CN101936988A - Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof - Google Patents
Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof Download PDFInfo
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- CN101936988A CN101936988A CN2010102356614A CN201010235661A CN101936988A CN 101936988 A CN101936988 A CN 101936988A CN 2010102356614 A CN2010102356614 A CN 2010102356614A CN 201010235661 A CN201010235661 A CN 201010235661A CN 101936988 A CN101936988 A CN 101936988A
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Abstract
The invention relates to a protein suspension chip for detecting a west nile antibody in a serum sample and a preparation method and a using method thereof. The method has the advantages of high detectability, high sensitivity, high specificity and wide dynamic range; and an open detection modular platform for the protein suspension chip to detect viral antibodies represented by the west nile antibody is established.
Description
Technical field
The present invention relates to a kind of protein suspension chip that detects serum sample Chinese and Western Buddhist nun sieve's antibody and preparation method thereof and using method.
Background technology
The west nile virus disease is that (West Nile Virus, the infectious disease that WNV) causes are a kind of Amphixenosises by west nile virus.Not propagate mutually behind the human infection west nile virus, be generally subclinical infection. be 3～15 days latent period, and most of the infected's symptom is slight, with heating, headache is had a sore throat, backache, myalgia, arthralgia, fatigue, conjunctivitis, fash, enlargement of lymph nodes, it is poor to receive, stomachache, diarrhoea and respiratory symptom etc.May cause high heat (〉=40 ℃) for the elderly and children, severe headache and central nervous system symptom and sign, as neck rigidity, lethargic sleep, stupor is twitched, paralysis etc., even cause death.
Utilize antigen and antibody specificity to react in the immunological method enzyme linked immunological experiment (ELISA) to detect antigen or antibody to mainly contain and survey antibody, double-antibody sandwich survey antigen, competition law, indirect method etc. by double antigens sandwich.The principle that indirect method is surveyed antibody is that specific antigen is attached on the solid phase carrier, then with serum to be checked in corresponding antibodies in conjunction with forming immune complex, the antibodies that adds again after the washing in enzymic-labelled antibody and the immune complex forms enzymic-labelled antibody-antibody-solid phase antigen compound, add the substrate colour developing, judge antibody content.
Suspending chip (suspension array) also claim liquid-phase chip, it is the biochip technology of new generation that the seventies in 20th century, U.S. Luminex company developed, the microsphere that utilizes the band coding is as carrier, flow cytometer is measured biomolecule such as nucleic acid, protein on a large scale as detection platform.At present, this technology has been widely used in the fields such as discriminance analysis of immunoassay, nucleic acids research, enzymatic analysis, antibody screening and acceptor and part.
The suspending chip of development mainly is based on foundation and the method evaluation and the optimization of laboratory detection method at present, to shorten detection time, the detection cost of reduction method.But whether suspension chip method can detect the Xi Niluo antibody in the human serum, and its detection by quantitative ability how, still lacks model and evaluation.
Summary of the invention
The object of the present invention is to provide a kind of protein suspension chip that detects serum sample Chinese and Western Buddhist nun sieve's antibody, this chip comprises: coding microball, be encoded western Buddhist nun sieve E albumen, biotin labeled two anti-and SPA of microballoon antigen as capture antibody as bag, streptavidin-phycoerythrin (SA-PE) and relevant buffer solution.
The invention provides the preparation method of the protein suspension chip of above-mentioned detection Xi Niluo antibody, this method is specially: in relevant buffer solution, coding microball with western Buddhist nun sieve E proteantigen bag by, with biotin labeled detection thing be goat anti-rabbit igg or/and Biotin-SPA, usefulness streptavidin-phycoerythrin (SA-PE) as signal detection, preferred No. 025 coding microball of described coding microball.
The invention provides a kind of indirect immunological detection method that adopts protein suspension chip to detect all this Chinese and Western of serum Buddhist nun sieve's antibody, this method adopts indirect method to detect the immunology detection principle of antibody, be specially: respond in testing process and can carry out on 96 hole filter plates or in microcentrifugal tube, comprising the following step: (1) wraps the microballoon that is encoded with western Buddhist nun sieve E proteantigen as capture antigen; (2) add in every hole or the pipe to contain and wrap the antigen that is hunted down, the working solution of the coding microball of promptly western Buddhist nun sieve E proteantigen cleans with cleaning fluid; (3) add the test serum sample, hatch the back and clean; (4) add with biotinylated detection thing, hatch the back and clean; (5) add streptavidin-phycoerythrin (SA-PE), hatch the back and clean, mixing after (6) adding detection damping fluid, (7) read MFI numerical value (being average fluorescent strength) with suspension chip system and analyze the data judging testing result negative or positive.
In this method, adopt biotin labeled SPA as detecting thing, and itself and western Buddhist nun sieve E protein combination composition indirect method detection architecture; The capture antigen west Buddhist nun sieve E proteantigen consumption that wraps the microballoon that is encoded is 0.1～240 μ g/1.25 * 10
6Individual coding microball or 0.02～960ng/2500～5000 microballoon/test, the biotin of marker detection antibody goat anti-rabbit igg is the biotin of carboxyl activity.Adopt the biotinylated detection antibody of 2mg/mL Bioin-SPA to detect as detecting thing in the step (4) with dilution in 1: 2500.The present invention also provides a kind of method that adopts protein suspension chip detection by quantitative serum sample Chinese and Western Buddhist nun sieve's antibody, this method comprises the following steps: positive detection sample that (1) adds or standard items through 4 times of gradient multiple proportions serial dilutions, and (2) are detected series of diluted samples and read corresponding fluorescent value (MFI) in suspension chip system; (3) the dose-response typical curve of the corresponding MFI value of making sample concentration, (4) with analysis software match dose-response curve and equation, (5) according to the dynamic detection range of dose-response curve decidable method, the unknown concentration sample can be judged the concentration of test sample according to the dose-response equation.
The ultimate principle of suspending chip is to utilize the microballoon of polystyrene (polystyrene) made, coat the ruddiness and the infrared light colour former of different proportion, and produce 100 kinds of different proportion colors, color numbers as 100 kinds of uniquenesses, every about 5.6 μ m of microballoon size, different research purposes such as immunoassay, nucleic acids research, enzyme analysis, acceptor and part discriminance analysis etc. be can comply with, and specific antibodies, nucleic acid probe and various acceptor probe demarcated according to different research purposes.It finishes detection in following suspension chip system, the microballoon of label probe and determinand react in 96 orifice plates, after the reaction, utilizes machine automatically reactant liquor to be picked up and by a microcapillary sense channel, only allows a microballoon to pass through sense channel at every turn.Being provided with twice laser in the sense channel, is red together, excites the color in the microballoon matrix, and the sorting code number of identification microballoon is to determine test item; Be green together, excite the color of reporter molecules, the tracer signal power is to detect the content of determinand.When the probe of sample to be tested and specific microballoon was attached together, the light that twice laser is excited all can be detected.And, then only have the exciting light in the microballoon to be detected if do not contain this subject matter in the sample.Microballoon kind and the quantity that is excited by machine and computing machine automatic statistical analysis twice laser again, thereby several test target things are arranged therein in the judgement sample to be tested, learn to have or not cause of disease to be measured to exist in the test sample book, or have several simultaneously to tens of kinds of cause of diseases.The suspending chip technology is owing to utilize microballoon to react in solution, overcome sheet film chip and when big Molecular Detection, be subjected to the influence to reaction kinetics such as surface tension, steric effect, utilize laser measuring technology simultaneously, improve the accuracy and the repeatability of sample detection greatly, had characteristics such as easy and simple to handle, the good reproducibility that is better than sheet film chip.
The inventor has done substantial improvement and innovation through a large amount of tests and deep research to the protein suspension chip preparation and the testing conditions thereof of Xi Niluo antibody, and it has following advantage:
1, the improvement of antigen coated amount
The package amount of Xi Niluo E proteantigen is the key that successfully detects, the present invention is carried out substantive optimization to bag by the antigen coated amount of microballoon makes the detection effect of serum sample very good, and wrap very lowly by the required antigen coated amount of suspending chip, antigen coated amount of the present invention is 0.1～240 μ g/1.25 * 10
6Individual coding microball or 0.02～960ng/2500～5000 microballoon/test, and the package amount of traditional immunology ELISA method is 1 μ g/ test.
2, biotin labeled improvement
Usually, labelled antibody need use excessive biotin.Theoretically, in testing process, excessive biotin is fallen by suction filtration during cleaning because of the unmarked antibody of going up is not connected by the antibody in conjunction with capture antibody on the microballoon, can not react with the SA-PE that adds subsequently, does not influence detection.But in the actual detected process, false positive appears in the phenomenon that the detection signal that fell in may be too high, so after advising biotin labeling antibody, removes unnecessary biotin as far as possible.IgG (molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ L of 10mM biotin solution.
3, sensitivity of Jian Ceing and dynamic range
The protein suspending chip quantitative detecting method of blood serum sample Xi Niluo antibody of the present invention and classical immunology ELISA detection method, the result has good anastomose property (coefficient R
2=0.964).Simultaneously, suspension chip method sensitivity is serum titer 4.9 * 10
-6, it is higher than the detection titre 7.8 * 10 of ELISA method
-5, sensitivity is increased more than 10 times; And the suspension chip method dynamic detection range is 4.9 * 10
-6～2 * 10
-2, it is higher than ELISA method dynamic detection range (promptly 7.8 * 10
-5～2 * 10
-2) 1 order of magnitude.
4, the specificity of method
The present invention is an envelope antigen by selecting western Buddhist nun sieve E albumen for use, is antibody to be measured with the anti-Xi Niluo antibody of rabbit, serves as to detect antibody with biotinylated goat-anti rabbit.This development test proves, exists mouse-anti dengue fever IgG, rabbit anti-avian influenza H5 serum, the anti-soil of rabbit to draw FopA polyclonal antibody, rabbit anti-dengue NS3 antibody etc. to disturb under the condition of antibody existence, and this method has good specificity.
5, the detectability of sample
The present invention has estimated the detectability of suspension chip method to human serum.By detection to human serum, tentative confirmation this method in the practicality that detects human serum Chinese and Western Buddhist nun sieve's antibody.
Description of drawings:
Fig. 1 is that No. 025 microballoon bag is by western Buddhist nun sieve E Protein Detection Xi Niluo antibody synoptic diagram;
Fig. 2 detects the anti-Xi Niluo serum dosage of rabbit-reaction normal curve map for the protein suspension chip method;
Fig. 3 detects the anti-Xi Niluo serum of rabbit canonical plotting for the ELISA method;
Fig. 4 is the correlativity that protein suspension chip and ELISA method detect the anti-Xi Niluo serum of rabbit.
Embodiment
As shown in Figures 1 to 4, protein suspension chip preparation method, detection and the quantivative approach of the detection Xi Niluo antibody that the present invention relates to are described further by following embodiment, but the present invention is subjected to the qualification of this embodiment never in any form.
One, material
1. the relevant damping fluid of protein suspension chip:
(1) PBS damping fluid (pH7.4): NaCl 137mmol/L; KCl 2.7mmol/L; Na
2HPO
410mmol/L; KH
2PO
42mmol/L.With 800mL dissolved in distilled water 8gNaCl, 0.2gKCl, 1.44g Na
2HPO
4With 0.24g KH
2PO
4PH value to 7.4 with the HCl regulator solution adds water to 1L.After the packing at 15psi (1.05kg/cm
2) high pressure steam 20 minutes, or filtration sterilization, be stored in room temperature.
(2) microballoon cleaning fluid: PBS (pH7.4), 0.05%TWEEN-20.
(3) microballoon activation damping fluid 100mM NaH
2PO
4: 3g NaH
2PO
4, 5N NaOH 1.5mL, constant volume are in 250mL, and pH 6.2.
(4) the microballoon bag is cushioned liquid 0.05M MES, pH 5.0:2.44g MES, and 5N NaOH0.15mL, constant volume is in 250mL.
(5) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, and 0.02%TWEEN, 0.05% azide, pH 7.4.
(6) microballoon confining liquid PBS-BN:PBS, 1%BSA, 0.05% azide, pH7.4.
(7) detect damping fluid: PBS, 1%BSA, pH7.4.
(8) antibody diluent PBS, pH7.4.。
(9) microballoon dilution: PBS, 1%BSA, pH7.4.
(10) sample diluting liquid PVX:PBS, 0.5%PVA, 0.8%PVP;
(11) biotinylation detect thing dilution: PBS-TBN (PBS, 0.1%BSA, 0.02%TWEEN-20,0.05%NaN3, pH7.4).
(12) SA-PE dilution: PBS (pH7.4), 1%BSA.
2. antigen and antibody
Antigen used in the present invention is western Buddhist nun sieve E albumen, can be available from microorganism epidemic research institute of military medicine research institute.
Detection antibody used herein is the anti-western Buddhist nun sieve IgG of rabbit, can be available from microorganism epidemic research institute of military medicine research institute, detecting antibody is biotinylation goat anti-rabbit igg and biotinylation SPA, and described goat anti-rabbit igg can be available from Beijing ancient cooking vessel state biotech company, SPA can be available from Sigma company.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is the anti-western Buddhist nun sieve IgG of rabbit, disturbed specimen or the sample of testing as the method specificity are other antibody or other protein of target detection beyond the region of objective existence, comprise mouse-anti dengue fever IgG, rabbit anti-avian influenza H5 serum, the anti-Tula antibody of rabbit, rabbit anti-dengue NS3 antibody, BSA, casein, tryptone etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid-4 ℃ of preservations.The storing solution titre of the anti-western Buddhist nun sieve IgG of rabbit is 0.08.In the comparative experiments, same sample is used for the detection of ELISA and suspending chip.
Anti-western Buddhist nun sieve IgG is the variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with rabbit to be analyzed, to draw the typical curve of sample detection dose-response, wherein several sample concentrations are lower than the susceptibility of detection, and enriched sample should make the binding site of coding microball be in state of saturation.
2, the processing of human serum sample
The human serum sample with diluted sample dilution in 1: 10, after for example human serum sample 5 μ L add sample dilution 50 μ L mixings, is carried out the detection of suspension chip method as sample to be checked.
The preparation of the protein suspending chip of embodiment 1, detection Xi Niluo antibody
1, the capture antigen bag microballoon that is encoded
No. 025 coding microball that the present invention adopts is available from U.S. Bio-Rad company, and coding microball is used for western Buddhist nun sieve E proteantigen that mark can be caught Xi Niluo antibody, promptly utilizes western Buddhist nun sieve E albumen bag by microballoon.
The activation of A, coding microball
Get 100 μ L (1.25 * 10
6Individual) coding microball is in the 1.5mL centrifuge tube, and 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.The microballoon cleaning buffer solution that adds 100 μ L suspends, and concussion and ultrasonic back 14000 * g are centrifugal, careful sucking-off and abandoning supernatant.The microballoon that adds 100 μ L activates damping fluid, then adds the EDC (50mg/mL) of the fresh configuration of 10 μ L earlier, adds the Sulfo-NHS of the 50mg/mL of the fresh configuration of 10 μ L again, shakes after 30 seconds at a high speed and wraps up with aluminium foil, jolts 20 minutes in room temperature.Add the PBS (pH7.4) of 150 μ L, after the concussion, 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.PBS (pH7.4) the suspended coding microballoon that adds 100 μ L.
B, use antigen coated coding microball
Get in the coding microball after capture antigen west Buddhist nun sieve E proteantigen 1～50 μ g joins activation, be settled to 500 μ L with the PBS damping fluid, room temperature jolts 2 hours.14000 * g is centrifugal, careful sucking-off and abandoning supernatant.PBS damping fluid with 500 μ L is washed once, and 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.Add the sealing damping fluid suspended coding microballoon of 250 μ L, jolt 30 minutes in room temperature, 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.The microballoon that adds 500 μ L is preserved liquid washing coding microball, and 16000 * g is centrifugal, careful sucking-off and abandoning supernatant.Preserve liquid suspended coding microballoon with the microballoon of 150 μ L at last, keep in Dark Place standby in 4 ℃.
C, bag are by the counting of microballoon
Draw an amount of microballoon, after the dilution, with blood counting chamber (0.10mm; 1/400mm
2) under simple microscope, count.According to formula (each big lattice number * 10
4* extension rate * volume (mL)) calculates microballoon quantity.
2, detect the substance markers biotin
The mark of A, biotin
Preparing concentration respectively is the antibody-solutions to be marked of 10mM biotin solution and 2mg/mL, and the biotin that has calculated volume is joined in the thing solution to be marked, jolts 30 minutes in room temperature (or 2 hours) on ice, packing after the post desalination excessively, and-20 ℃ are frozen standby.
The consumption of B, antibody
IgG (molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ l of 10mM biotin solution.
The optimization of embodiment 2, suspending chip preparation method condition
1, the selection of microballoon envelope antigen package amount
Amount bag with 1 μ g, 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 16 μ g, 20 μ g, 24 μ g is encoded to No. 025 microballoon by 100 μ L respectively.Effect compares after testing, with 1～24 μ g/1.25 * 10
6Individual coding microball or 0.2～96ng/2500～5000 microballoon/test is wrapped preferably by effect, and microscopically counting back lucifuge stored refrigerated is stand-by.As shown in Figure 1, bag is all dropped in its correct surveyed area by No. 025 microballoon of western Buddhist nun sieve E proteantigen, and obtains high s/n ratio result (the MFI value is much larger than 2000), illustrates that the suspending chip detection system of optimizing can successfully be used for the Xi Niluo detection of antibodies.
2, biotinylation detects the optimization of thing
The present invention uses amino active biotin respectively, the biotin of LC-hydrazides (hydrazide)-biotin and carboxyl activity for example, for example the biotin labeling of Sulfo-NHS-biotin and SH-activity detects antibody, detect effect relatively through the Quality Control process, the Sulfo-NHS-biotin labeling detects quality testing to survey effect better in this experiment, and the method that detects effect adopts the conventional method of this area or according to the described method of the product description of manufacturer.
The inventor is through verification experimental verification, find that the biotinylated antibody goat-anti of 2mg/mL rabbit detects Xi Niluo antibody in the rabbit anteserum with dilution in 1: 500 as detecting thing, testing result shows that biotinylation detects the detection effect that thing Bio-goat anti-rabbit antibody can obtain the Supreme People's Procuratorate's measured value and low background; As detecting human serum Chinese and Western Buddhist nun sieve's detection of antibodies thing, testing result showed that biotinylation detects the detection effect that thing Biotin-SPA can obtain the Supreme People's Procuratorate's measured value and low background to the biotinylated detection thing of 2mg/mL Biotin-SPA with dilution in 1: 2500.
1, testing sample preparation
With sample diluting liquid sample to be tested is formulated as the variable concentrations sample and carries out the protein suspension chip detection.
2, the detection of sample and result judge
The testing process total overall reaction is all carried out on 96 hole filter plates, and testing process is as follows:
1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washs and use the vacuum pump suction filtration with cleaning fluid;
2) add 50 μ L test sample, the room temperature lucifuge jolts 30 minutes behind the mixing, with cleaning fluid washing and suction filtration;
3) biotinylation with after the detection thing diluted that adds 50 μ L debita spissitudos detects thing, and the room temperature lucifuge jolts 30 minutes behind the mixing, washing lotion washing and vacuum pump suction filtration;
4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10 minutes behind the mixing.Washing lotion washing and vacuum pump suction filtration;
5) the detection damping fluid of adding 125 μ L is through the resuspended mixing of spiral;
6) read MFI numerical value and analyze data with suspension chip system.
3, testing result is judged
According to experimental study result and correlative study report, the present invention defines protein suspension chip method and detects the detection substrate concentration of the minimum detectability (LOD value) of Xi Niluo antibody for fluorescence intensity critical value (Cutoff) correspondence.Wherein, the definition of Cutoff is to adopt blank sample (Blank) fluoroscopic examination signal MFI average to add 3 times of standard deviations (SD), and promptly the Cutoff value is=MFI (blank)+3 times standard deviation.Corresponding fluorescence intensity level then is judged to be the Xi Niluo antibody test positive as a result if testing result is higher than LOD; Corresponding fluorescence intensity level then is judged to be Xi Niluo antibody test feminine gender as a result if testing result is lower than LOD.
Embodiment 4, suspending chip are to Xi Niluo proteantigen specific detection
The using suspending chip detection method detects rabbit anti-western Buddhist nun sieve IgG, mouse-anti dengue fever IgG, rabbit anti-avian influenza H5 serum, the anti-Tula antibody of rabbit, rabbit anti-dengue NS3 antibody, BSA, casein, tryptone etc. respectively, according to testing result criterion among the embodiment 3, only the anti-western Buddhist nun sieve IgG of rabbit is positive, and all the other interference antibody or albumen are all negative.Cross reaction or non-specific responding all do not take place in method for detecting suspension chip and other test antibody that the Xi Niluo antibody that the present invention sets up is described.
The foundation of embodiment 5, suspending chip detection by quantitative model
1, the anti-western Buddhist nun sieve IgG typical curve specimen preparation of rabbit
Is 0.08 with 4 times multiple proportions gradient dilutions to titre to be 3.125 * 10 with the anti-western Buddhist nun sieve IgG standard items of rabbit by titre with sample diluting liquid
-7Become the series concentration sample.
2, the drafting of dose-response typical curve
Detect above-mentioned series concentration sample according to the detection method among the embodiment 3, and draw dose-response typical curve (as shown in Figure 2) according to the suspension chip system testing result.Wherein, X-axis is represented titre, the fluorescent value (MFI) that on behalf of the suspending chip instrument, Y-axis detect.The testing result mean value that each is data represented 3 times, coordinate axis is set with logarithm-logarithmic relationship, and the titre of the anti-Xi Niluo serum of rabbit is respectively among Fig. 2: S1:3.125 * 10
-7, S2:1.25 * 10
-6, S3:5 * 10
-6, S4:2 * 10
-5, S5:8.0 * 10
-5, S6:3.12 * 10
-4, S7:1.25 * 10
-3, S8:5 * 10
-3, S9:2 * 10
-2, S10:0.08.
Suspension chip system resists western Buddhist nun sieve IgG dose-response typical curve equation according to testing result match rabbit:
FI＝-2.07898+(13723.6+2.07898)/[1+(Conc/0.141595)
-1.37795]
0.715313
3, suspending chip detects lowest detectable limit and the dynamic range of the anti-western Buddhist nun sieve IgG of rabbit
According to minimum detectability definition and dose-response typical curve equation, the present invention is that the lowest detectable limit of the anti-western Buddhist nun sieve IgG of protein suspension chip method detection rabbit is that serum titer is: 0.000035.
It is to make the binding site of coding microball be in the detection substrate concentration of state of saturation that the present invention defines Supreme Procuratorate's rising limit.According to the rising of typical curve along with the anti-western Buddhist nun sieve IgG concentration of rabbit, its corresponding MFI value also increases progressively thereupon, when the anti-western Buddhist nun sieve IgG titre degree of rabbit surpasses 0.08, the immune response of the antigen-antibody state that reaches capacity, the MFI value begins to enter plateau, substrate concentration to be checked in the interpret sample is too high, need will detect behind the diluted sample again.
Therefore, can judge that according to minimum detectability and Supreme Procuratorate's rising limit the present invention is that the dynamic range of the anti-Xi Niluo serum of suspending chip detection by quantitative rabbit is 8.0 * 10
-50.08.
Embodiment 6, protein suspension chip method and ELISA method detect the comparison of Xi Niluo serum
1, biotin-avidin ELISA (BAS-ELISA) detection method
As a comparison, biotin-avidin ELISA adopts and comprises the following steps: 1) add western Buddhist nun sieve E albumen 1～50 μ g of 50 μ L with the dilution of PBS damping fluid, 4 ℃ of static spending the night to the every hole of common ELISA microwell plate;
2) add confining liquid, 37 ℃ of sealings 2 hours;
3) washing lotion is washed 3 times, pats dry;
4) add test sample, every hole 50 μ L, room temperature was placed 40 minutes; Washing lotion is washed 3 times, pats dry;
5) add an amount of biotinylation and detect thing, every hole 50 μ L, room temperature was placed 30 minutes; Washing lotion is washed 3 times, pats dry;
6) add an amount of SA/HRP (horseradish enzyme labeling Streptavidin), 50 μ L/ holes, room temperature was placed 3 minutes; Washing lotion is washed 3 times, pats dry;
7) add TMB colour developing liquid (solvable type single component tmb substrate solution) colour developing, 50 μ L/ holes, room temperature was placed 10 minutes;
8) use 2M H
2SO
4Cessation reaction;
9) use the microplate reader survey and read A450nm numerical value.
2, the method for detecting suspension chip of sample
Adopt indirect method to survey the immunology detection pattern of antibody, total overall reaction is all carried out on 96 hole filter plates in the testing process.
1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washing lotion washing and with vacuum pump suction filtration 2 times;
2) add 50 μ L test sample, the room temperature lucifuge jolts 30 minutes behind the mixing, with cleaning fluid washing and suction filtration 3 times;
3) add 50 μ L debita spissitudos with the biotinylated antibody after the antibody diluent dilution, the room temperature lucifuge jolts 30 minutes behind the mixing, washing lotion washing and vacuum pump suction filtration 3 times;
4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10 minutes behind the mixing.With cleaning fluid washing and vacuum pump suction filtration 3 times;
5) the detection damping fluid of adding 125 μ L is through the resuspended mixing of spiral;
6) read MFI numerical value and analyze data with the Bio-Plex suspension chip system.
3, the comparison of the sensitivity of two kinds of detection methods and dynamic range and correlativity
The anti-Xi Niluo blood of the mutually same group rabbit E albumen blood serum sample of preparation detects with sample diluting liquid 4 doubling dilutions while using suspending chip and ELISA method.Draw the typical curve (horizontal ordinate is the sample titre among the figure, and ordinate is an absorbance, and coordinate axis is taken the logarithm-logarithmic relationship) that the ELISA method detects the anti-Xi Niluo serum of rabbit, and compare with the suspension chip method result.
According to two kinds of method standard curves: suspension chip method as, sensitivity is 4.9 * 10
-6(serum titer), linear detection range 4.9 * 10
-6～2 * 10
-2The ELISA method as shown in Figure 3, sensitivity is 7.8 * 10
-5(serum titer), linear detection range 7.8 * 10
-5～2 * 10
-2Can reach a conclusion: detect the anti-Xi Niluo blood serum sample of identical rabbit, the protein suspension chip method has higher detection sensitivity than ELISA method, and is promptly high 10 times; And has wideer dynamic detection range, promptly high 1 order of magnitude.
In addition, with two kinds of method testing results 10
-6～10
-2Compare in the scope, can judge that as shown in Figure 4 protein suspension chip method and ELISA method have good correlativity, related coefficient is 0.964.
Embodiment 7, human serum sample's detection
1, human serum sample's preparation
Human serum is used for protein suspension chip after with sample diluting liquid 1: 10 dilution (human serum sample 5 μ L add sample dilution 50 μ L mixings) to be detected.
2, human serum sample's detection
1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washs and use the vacuum pump suction filtration with cleaning fluid;
2) add 50 μ L human serum sample to be detected, the room temperature lucifuge jolts 30 minutes behind the mixing, with cleaning fluid washing and suction filtration;
3) add 50 μ L debita spissitudos with the biotinylation SPA after the antibody diluent dilution, the room temperature lucifuge jolts 30 minutes behind the mixing, washing lotion washing and vacuum pump suction filtration;
4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10 minutes behind the mixing.Washing lotion washing and vacuum pump suction filtration;
5) the detection damping fluid of adding 125 μ L is through the resuspended mixing of spiral;
6) read MFI numerical value with suspension chip system,, determine the concentration of human serum to be detected Chinese and Western Buddhist nun sieve's antibody according to typical curve.
Through revision test repeatedly, biotinylation SPA dilution ratio is selected dilution in 1: 2500 for use, the SA-PE dilution ratio is selected dilution in 1: 300 for use, through detection to 94 parts of human serums, 3 times of sums of the average of the MFI value of gained and its standard deviation are as the dividing value of judging yin and yang attribute, be that C utoff value is 1103, if the MFI value that detection obtains is greater than 1103, it is the Xi Niluo excessive concentration in the serum, can be considered the Xi Niluo antibody positive may be infected by west nile virus, if the MFI value is less than 1103, promptly the low normal value of the Xi Niluo antibody concentration in the serum can be judged as the Xi Niluo negative antibody, does not infect the recessive carrier of west nile virus or west nile virus.
Claims (10)
1. protein suspension chip that detects serum sample Chinese and Western Buddhist nun sieve's antibody, it is characterized in that this chip comprises: coding microball, as be encoded western Buddhist nun sieve E albumen of microballoon antigen of bag, biotin labeled two anti-and SPA are as detecting thing, streptavidin-phycoerythrin and relevant buffer solution.
2. preparation method who detects the protein suspension chip of Xi Niluo antibody, it is characterized in that, this method is specially: in relevant buffer solution, coding microball is with western Buddhist nun sieve E proteantigen bag quilt, with biotin labeled detection thing be goat anti-rabbit igg or/and Biotin-SPA, with streptavidin-phycoerythrin as signal detection.
3. the preparation method of the protein suspension chip of detection Xi Niluo antibody as claimed in claim 2 is characterized in that described coding microball is preferably coding microball No. 025.
4. an indirect immunological detection method that adopts protein suspension chip to detect serum sample Chinese and Western Buddhist nun sieve's antibody is characterized in that this method comprises the following steps:
1) western Buddhist nun sieve E proteantigen is wrapped the microballoon that is encoded as capture antigen;
2) adding contains the working solution that wraps the antigen encoding microballoon that is hunted down in detecting carrier, cleans with cleaning fluid;
3) add the test serum sample, hatch the back and clean;
4) adding is anti-with biotin labeled two, hatches the back and cleans;
5) add streptavidin-phycoerythrin, hatch the back and clean;
6) adding mixes after detecting damping fluid;
7) read average fluorescent strength numerical value and analyze the data judging testing result with suspension chip system.
5. detection method as claimed in claim 4 is characterized in that, adopts biotin labeled SPA as detecting thing, and itself and western Buddhist nun sieve E protein combination composition indirect method detection architecture.
6. detection method as claimed in claim 4 is characterized in that, bag is 0.1～240 μ g/1.25 * 10 by western Buddhist nun sieve E proteantigen consumption of described coding microball in the described step 1)
6Individual coding microball or 0.02～960ng/2500～5000 microballoon/test.
7. a method that adopts protein suspension chip detection by quantitative serum sample Chinese and Western Buddhist nun sieve's antibody is characterized in that this method comprises the following steps:
1) positive detection sample of Jia Ruing or standard items are through the gradient doubling dilution;
2) series of diluted samples is detected in suspension chip system read corresponding fluorescent value;
3) dose-response curve of the corresponding fluorescent value of making sample concentration;
4) with analysis software match dose-response curve and equation;
5) judge dynamic detection range according to dose-response curve, according to the concentration of dose-response equation judgement sample Chinese and Western Buddhist nun sieve's antibody.
8. method as claimed in claim 7 is characterized in that, the gradient doubling dilution is 4 times in the described step 1).
9. method as claimed in claim 7 is characterized in that, the positive detection sample that adds in the described step 1) is the anti-Xi Niluo antibody of rabbit.
10. method as claimed in claim 7 is characterized in that, described step 2) in adopt the biotinylated detection antibody of 2mg/mL Bio-goat anti-rabbit antibody to detect as detecting thing with dilution in 1: 500.
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| CN2010102356614A CN101936988A (en) | 2010-07-23 | 2010-07-23 | Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof |
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| CN2010102356614A CN101936988A (en) | 2010-07-23 | 2010-07-23 | Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof |
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Application publication date: 20110105 |