dot matrix techniques (Multi-AnalyteSuspensionArray, MASA), Luminex technology or xMAP(flexiblemultiple-analyteprofiling) technology.Liquid-phase chip technology adopt diameter be the poly-third ethene microballoon of 5.6um as detection carrier, each microballoon redness and orange two kinds of fluorescent dyes are encoded, and can be obtained the microballoon of different fluorescent dye in 100 by the ratio adjusting these two kinds of fluorescent dyes.After microballoon is irradiated by the laser of 635nm, microballoon can launch the fluorescence of 658nm and 712nm.The address of often kind of microballoon is uniquely determined by the ratio of two kinds of fluorescent dyes, by often kind of coding microball covalent coupling capture molecules so often kind of coding microball have the test item of its correspondence, when detecting, the microballoon and sample to be tested that carry out different determinand are mixed to form the potpourri of microballoon-capture molecules-determinand, finally add Streptavidin phycoerythrin (Strepavidin-phycoerythrin, SA-PE) as reporter molecules and potpourri specific binding, in a reacting phase, 100 kinds of reactions can be detected at most.New Luminex liquid-phase chip system takes three kinds of fluorescent materials, by adjusting the ratio of three kinds of fluorescent dyes, microballoon can be divided into 500 kinds.

When Luminex-100/200/xMAP detects, instrument abstraction reaction sample excites two kinds/tri-kinds fluorescent material signal receivers of microballoon inside to receive fluorescence signal by red laser, distinguishes microballoon numbering, determines the capture molecules on microballoon; Green fluorescence excites in conjunction with the reporter molecule (phycoerythrin) on microballoon, in conjunction with more fluorescence signals stronger, result by average fluorescent strength (MeanFluorescentIntensity, MFI) judge.The signal of microballoon and phycoerythrin signal are simultaneously by machine recognition, and system just thinks useful signal, and the interference that doing so avoids other materials improves the specificity of experiment.

Summary of the invention

The present invention is exactly the circumscribed problem detected to overcome the high and WB of HIV third generation reagent false positive rate, a kind of Luminex HIV antigen-antibody combined detection kit proposed and detection method.

The present invention realizes according to following technical scheme.

A kind of Luminex HIV antigen-antibody combined detection kit, comprise the microballoon being wrapped quilt by Human Immunodeficiency Virus antigen gp41, p17, p24, p31, p66, gp120 respectively, biontinylated anti-human two resists, SA-PE solution; The microballoon of p24 monoclonal antibody bag quilt, fluorescein-labeled monoclonal antibody of matching with p24 monoclonal antibody.

The bag of arbitrary described Human Immunodeficiency Virus antigen gp41, p17, p24, p31, p66, gp120 and p24 monoclonal antibody and microballoon is that the antigen of 2-5ug or antibody bag are by 1 × 10 by ratio ⁶individual microballoon.

Described biontinylated anti-human two resists for biotinylation goat anti-human antibody, and the anti-concentration of biontinylated anti-human two is 4-8ug/ml.

Described monoclonal antibody of matching with p24 monoclonal antibody is the anti-p24KC57 antibody that RD1 marks; The concentration of fluorescein-labeled monoclonal antibody of matching with p24 monoclonal antibody is 0.5-1ug/ml.

The concentration of described SA-PE solution is 13-15ug/ml; The reaction time adding SA-PE solution is 20-30min.

A kind of detection method of Luminex HIV antigen-antibody combined detection kit

(1) antibody is detected: antigen coated microballoon and sample are reacted 40-60min, biontinylated anti-human two anti-reflective adding 4-8ug/ml again answers 30-40min, finally add the SA-PE solution reaction 20-30min of 13-15ug/ml, then carry out the analysis of instrument inspection software;

(2) detectable antigens: the microballoon of p24 monoclonal antibody bag quilt and sample are reacted 40-60min, then the monoclonal antibody of matching with p24 monoclonal antibody adding 0.5-1ug/ml hatches 40-60min, finally carries out the analysis of instrument inspection software.

Every 1ul sample adds 4-8 antigen coated microballoon and reacts.

The microballoon that every 1ul sample adds 4-8 p24 monoclonal antibody bag quilt reacts.

Present invention obtains following beneficial effect.

The present invention only needs a small amount of sample can carry out combine detection to HIV6 kind antibody and P24 antigen simultaneously.And the present invention has very high sensitivity and specificity compared with Western blot: carrier microballoons surface area is large, each microballoon can wrap by 100000 capture antibody/antigens, highdensity capture antibody/antigen like this ensure that and can be combined by the antigen/antibody molecule farthest in sample, improves detection sensitivity.The present invention can detect HIV antibody in early days in time in infection, and the morning realizing HIV finds, early intervention, reduces HIV spread, ensures transfusion safety, have broad application prospects.

Accompanying drawing explanation

Fig. 1 is P31, P24, P17, P66 of the present invention tetra-kinds of protein SDS-PAGE electrophoretograms;

Fig. 2 is that the anti-concentration of biotinylation two of the present invention is to the effect diagram of MFI;

Fig. 3 is that SA-PE of the present invention adds the effect diagram of concentration to MFI;

Fig. 4 is that the SA-PE reaction time of the present invention is to the effect diagram of MFI;

Fig. 5 is that the present invention tests the effect diagram of sample loading alternative to MFI.

Embodiment

Below in conjunction with drawings and Examples, the present invention is further detailed.

The structure of 1.gp17, p31, p66, p24 expression plasmid and qualification (antigen gp120, gp41 are synthesized by ten thousand safe biological medicine company incorporated company)

1.1 the amplification of object fragment

With reference to the HIV-1CRF01-AE hypotype gp17 on NCBI, p31, p66, p24(TaxonomyID:471721) full length gene, primer is designed with reference to pertinent literature analysis software Primer5, P24 upstream is introduced NdeI restriction enzyme site and is introduced initiation codon ATG simultaneously, and BamHI restriction enzyme site is introduced in downstream.BamHI restriction enzyme site is introduced in P17, P31, P66 upstream, and HindIII restriction enzyme site is introduced in downstream.Each primer sequence is as follows: (primer is synthesized by Ying Weijie base trade Co., Ltd)

P24

Upstream: 5`-CGCATATGCAAGGGCAAATGGTAC-3`

Downstream: 5`-CCCGGATCCTGCTGTCATCATTTCT-3`

P17

Upstream: 5`-CGGATCCGGTGCGAGAGCGTCGGTATT-3`

Downstream: 5`-CCAAGCTTATTTTGGCTGACC-3`

P31

Upstream: 5`-CGCGGATCCATGGAATAGATAAGGC-3`

Downstream: 5`-CCCAAGCTTAATCCTCATCCTGTCT-3`

P66

Upstream: 5`-CGGATCCCCCATTAGTCCTATTGAG-3`

Downstream: 5`-CCAAGCTTAAATAGTACTTTCCTGATTCC-3`

With HIV-1 Chinese epidemic strain CRF01-AE gene (TaxonomyID:471721) total length be template on GeneAmpPCRsystem9700PCR instrument (PE company of the U.S.) according to precious bioengineering company limited PyrobestDNAPolymerase reagent instructions preparation PCR reaction system, as follows:

10×PyrobestBufferII(Mg ²⁺Plus)5ul

DNTPMixture (each 2.5mM) 4ul

Upstream primer (20uM) 1ul

HIV-1-1CRF01-AE1ul

PyrobestDNAPolymerase(5U/ul)0.5ul

Sterile purified water upto50ul

PCR reaction conditions:

95 DEG C of denaturation 5min

94 DEG C of sex change 30s

56 DEG C of annealing 30s

72 DEG C extend 2min

Totally 30 circulations

72 DEG C extend 5min again

1.2 Normal Agarose Gels and target DNA fragment reclaim

Taking 0.3g agarose is dissolved in 30ml1 × tbe buffer liquid, and peptization solution is treated in heating, and the EB solution adding 3ul10mg/ml after being cooled to 60 ° of about C mixes gently, pours in glue box, after glue thoroughly cools, firmly extract comb uniformly.Get 5ulPCR product and add 6 × LoadingBuffer1ul mixing, 150V, 30min electrophoresis after loading.

Use plain agar sugar gel to reclaim the recovery that kit (Tian Gen biochemical technology company limited, DP309) carries out object fragment, concrete steps are as follows:

Before using first, in BufferWB, add the absolute ethyl alcohol of 56ml;

1) if the BufferDC(adding 3 times amount in PCR reactant liquor need add BufferDC quantity not sufficient 100 μ l time should add 100 μ l), then Homogeneous phase mixing;

2) SpinColumn in kit is placed on CollectionTube;

3) by aforesaid operations 1) solution be transferred to room temperature 12,000rpm in SpinColumn centrifugal 1 minute, abandon filtrate.Filtrate is added in SpinColumn more once centrifugal, the recovery of DNA can be improved;

4) add in SpinColumn by the BufferWB of 700 μ l, the centrifugal 30s of room temperature 12,000rpm, abandons filtrate;

5) repetitive operation step 4;

6) SpinColumn is placed on CollectionTube, centrifugal 1 minute of room temperature 12,000rpm;

7) SpinColumn is placed on the centrifuge tube of new 1.5ml, adds the aqua sterilisa of 30 μ l or ElutionBuffer room temperature leaves standstill 1 minute in the centre of SpinColumn film.Be conducive to when aqua sterilisa or ElutionBuffe being heated to 60 DEG C of uses improving elution efficiency;

8) room temperature 12,000rpm centrifugal 1 minute eluted dna.-20 ° of C are frozen for DNA product.

PCR primer after 1.3 purifying and the double digestion of empty plasmid

PCR primer after purifying, PET-15b, PET-32a empty carrier carry out the reaction of NdeI and BamHI, BamHI and HindIII double digestion respectively, and reaction system is prepared according to restriction enzyme instructions.

PCR primer double digestion after purifying:

DNA2ug

10×FastDigestbuffer3ul

BamHI1ul

NdeI/HindIII1ul

Sterilizing distilled water upto30ul

37 DEG C of incubator enzymes cut >1 hour, and 80 DEG C of water-baths stop endonuclease reaction.

The double digestion of empty carrier:

PET-15b/32a1ug

10×FastDigestbuffer3ul

BamHI1ul

NdeI/HindIII1ul

Sterilizing distilled water upto30ul

37 DEG C of incubator enzymes cut 5 minutes, and 80 DEG C of water-baths stop endonuclease reaction.Use common DNA Product Purification Kit (Tian Gen biochemical technology company limited, DP204) to reclaim digestion products, recycling step is as described in front " 1.2 ".

The structure of 1.4PET-32a-17, PET-32a-31, PET-32a-66, PET-15b-24 recombinant plasmid

Linked system is carried out according to precious biotech firm coupled reaction operation instructions:

10×T4DNALigasebuffer5ul

P17/p24/p31/p66 object fragment 0.3pmol

PET-15b/32a0.03pmol

T4DNALigase1ul

Sterilizing distilled water upto50ul

16 DEG C of connections are spent the night

Product conversion will be connected to competent escherichia coli cell DH5a(Tian Gen biochemical technology company limited, CB101) in, 37 DEG C of incubator overnight incubation, the positive single colony inoculation of picking contains 37 DEG C of air bath shaking table 200rpm concussion cultivation 12-16 hour in the LB fluid nutrient medium of 100ug/ml ampicillin in 3ml respectively.The little extraction reagent kit of plasmid (Tian Gen biochemical technology company limited, DP103) is used to extract plasmid, then double digestion qualification.

Recombinant plasmid double digestion identification system is as follows:

Recombinant plasmid 30ul

10×FastDigestbuffer5ul

BamHI1ul

NdeI/HindIII1ul

Sterilizing distilled water upto50ul

37 DEG C of incubator enzymes cut >1 hour, and 80 ° of C water-baths stop endonuclease reaction.1% agarose gel electrophoresis, 150V30 minute.

2.HIV-1p24, p17, p31, p66 protein expression and purifying

2.1 bacterium BL21(DE3 is expressed in being transformed into of recombinant plasmid) (Tian Gen biochemical technology company limited, CJ105)

1) get the competent cell that 50ul ice bath melts, add recombinant plasmid 1ul, mix gently, place 30 minutes in ice bath;

2) place 45 seconds in 42 DEG C of water-baths, pipe to be transferred in ice bath 2 minutes then fast, this process does not shake centrifuge tube;

3) in each centrifuge tube, add the aseptic LB nutrient culture media of 500ul (not containing microbiotic), as 37 DEG C after mixing, 200rpm cultivates 1 hour, makes cell recovery;

4) draw the competent cell transformed in right amount and be added to (ampicillin 100ug/ml) on LB agar medium, cell is coated with out uniformly, flat board is placed in 37 DEG C of incubators until liquid is all absorbed, be inverted dull and stereotyped 37 DEG C of incubated overnight.

2.2 the induction of recombinant protein and qualification

The single bacterium colony of picking recombinant plasmid transformed e. coli bl21 (DE3), be inoculated into 3mlLB(ampicillin 100ug/ml) in nutrient solution, 37 DEG C of 200rpm shake overnight incubation, by spending the night, the ratio of bacterium liquid in 1:100 is seeded to LB(ampicillin 100ug/ml) in nutrient solution, 37 DEG C of 200rpm concussions are cultured to bacterium liquid OD600 and reach about 0.6, add IPTG(mother liquid concentration 1M) to final concentration 0.5mmol/L, abduction delivering 4 hours, gets that 1ml bacterium liquid 12000rpm is centrifugal abandons supernatant.Thalline processes and carries out SDS-PAGE electrophoresis, and the complete SDS-PAGE glue of electrophoresis, through the dyeing of Coomassie brilliant blue-R250 dyeing liquor, observes electrophoresis result after the decolouring of methanol-acetic acid solution.

Confirm through SDS-PAGE electrophoresis the expression having order albumen after expressing in a small amount, the single positive colony of picking is to 100mlLB(ampicillin 100ug/ml) in nutrient solution, 37 DEG C of 200rpm shake cultivation 12 hours, transfer in 1L containing in the LB nutrient solution of amicillin resistance with 10% inoculum concentration.37 DEG C of 200rpm concussions are cultivated, and reach about 0.6 to OD600, adding IPTG to final concentration is 0.5mmol/L, continue 16 ° of C and shake overnight incubation.5000rpm, 4 DEG C centrifugal, receives bacterium.

The purifying of 2.3 recombinant proteins

2.3.1 ultrasonication thalline

1) the centrifugal bacterial sediment obtained is resuspended in the BindingBuffer of 40ml.And proceed in small beaker;

2) clean the probe of Ultrasound Instrument (Kunshan Ultrasonic Instruments Co., Ltd., KQ-700DV type), and dry with thieving paper.Thalline suspension beaker will be filled and be placed in mixture of ice and water, carry out ultrasonic;

3) with 40% ultrasound intensity, ultrasonic frequency of stopping for 2 seconds 4 seconds starts ultrasonic process;

4) ultrasonic about 5 minutes, clarify to cellular lysate;

5) 4 DEG C of 12000rpm, ultracentrifugation is about 30-60 minute, cleer and peaceful precipitation in separation.

2.3.2 the Ni-NTA affinity chromatography of recombinant protein

Series of strata that Ni-NTA is affine mainly utilize histidine energy and metallic ion Ni to carry out specific combination, thus be fixed on Ni-NTA resin, wash-out is carried out successively with the imidazole buffer of variable concentrations, can compete containing the protein of His label, thus realize being separated of destination protein and foreign protein.Concrete operation step is as follows:

1) get 3mLNi-NTA filler to join in gravity post, add the ddH of 5-10 column volume ₂o washes away and preserves column packing 20% ethanol used, then adds the BingingBuffer balance nickel post of 3-5 column volume;

2) supernatant of the centrifugal gained of carrying out ultrasonic bacteria breaking adds in the nickel post balanced after 0.22um membrane filtration, for improving the joint efficiency of albumen and nickel post, filtered solution can be repeated upper prop 2-3 time again, wash nickel post with the WashBuffer containing different imidazole concentration, be combined more weak foreign protein to wash away with nickel post.Can get 10ul eluent in interval in elution process, add 100ulBradford solution, the constant indigo plant of thing to be mixed stops wash-out;

3) eluent is collected with the destination protein that 500mmol imidazole elution buffer eluting nickel post combines;

4) wash-out complete after the imidazole elution buffer of continuation 500mmol rinse column packing, then with the sterilizing distilled water of 5-10 column volume flushing nickel post;

5) by column packing not in sterilizing distilled water, 4 DEG C save backup;

6) above-mentioned steps sampling respectively, carries out SDS-PAGE.

As shown in Figure 1, the first swimming lane is Protein Marker; Second swimming lane is the p31 albumen after ni-sepharose purification; 3rd swimming lane is p24 albumen after ni-sepharose purification; 4th swimming lane is p17 albumen after ni-sepharose purification; 5th swimming lane is the p66 albumen after ni-sepharose purification; 6th swimming lane is bacterial sediment after p31 protein expression; 7th swimming lane is bacterial sediment after p24 protein expression; 8th swimming lane is bacterial sediment after p17 protein expression; 9th swimming lane is bacterial sediment after p66 protein expression.

The concentration determination (microplate reader method) of 2.4 protein

1) measure Folin phenol first reagent A and Folin phenol first reagent B as required by 50:1 mixing (term of validity is 24 hours);

2) dilution standard product: get 10 microlitre BSA standard items PBS and be diluted to 100 microlitres, make final concentration be 0.5mg/mL.Join in the protein standard sample wells of 96 orifice plates by standard items by 0,2,4,6,8,12,16,20 microlitres, every hole adds PBS and supplies 20 microlitres, drawing standard curve;

3) sample is done suitable dilution (diluting 2 times, 4 times, 8 times respectively), add in the sample well of 20 microlitre to 96 orifice plates;

4) the every hole of Folin phenol first reagent prepared is added 200 microlitres, concussion mixing room temperature places 10 minutes gently;

5) add Folin phenol second reagent in every hole, mix rapidly, hatch 30 minutes for 37 DEG C, measure A650 by microplate reader (U.S. Bole, Bio-rad680), calculate protein concentration (see table 1) according to typical curve.

2.5 indirect elisa methods detect HIV-1p17, p24, p31, p66 antibody

10 parts of HIV-1 positive serums used, all confirm through WB, containing the antibody that the HIV-1 recombinant antigen of expressing is corresponding, and 10 parts of HIV-1 negative serums.

1) ddH is used ₂o soaks 96 orifice plates, and thieving paper pats dry;

2) HIV-1p17, p24, p31, p66 antigen of variable concentrations wraps by 96 orifice plates respectively, places 12 hours for 4 DEG C;

3) 1 × PBS-0.05%tweenPH7.4 washs 96 orifice plate three times, each 5 minutes, 200ul/ hole;

4) 3 hours are closed for 1 × PBS-2%BSA37 DEG C;

5) 1 × PBS-0.05%tweenPH7.4 washs 96 orifice plate three times, each 5 minutes, 200ul/ hole;

6) add known positive serum and the known negative serum 100ul/ hole of variable concentrations, blank well adds 1 × PBS100ul/ hole.37 DEG C of water-baths 1 hour;

7) 1 × PBS-0.05%tweenPH7.4 washs 96 orifice plate three times, each 5 minutes, 200ul/ hole;

8) every hole adds the ELIAS secondary antibody (PH7.41 × PBS-0.05%tween dilution) of variable concentrations, 37 ° of C water-baths 1 hour;

9) 1 × PBS-0.05%tweenPH7.4 washs 96 orifice plate three times, each 5 minutes, 200ul/ hole;

10) nitrite ion A and nitrite ion B1:1 mixes, 100ul/ hole, 37 DEG C of lucifuge water-baths 20 minutes;

11) every hole adds 50ul stop buffer cessation reaction;

12) microplate reader reads 450nm absorbance, the results are shown in Table 2.

3. Luminex detects the application in HIV-1

The bag quilt of 3.1 microballoons

Human Immunodeficiency Virus antigen gp41, p17, p24, p31, p66, gp120 are wrapped respectively by 50,56,65,71,83, No. 77 microballoons (the leading Science and Technology Ltd. in Central Plains, Beijing, model is respectively LC10050, LC10056, LC10065, LC10071, LC10083, LC10077) on, p24 monoclonal antibody (Wo Ben immunodiagnosis company, 1103-clone4F6) bag is by No. 37 microballoons (the leading Science and Technology Ltd. in Central Plains, Beijing, LC10037).The coupling method of albumen and microballoon carries out with reference to Luminex company operation manual.

1) from refrigerator, kit is got, equilibrium at room temperature 20-30 minute;

2) equilibrium at room temperature during this period of time, the volume number needed for calculation procedure 4,8,11,13, so that each reaction is carried out smoothly;

3) microballoon of resuspended non-coupling: vortex microballoon for subsequent use 10 seconds, ultrasonic 10 seconds with dispersion microsphere;

4) 100ul microsphere suspension (about 1.25 × 10 is drawn ⁶individual) in the low Percentage bound centrifuge tube of 1.5mL.The centrifugal 1-2min of >=8000 × g, carefully siphons away supernatant with suction pipe;

5) microballoon is washed: in low Percentage bound centrifuge tube, add 125ul activation buffer, vortex 10 seconds, ultrasonic 10 seconds, the centrifugal 1-2 minute of >=8000 × g.Carefully supernatant is siphoned away with suction pipe;

6) repeat step 5, once wash twice altogether;

7) carefully supernatant is siphoned away with suction pipe;

8) 480 μ L activation buffer join reaction tube;

9) vortex reaction tube 10 second, then ultrasonic process 10 seconds, with dispersion microsphere;

10) sulfonic group-NHS that vortex provides manages at least 10 seconds;

11) sulfonic group-NHS solution of 10 μ L is added to reaction tube;

12) activation buffer of 250 μ L is added in EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) in bottle (10 milligrams, now with the current);

13) the EDC solution of 10 μ L is added to reaction tube;

14) reaction tube at least 10 seconds of vortex;

15) microballoon room temperature lucifuge hatches 20 ± 1 minutes, and once, the centrifugal 1-2 minute of >=8000 × g, carefully siphons away supernatant with suction pipe to 10 minutes vortex microballoons;

16) microballoon is washed: get 500 μ L activation buffer and join reaction tube.Vortex reaction tube 10 seconds, then ultrasonic process 10 seconds, with dispersion microsphere;

17) repeat step 16 twice, wash three times altogether;

18) the centrifugal 1-2 minute of >=8000 × g, carefully siphons away supernatant with suction pipe;

19) in reaction tube, 900ul activation buffer is added;

20) in reaction tube, the antigen that 100ul final concentration is 0.25mg/ml and antibody is added;

21) reaction tube of vortex is at least 10 seconds;

22) microballoon lucifuge rotates 2 hours (rotational speed 15-30 turns) on spinner;

23) microballoon is cleaned: in reaction tube, add 500 μ L lavation buffer solutions.Vortex reaction tube 10 seconds, then ultrasonic process 10 seconds, with dispersion microsphere;

24) repeat step 23 twice, wash three times altogether;

25) the centrifugal 1-2 minute of >=8000 × g, carefully siphons away supernatant with suction pipe;

26) in reaction tube, 125ul lavation buffer solution is added;

In 27 oscillating reactions pipe 10 seconds, then ultrasonic process 10 seconds, with dispersion microsphere.Count microballoon with haemocytometer, determine the concentration of microballoon;

28) keep in Dark Place at 2-8 DEG C.

3.2HIV-1 antibody positive, uncertain, the antibody test of ' negative ' specimens and the optimization of reaction conditions

1) 96 orifice plates are soaked by the analysis buffer in 100ul/ hole before application of sample;

2) add negative serum and dilute the positive serum of 25 times by analysis buffer, 25ul/ hole;

3) the good microballoon of the dilution of mixing (guaranteeing 100/ul) 25ul/ hole is added;

4) room temperature concussion cultivation 1 hour;

5) room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant;

6) 125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant;

7) the 6th step is repeated, twice;

8) biotinylation two anti-(Beijing DingGuo ChangSheng Biology Technology Co., Ltd, SB-0052) diluted is added, 2ug/ml, 25ul/ hole;

9) room temperature concussion cultivation 1 hour;

10) room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant;

11) 125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant;

12) SA-PE25ul of 20ug/ml is added in every hole;

13) room temperature concussion hatches 30 minutes;

14) room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant;

15) 125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant;

16) add the resuspended microballoon of 125ul/ hole lavation buffer solution, Luminex-200 detects (luminex company of the U.S., luminex200), and Luminex-100 software (luminex company of the U.S.) is analyzed.

3.2.1 the optimization of the anti-concentration of biotinylation two

Remaining reaction condition is constant, by anti-for biotinylation two doubling dilution (8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml), best reaction density is determined according to average fluorescent strength (MeanFluorescentIntensity, MFI).

As shown in Figure 2, MFI improves, the MFI the highest (MFI:6799) when the anti-concentration of biotinylation two is 4ug/ml along with the raising of the anti-concentration of biotinylation two experimental result.

3.2.2SA-PE the optimization of concentration is added

Remaining reaction condition is constant, SA-PE is added each hole 20ug/ml, 18ug/ml, 16ug/ml, 14ug/ml, 12ug/ml, 10ug/ml, 8ug/ml, 6ug/ml, 4ug/ml, 2ug/ml respectively by following concentration, determines best reaction density according to average fluorescent strength.

As shown in Figure 3, when to add concentration be 14ug/ml to SA-PE, MFI is 10857 to experimental result, and in several concentration that experiment is used, MFI is the highest, therefore selects 14ug/ml to be the optimum concentration of SA-PE.

3.2.3SA-PE the optimization of reaction conditions

Remaining reaction condition is constant, and the SA-PE reaction time was respectively at 10 minutes, 20 minutes, 30 minutes cessation reactions.The reaction time of SA-PE the best is determined according to average fluorescent strength.

The SA-PE reaction time on the impact of experiment as shown in Figure 4, as can be seen from figure obviously, the MFI difference that SA-PE reacts 10min, 20min, 30min is not too large, but be totally the trend risen, in order to ensure the quality of testing and can obtain a higher MFI value, the SA-PE reaction time have finally chosen 30min.

3.2.4 the optimization of sample loading alternative

Determine biotinylation two to resist, SA-PE add concentration after, inquire into sample loading alternative whether to have an impact to experiment, devise following several sample loading alternative (all the other experiment conditions are all identical), best sample loading alternative is determined according to average fluorescent strength (MeanFluorescentIntensity, MFI):

1) anti-, the SA-PE of microballoon, serum specimen, biotinylation two adds 96 orifice plates simultaneously, and room temperature concussion cultivation 1 hour, room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times), adds the resuspended microballoon of 125ul/ hole lavation buffer solution, and Luminex-200 detects, Luminex-100 software analysis.

2) microballoon, serum specimen, biotinylation two is anti-adds 96 orifice plates simultaneously, room temperature shakes 1 hour room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.Add optimized concentration SA-PE in each hole, room temperature concussion hatch 30 minutes.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times).Add the resuspended microballoon of 125ul/ hole lavation buffer solution, Luminex-200 detects, Luminex-100 software analysis.

3) microballoon and seroreaction 1 hour, room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times), and the biotinylation two having optimized concentration resists and adds each hole with SA-PE simultaneously, room temperature concussion reaction 1 hour.Room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times).Add the resuspended microballoon of 125ul/ hole lavation buffer solution, Luminex-200 detects, Luminex-100 software analysis.

4) microballoon and seroreaction 1 hour, room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times).Add the biotinylation two having optimized concentration to resist, room temperature concussion reaction 1 hour, room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times).Add the SA-PE having optimized concentration, room temperature concussion reaction 30 minutes.Room temperature 3500rpm, centrifugal 5 minutes, carefully abandons supernatant.125ul/ hole lavation buffer solution washing microballoon, 3500rpm, centrifugal 5 minutes, carefully abandons supernatant (washing 3 times).Add the resuspended microballoon of 125ul/ hole lavation buffer solution, Luminex-200 detects, Luminex-100 software analysis.

The MFI result of various sample loading alternative reaction as shown in Figure 5, through the MFI value of more different sample loading alternative, namely discovery only has sample loading alternative 4(: microballoon and sample react 1 hour, add biotinylation two anti-reflective again and answer 1 hour, finally add SA-PE and shake reaction 30 minutes) obtain desirable MFI value, the MFI of sample loading alternative 1 and 2 almost can be regarded as blank value, and sample loading alternative 3 is slightly better than sample loading alternative 1,2, but not as good as sample loading alternative 4.

The multi-channel detection of 3.3HIV-1 antibody

Use Luminex-100 software editing multi-channel detection application program, the experiment condition optimized according to preliminary experiment (detects antibody: microballoon and sample react 1 hour, add biotinylation two anti-reflective again and answer 1 hour, finally add SA-PE and shake reaction 30 minutes, detect finally by luminex200; Detectable antigens: microballoon and sample reaction 1h, then add the antibody incubation 1h of RD1 mark, detect finally by luminex200.) operate, 50#, 56#, 65#, 71#, 77#, 83# pearl dilution mixing is guaranteed every number pearl every hole counting is no less than 100,138 routine samples are detected, calculates cut-off value and analyze data.

Microballoon MFI numerical value is greater than cut-off value and is defined as the positive, otherwise is then negative.Examination criteria by CDC specifies: form at least two Env and be with (gp41, gp120/gp160) or an Env band to add that p24 is with result to be judged to be the positive, occur that banding pattern can not meet Positive judgement standards result and be judged to be suspicious, form result without any HIV band and be judged to be feminine gender.LuminexxMAP detects positive findings 91 example, uncertain result 42 example, negative findings 5 example.

The multi-channel detection of 3.4HIV-1p24 antigen

No. 37 pearls having marked p24 monoclonal antibody are added to 96 orifice plates of PBS rinse, add sample incubated at room 60min; 125ulPBS – 20% Tween-20 cleaning twice, the RD1 then adding 25ul pairing marks anti-p24KC57 antibody (Beckman Ku Erte, catalog#6604667).125ulPBS – 0.02%Tween-20 to clean after twice and resuspended, and then Luminex-200 detects, Luminex-100 software analysis.

To 138 routine analyzing specimens, wherein 3 routine LuminexxMAP antibody uncertain sample HIVp24 antigen is positive, and all the other are feminine gender, it should be noted that the sample HIVp24 antigen of wherein 2 routine LuminexxMAP antibody positives is for negative.

The comparison of 4LuminexxMAP method and WB method testing result

Embodiment 1:

The sample of 105 routine Tianjin Blood Center is analyzed, 19 routine WB uncertain but in the sample of the RNA positive luminexxMAP detect 8 routine positive samples, 18 routine WB uncertain but in the sample that RNA is negative sexual luminexxMAP detect 3 routine negative sexual samples, illustrate that luminexxMAP decreases the appearance of uncertain sample to a certain extent.3 routine WB negative but in the sample of the RNA positive 1 routine luminexxMAPHIV-1 antibody test result be uncertain, avoid the undetected of WB to a certain extent, the specificity of luminexxMAP is described, and comparatively WB is high.The sensitivity 82.7%(72/87 that LuminexxMAP detects) higher than the sensitivity 74.7%(65/87 of WB).

Embodiment 2:

Analyze the positive male faggotry's detection result of specimen of 33 routine RNA, WB detects HIV-1 antibody positive 13 example, uncertain sample 18 example of HIV-1 antibody, and HIV-1 negative antibody is 2 examples; LuminexxMAP detects positive 19 examples, and uncertain sample is 14 examples.Contrast finds, the sensitivity 57.8%(19/22 that luminexxMAP detects) higher than the sensitivity 39.4% of WB.

Embodiment 3:

The criterion of HIV immunoblot experiment is reference with antibody test always: form at least two Env and be with (gp41, gp120/gp160) or an Env band to add that p24 is with result to be judged to be the positive, occur that banding pattern can not meet Positive judgement standards result and be judged to be suspicious, form result without any HIV band and be judged to be feminine gender, the false positive results that primary dcreening operation is tested can be differentiated.Fig. 3 result illustrate the detection of luminexxMAP antigen before to a certain degree pointing out sample to be in antibody male rotary or sun turn in journey, but HIV-1p24 antigen increases in transient in the infected's body, so also there is the routine HIV-1 antibody positive of M1126, M1127 two, the situation of HIV-1p24 antigen negative.

Claims

1. a Luminex HIV antigen-antibody combined detection kit, comprises the microballoon being wrapped quilt by Human Immunodeficiency Virus antigen gp41, p17, p24, p31, p66, gp120 respectively, and biontinylated anti-human two resists, SA-PE solution; The microballoon of p24 monoclonal antibody bag quilt, fluorescein-labeled monoclonal antibody of matching with p24 monoclonal antibody.

2. a kind of Luminex HIV antigen-antibody combined detection kit according to claim 1, is characterized in that: the bag of arbitrary described Human Immunodeficiency Virus antigen gp41, p17, p24, p31, p66, gp120 and p24 monoclonal antibody and microballoon is that the antigen of 2-5ug or antibody bag are by 1 × 10 by ratio ⁶individual microballoon.

3. a kind of Luminex HIV antigen-antibody combined detection kit according to claim 1, is characterized in that: described biontinylated anti-human two resists for biotinylation goat anti-human antibody, and the anti-concentration of biontinylated anti-human two is 4-8ug/ml.

4. a kind of Luminex HIV antigen-antibody combined detection kit according to claim 1, is characterized in that: described monoclonal antibody of matching with p24 monoclonal antibody is the anti-p24KC57 antibody that RD1 marks; The concentration of fluorescein-labeled monoclonal antibody of matching with p24 monoclonal antibody is 0.5-1ug/ml.

5. a kind of Luminex HIV antigen-antibody combined detection kit according to claim 1, is characterized in that: the concentration of described SA-PE solution is 13-15ug/ml; The reaction time adding SA-PE solution is 20-30min.

6. a detection method for Luminex HIV antigen-antibody combined detection kit, is characterized in that:

7. the detection method of a kind of Luminex HIV antigen-antibody combined detection kit according to claim 6, is characterized in that: every 1ul sample adds 4-8 antigen coated microballoon and reacts.

8. the detection method of a kind of Luminex HIV antigen-antibody combined detection kit according to claim 6, is characterized in that: the microballoon that every 1ul sample adds 4-8 p24 monoclonal antibody bag quilt reacts.