CN101533020A - New method for detecting SARS antibody in serum sample and a product thereof - Google Patents
New method for detecting SARS antibody in serum sample and a product thereof Download PDFInfo
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- CN101533020A CN101533020A CN200910082600A CN200910082600A CN101533020A CN 101533020 A CN101533020 A CN 101533020A CN 200910082600 A CN200910082600 A CN 200910082600A CN 200910082600 A CN200910082600 A CN 200910082600A CN 101533020 A CN101533020 A CN 101533020A
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Abstract
The invention relates to a new method for quantitatively detecting SARS antibody of protein suspended chip in SARS antibody and a product thereof. The method of the invention has good detection ability, high flexibility, strong specificity and wide dynamic range, and establishes open detection modelization platform detected by virus antibody protein suspended chip using SARS antibody as representative.
Description
Technical field
The present invention relates to the quantitative detecting method of SARS antibody in a kind of new detection serum sample and product and preparation method thereof.
Background technology
Wreak havoc serious acute respiratory syndrome (the Severe AcuteRespiratory Syndrome in the whole world in year spring winter to 2003 in 2002, SARS, severe acute respiratory syndrome) pathogen sars coronavirus (SARS-CoV) is exactly a coronaviridae, a kind of in the coronavirus genus.Coronavirus is one of main pathogen of adult's common cold, and the childhood infection rate is higher, mainly is the infection of the upper respiratory tract, generally seldom involves lower respiratory tract.In addition, also can cause baby and neonate's acute gastroenteritis, cardinal symptom is water sample stool, heating, vomiting, can draw 10 every day surplus time, severe patient even the blood watery stool occurs also causes nervous system syndrome under the rare occasion.SARS-CoV is the sub-thread positive chain RNA virus, belongs to the nido virales, coronaviridae, coronavirus genus.According to serotype, coronavirus genus mainly is divided into 3 Group, comprises multiple mammal coronavirus and bird infectious bronchitis virus.SARS-CoV is first coronavirus that causes human serious disease.SARS-CoV primary structure albumen has: S albumen (spike glycoprotein), M albumen (transmembrane protein), E albumen (envelope protein), N albumen (NP).N albumen is wherein the most stable structural proteins, also is virus protein the abundantest in the course of infection, and confirmation is desirable diagnostic antigen.
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 the detection for cell factor at present.But whether suspending chip can detect the SARS antibody in the human serum, still lacks model and evaluation.
Summary of the invention
The present invention relates to the protein suspension chip of SARS antibody in a kind of new detection serum, this chip comprises: coding microball, as wrapping by the SARS-CoV N albumen of microballoon antigen, biotin labeled goat anti-rabbit igg is as detecting antibody, streptavidin-phycoerythrin and suitable buffer solution, described coding microball is No. 044 coding microball preferably.Described SARS-CoV N albumen for example is reorganization SARS-CoV total length nucleoprotein-N32 (aa1-422) and reorganization SARS-CoV nucleoprotein fragment N13 (aa221-422), all through nickel affinity column purifying.
The invention provides the preparation method of the protein suspension chip of above-mentioned detection SARS antibody, it is characterized in that, in relevant buffer solution, is goat anti-rabbit igg, streptavidin-phycoerythrin (SA-PE) with the coding microball bag by SARS-CoV N albumen, biotin labeled detection antibody, described coding microball is No. 044 coding microball preferably, described SARS-CoV N albumen for example is reorganization SARS-CoV total length nucleoprotein-N32 (aa1-422) and reorganization SARS-CoV nucleoprotein fragment N13 (aa221-422), all through nickel affinity column purifying.
The invention provides a kind of protein suspension chip quantitative detecting method of SARS antibody, this method adopts indirect method to detect the immunology detection principle of antibody, it is characterized in that, in testing process, respond and on 96 hole filter plates or in microcentrifugal tube, to carry out, comprising the following step: add to contain in (1) every hole or the pipe and wrap the antigen that is hunted down, be the working solution of the coding microball of SARS-CoV N albumen bag quilt, clean with cleaning fluid; (2) add the test serum sample, hatch the back and clean; (3) add with biotinylated detection antibody, hatch the back and clean; (4) add streptavidin-phycoerythrin (SA-PE), hatch the back and clean, mixing after (5) adding detection damping fluid, (6) read MFI numerical value (being average fluorescent strength) with suspension chip system and analyze the data judging testing result negative or positive.
The present invention also provides a kind of protein suspension chip quantitative detecting method that detects blood serum sample kind SARS antibody, it is characterized in that, this method comprises the following steps: that positive detection sample that (1) adds or standard items are through 4 times of gradient multiple proportions serial dilutions, (2) the dose-response typical curve of the corresponding MFI value of making sample concentration, (3) with analysis software match dose-response curve and equation, (4) can judge dynamic detection range according to dose-response curve, the unknown concentration sample can go out sample detection concentration according to the dose-response Equation for Calculating, goes back decidable method detectability.
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 5.5 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.The microballoon of label probe and determinand react in 96 orifice plates.After the reaction, utilize machine automatically reactant liquor to be picked up and by a microcapillary sense channel, only allow 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 and deep research to the protein suspension chip preparation and the testing conditions thereof of SARS antibody, and it has following advantage:
1, the improvement of antigen coated amount
The package amount of SARS-CoV N albumen 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 3-9 μ g/1.25 * 10
6Individual microballoon, i.e. 6-36ng/2500-5000 microballoon/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 quantitative detecting method of blood serum sample SARS antibody of the present invention is compared with the immunology ELISA detection method of classics with chip, and the result has good anastomose property (coefficient R
2=0.9994).Simultaneously, suspension chip method sensitivity is 402.9pg/mL, is higher than the 600ng/mL of ELISA method, and sensitivity is increased more than 1000 times; And suspension chip method dynamic detection range of the present invention is 146.48-2400000pg/mL L, is higher than ELISA method dynamic detection range 0.6-38.4ug/mL and reaches 2 orders of magnitude.
4, the specificity of method
The present invention is an envelope antigen by selecting SARS-CoV N albumen for use, is antibody to be measured with the anti-SARS antibody of rabbit, serves as to detect antibody with biotinylated goat-anti rabbit.This development test proves, exists mouse-anti influenza NPIgG, rabbit antitubercle serum, rabbit approach antibody, the anti-pestis F 1 IgG of rabbit to disturb that this method has good specificity under the antibody existence condition.
5, the detectability of sample
The present invention has estimated suspension chip method to SARS detection of antibodies ability in the human serum.By detection to human serum, tentative confirmation this method SARS antibody practicality in detecting human serum.
Description of drawings:
Fig. 1: No. 044 microballoon bag is by SARS-CoV N Protein Detection SARS antibody synoptic diagram;
Fig. 2: the protein suspension chip method detects the anti-SARS antibody dosage of rabbit-reaction normal curve map;
Fig. 3: the ELISA method detects the anti-SARS antibody of rabbit canonical plotting;
Fig. 4: protein suspending chip and ELISA method detect the correlativity of the anti-SARS antibody of rabbit.
Embodiment
Protein suspension chip preparation method, detection and the quantivative approach of the detection SARS 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, 1.44gNa
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 be in 250mL, pH6.2.
(4) the microballoon bag is cushioned liquid 0.05M MES, pH5.0:2.44g MES, and 5N NaOH 0.15mL, constant volume is in 250mL.
(5) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, 0.02%TWEEN, 0.05% azide, pH7.4.
(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) biotinylated antibody 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 SARS-CoV N proteantigen used herein for example is reorganization SARS-CoV total length nucleoprotein-N32 (aa1-422) and reorganization SARS-CoV nucleoprotein fragment N13 (aa221-422), all through nickel affinity column purifying.
Antibody to be measured used herein is the anti-SARS IgG of rabbit.Detecting antibody is biotinylation goat anti-rabbit igg and biotinylation goat anti-human igg, described goat anti-rabbit igg and goat anti-rabbit igg.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is the anti-SARS 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 rabbit antitubercle serum, rabbit anti influenza NP IgG, rabbit anti-avian influenza H5N1 serum, the anti-pestis F 1 IgG of rabbit, BSA, casein, tryptone etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid 4 ℃ of preservations.The storing solution concentration of the anti-SARS N of the anti-rabbit of rabbit IgG is 0.036mg/mL.In the comparative experiments, same sample is used for the detection of ELISA and suspending chip.
Anti-SARS 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
Respectively the human serum sample is diluted with sample diluting liquid 1:10, after for example human serum sample 5 μ L add sample dilution 50 μ L mixings, carry out the detection of suspension chip method as sample to be checked.
The preparation of the protein suspension chip of embodiment 1, detection SARS antibody
1, the capture antigen bag microballoon that is encoded
No. 044 coding microball that the present invention adopts is available from U.S. BIO-RAD company, and coding microball is used for the SARS-CoV N proteantigen that mark can be caught SARS antibody, promptly utilizes SARS-CoV N 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 SARS-CoV N proteantigen 4-50 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 antibody labeling 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 antibody-solutions 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.
B, antibody consumption calculate
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, 3 μ g, 6 μ g, 9 μ g, 12 μ g, 15 μ g, 20 μ g is encoded to No. 027 microballoon by 100 μ L respectively.Effect compares after testing, with 6 μ g/1.25 * 10
6Individual microballoon is that 12-24ng/2500-5000 microballoon/test pack is best 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. 044 microballoon of SARS-CoV N 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 SARS detection of antibodies.
2, the optimization of biotinylated antibody
The present invention uses for example biotin of LC-hydrazides (hydrazide)-biotin and carboxyl activity of amino active biotin respectively, for example the biotin labeling of Sulfo-NHS-biotin and SH-activity detects antibody, detect effect relatively through the Quality Control process, it is better that the biotin labeling of SH-activity detects the antibody test effect in this experiment, and the method that detects effect adopts the conventional method of this area or the described method of product description of installation manufacturer.
The inventor is through verification experimental verification, find that the biotinylated antibody goat-anti of 2mg/mL rabbit detects as detecting antibody with dilution in 1: 250, testing result shows that biotinylation detects the detection effect that antibody Bio-goat anti-rabbit antibody can obtain the Supreme People's Procuratorate's measured value and low background; As detecting plague antibodies in the human serum, testing result showed that biotinylation detects the detection effect that antibody Bio-goat anti-human antibody can obtain the Supreme People's Procuratorate's measured value and low background to the biotinylated antibody goat-anti of 2mg/mL people with dilution in 1: 2500.
Embodiment 3, suspending chip specimen preparation, detection and result judge
1, testing sample preparation
With sample diluting liquid sample to be tested is configured to 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) 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;
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 the protein suspension chip method and detects the detection substrate concentration of the minimum detectability (LOD value) of SARS 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 SARS antibody test positive as a result if testing result is higher than Cutoff; Corresponding fluorescence intensity level then is judged to be SARS antibody test feminine gender as a result if testing result is lower than Cutoff.
The using suspending chip detection method detects rabbit approach IgG, mouse-anti influenza NP IgG, the anti-SARSIgG of rabbit, rabbit anti-avian influenza H5N1 serum, the anti-pestis F 1 IgG of rabbit, BSA, casein, tryptone etc. respectively, according to testing result criterion among the embodiment 3, only the anti-SARS 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 protein suspension chip detection method and other test antibody that the SARS antibody that the present invention sets up is described.
The foundation of embodiment 5, suspending chip detection by quantitative model
1, the anti-SARS IgG of rabbit typical curve specimen preparation
With sample diluting liquid the anti-SARS IgG of rabbit standard items are become series concentration sample with 4 times of multiple proportions gradient dilutions to 0.55ng/mL by 0.036mg/mL.
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 the concentration (ng/mL) of antibody, 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, the concentration (ng/mL) of the anti-SARS antibody of rabbit is respectively S1:0.549 among Fig. 2, S2:2.197, S3:8.789, S4:35.156, S5:140.625, S6:562.5, S7:2250.0, S8:9000.0.
Suspension chip system is according to the anti-SARS IgG of testing result match rabbit dose-response typical curve equation: FI=0.774363+ (15544.5-0.774363)/[1+ (Conc/2884.69)
-7.77675]
0.0999461
3, suspending chip detects sensitivity and the dynamic range of the anti-SARS IgG of rabbit
According to minimum detectability definition and dose-response typical curve equation, the present invention is that the sensitivity of the anti-SARS IgG of protein suspension chip method detection rabbit is: 105.57pg/mL (Cutoff=6.31066, MFI (blank)=5.25, standard deviation=0.35355).
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-SARS IgG of rabbit concentration, its corresponding MFI value also increases progressively thereupon, when the anti-SARS IgG of rabbit concentration reaches 0.025mg/mL, the immune response of the antigen-antibody state that promptly reaches capacity, the MFI value also enters plateau, substrate concentration height to be checked in the interpret sample need will detect after the enriched sample dilution 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-SARS IgG of suspending chip detection by quantitative rabbit is 4.883～25000ng/mL.
1, biotin-avidin ELISA (BAS-ELISA) detection method
As a comparison, biotin-avidin ELISA employing comprises the following steps:
As to this, adopt the indirect method ELISA that comprises the following steps to detect:
1) adds the SARS-CoV N albumen 5-50 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 antibody, 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-SARS antibody of the mutually same group rabbit 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 a sample concentration among the figure, and ordinate is an absorbance, and coordinate axis is taken the logarithm-logarithmic relationship) that the ELISA method detects the anti-SARS antibody of rabbit, and compare with the suspension chip method result.
According to two kinds of method standard curves: suspension chip method sensitivity as shown in Figure 2 is 402.9pg/mL, linear detection range 146.48-2400000pg/mL; ELISA method sensitivity as shown in Figure 3 is 600ng/mL, linear detection range 0.6-38.4 μ g/mL.Can reach a conclusion: detect the anti-SARS antibody of identical rabbit sample, the protein suspension chip method has higher detection sensitivity than ELISA method, and is promptly high 1000 times; And has wideer dynamic detection range, promptly high 3 orders of magnitude.
In addition, two kinds of method testing results are compared as shown in Figure 4 in 0.6-38.4 μ g/mL scope, can judge that protein suspension chip method and ELISA method have good correlativity, related coefficient is 0.9518.
1, human serum sample's preparation
Detect being used for protein suspension chip after human serum sample's dilution 1: 10 dilution (human serum sample 5 μ L add sample dilution 50 μ L mixings).
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 goat anti-human antibody 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 SARS antibody in the human serum to be detected according to typical curve.
Through revision test repeatedly, biotinylation goat anti-human igg dilution ratio is selected the 1:2500 dilution for use, the SA-PE dilution ratio is selected the 1:300 dilution 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 3123.355, being scaled concentration value is 366ng/mL, if the MFI value that detection obtains is greater than 3123.355, be that SARS antibody concentration in the serum surpasses 366ng/mL and can be considered the SARS antibody positive and may be infected by SARS virus, if the MFI value is less than 3123.355, be that SARS antibody concentration in the serum is lower than 9.6ng/mL and can be judged as the SARS negative antibody, do not infect SARS virus.
Claims (8)
1, a kind of indirect immunology quantitative detecting method that adopts protein suspension chip to detect SARS antibody in the blood serum sample is characterized in that total overall reaction can be carried out in the testing process in 96 hole filter plates or microcentrifugal tube, and this method comprises the following steps:
(1) bag is a SARS-CoV N proteantigen by the capture antigen of microballoon;
(2) adding contains the working solution that wraps the antigen encoding microballoon that is hunted down in the hole, cleans with cleaning fluid;
(3) add the test serum sample, hatch the back and clean;
(4) add with biotinylated detection antibody, hatch the back and clean;
(5) add streptavidin-phycoerythrin, hatch the back and clean;
(6) mixing after the adding detection damping fluid;
(7) read average fluorescent strength (MFI) numerical value and analyze the data judging testing result with suspension chip system.
2, the method for claim 1 is characterized in that described SARS-CoV N proteantigen is for reorganization SARS-CoV total length nucleoprotein-N32 (aa1-422) and reorganization SARS-CoV nucleoprotein fragment N13 (aa221-422), all through nickel affinity column purifying.
3, the method for claim 1 is characterized in that, adopts biotin labeled goat anti-human igg as detecting antibody, and itself and the combination composition indirect method detection architecture of SARS-CoV N albumen.
4, the method for claim 1 is characterized in that, described bag is 3-9 μ g/1.25 * 10 by the anti-SARSN proteantigen of catching of microballoon consumption
6Individual coding microball or 6～36ng/2500-5000 microballoon/test.
5, the method for claim 1 is characterized in that, the biotin of the marker detection antibody goat anti-human igg in the described method is the biotin of carboxyl activity.
6, the method for claim 1 is characterized in that, the biotinylated detection antibody of 2mg/mL Bio-goat anti-human igg detects as detecting antibody with the 1:2500 dilution.
7, the protein suspension chip method of SARS antibody in a kind of detection by quantitative blood serum sample 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, and described gradient doubling dilution is 4 times;
(2) series of diluted samples is detected in suspension chip system read corresponding fluorescent value (MFI);
(3) dose-response curve of the corresponding MFI value of making sample concentration;
(4) with analysis software match dose-response curve and equation;
(5) the unknown concentration sample can be according to the concentration of SARS antibody in dose-response curve and the equation judgement unknown sample.
8, a kind of protein suspension chip that detects SARS antibody in the blood serum sample that adopts, this chip comprises: coding microball, as wrapping by the SARS-CoV N proteantigen of microballoon antigen, biotin labeled goat anti-rabbit igg and goat anti-human igg are as detecting antibody, streptavidin-phycoerythrin and suitable buffer solution.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101936990A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Preparation method and using method of protein suspension chip for detecting tick borne encephalitis antibody in serum sample |
| CN111518176A (en) * | 2020-07-06 | 2020-08-11 | 北京金智准科技有限公司 | Paired antigen for novel coronavirus antibody double-antigen sandwich detection, detection test paper and preparation method thereof |
| WO2021232713A1 (en) * | 2020-05-18 | 2021-11-25 | 博奥赛斯（天津）生物科技有限公司 | Enzyme-linked immunosorbent assay detection kit for novel coronavirus igg antibody |
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| DIRK DEREGT等: "A microsphere immunoassay for detection of antibodies to avian influenza virus", <JOURNAL OF VIROLOGICAL METHODS> * |
| 杜培革等: "SARS 冠状病毒N 蛋白基因的表达及其在SARS 诊断中的应用", <中华实验和临床病毒学杂志> * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101936990A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Preparation method and using method of protein suspension chip for detecting tick borne encephalitis antibody in serum sample |
| WO2021232713A1 (en) * | 2020-05-18 | 2021-11-25 | 博奥赛斯（天津）生物科技有限公司 | Enzyme-linked immunosorbent assay detection kit for novel coronavirus igg antibody |
| CN111518176A (en) * | 2020-07-06 | 2020-08-11 | 北京金智准科技有限公司 | Paired antigen for novel coronavirus antibody double-antigen sandwich detection, detection test paper and preparation method thereof |
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