CN201903543U - Protein suspension array system for detecting avian influenza antibody in blood serums - Google Patents
Protein suspension array system for detecting avian influenza antibody in blood serums Download PDFInfo
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- CN201903543U CN201903543U CN2010206284428U CN201020628442U CN201903543U CN 201903543 U CN201903543 U CN 201903543U CN 2010206284428 U CN2010206284428 U CN 2010206284428U CN 201020628442 U CN201020628442 U CN 201020628442U CN 201903543 U CN201903543 U CN 201903543U
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Abstract
The utility model discloses a protein suspension array system for detecting avian influenza antibody in blood serums. The protein suspension array system comprises an array base body, a liquid adding system, a suspension array detector and a computer, wherein the array base body is filled with a related buffer solution and an antibody to-be-detected, a code microsphere is arranged in the related buffer solution and coated with a captured antigen, the liquid adding system is used for sequentially adding a second antibody and/or SPA marked by biotins and a fluorescent signal detecting object into the array base body, the suspension array detector comprises micro tubule detection passages and a detection laser, the code microsphere sequentially passes the micro tubule detection passages, the detection laser is used for exciting and identifying color of the code microsphere and an object to be detected, and the computer is connected with the suspension array detector, and is used for recording and analyzing measurement result of the suspension array detector. The suspension array technique utilizes the microsphere reacting in the solution; the accuracy and the repeatability of sample detection are improved by adopting the laser detection technique; and superior to a metal-film array, the protein suspension array system has the characteristics of simplicity and convenience in operation, good repeatability, and the like.
Description
Technical field
The utility model relates to a kind of protein suspension chip system that detects avian influenza antibody in the serum sample.
Background technology
Avian influenza virus belongs to influenza A virus, and influenza A virus is mainly in bird, and in general, there are receptor-specific difference in avian influenza virus and human influenza virus, and avian influenza virus is not easy to infect to the people.The avian influenza virus that causes people's infection morbidity individually may be the virus that variation has taken place, but obstacle direct infection people had 18 Hong Kong residents and has infected H5N1 subtype influenza virus between Hong Kong avian influenza virus in 1997 was broken through kind first, the wherein generation of 6 people's death incidents, broken the traditional understanding pattern of people to avian influenza virus, studies show that, the avian influenza virus subtype of infected person is mainly H5N1, H9N2, H7N7, and the conditions of patients that wherein infects H5N1 is heavy, the case fatality rate height.
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 suspending chip can detect the avian influenza antibody in the human serum, and its detection by quantitative ability how, still lacks model and evaluation.
The utility model content
The purpose of this utility model is to provide a kind of protein suspension chip system that detects avian influenza virus antibody in the serum, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, relevant buffer solution and antibody to be measured are housed in the chip basal body, be provided with coding microball No. 032 in the relevant damping fluid, be coated with capture antigen on the coding microball, add liquid systems and in chip basal body, add biotin labeled two anti-and/or SPA successively, fluorescence signal detects thing, the suspending chip detector comprises microcapillary sense channel and detection laser, reacted solution is drawn in the microcapillary sense channel, so that each coding microball is successively by the microcapillary sense channel, detection laser excites and the color of recognition coding microballoon and the fluorescence signal of last determinand thereof, computing machine links to each other with the suspending chip detector, and record, analyze the measurement result of suspending chip detector.
Further, described relevant buffer solution washs the used microballoon cleaning fluid of coding microball, SA-PE dilution after comprising the used microballoon dilution of the sample diluting liquid that is used for described antibody dilution to be measured, the described coding microball of dilution, dilution described biotin labeled two are anti-and/or SPA is used antibody diluent, each reaction, detects damping fluid.
Further, described capture antigen is preferably bird flu H5 albumen.
Further, described antibody to be measured is blood serum sample.
Further, described biotin labeled two anti-and/or SPA are preferably Biotin-goat anti-rabbit igg and/or Biotin-SPA.
Further, described fluorescence signal detection thing is streptavidin-phycoerythrin.
Further, described chip basal body is 96 hole filter plate or microcentrifugal tubes.
Further, described detection laser comprises with exciting color in the described coding microball matrix, the sorting code number of recognition coding microballoon to determine the red laser of test item, and the color signal, tracer signal power that is used to excite and discern testing molecule is with the green laser of the content that detects described antibody to be measured.
Through a large amount of and deep research, the protein suspension chip preparation and the testing conditions thereof of avian influenza antibody have been done substantial improvement and innovation, it has following advantage:
1, the improvement of antigen coated amount
The package amount of bird flu H5 albumen is the key that successfully detects, the utility model is carried out substantive optimization to bag by the antigen coated amount of microballoon makes the detection effect of serum sample very good, and the required antigen coated amount of coating protein suspending chip is very low, bird flu H5 proteantigen package amount of the present utility model is 0.1～100 μ g/1.25 * 106 coding microball or 0.02～400ng/2500～5000 microballoon/test, and general ELISA to test the package amount of required antigen be 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 antigen 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, the phenomenon that the detection signal that fell in may be too high, so after advising biotin labeling antibody, remove 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 suspension chip quantitative detecting method of blood serum sample avian influenza antibody of the present utility model is compared with the immunology ELISA detection method of classics, and the result has good anastomose property (coefficient R 2=0.9863).Simultaneously, suspension chip method sensitivity is higher than the detection titre 10-5 of ELISA method for detecting titre 4.767 * 10-6, and sensitivity improves more than 10 times; And the suspension chip method dynamic detection range is 2 * 10-7～2 * 10-2, is higher than ELISA method dynamic detection range 10-5～10-2 and reaches 1 order of magnitude.
4, the specificity of method
The utility model is an envelope antigen by selecting bird flu H5 albumen for use, is antibody to be measured with rabbit anti-avian influenza H5N1 antibody, serves as to detect thing with biotinylation-goat anti-rabbit igg.This development test proves, this method has good specificity under the interference antibody existence conditions such as mouse-anti influenza NP IgG, rabbit antitubercle serum, the anti-F 1 antibody of plague bacterium polyclonal antibody of rabbit, the anti-SARS IgG of rabbit existing.
5, the detectability of sample
The utility model has been estimated the detectability of protein suspension chip method to human serum.By the detection of human serum, tentative confirmation the practicality of this method avian influenza antibody in detecting human serum.
Description of drawings:
Fig. 1 is the utility model structural representation;
Fig. 2 is that No. 032 microballoon bag is by bird flu H5 Protein Detection rabbit anti-avian influenza H5N1 serum synoptic diagram;
Fig. 3 detects rabbit anti-avian influenza H5N1 serum dosage-reaction normal curve for the protein suspension chip method.
Fig. 4: the ELISA method detects rabbit anti-avian influenza H5N1 serum canonical plotting;
Fig. 5: protein suspension chip and ELISA method detect the correlativity of rabbit anti-avian influenza H5N1 serum.
Embodiment
Extremely shown in Figure 5 as Fig. 1, a kind of protein suspension chip system that detects avian influenza antibody in the serum sample of the utility model, comprise chip basal body 1, add liquid systems 2, suspending chip detector 3 and computing machine 4, wherein, chip basal body 1 is 96 hole filter plate or microcentrifugal tubes, relevant buffer solution and antibody to be measured are housed in it, be provided with the coding microball (not shown) in the relevant damping fluid, be coated with bird flu H5 albumen on the coding microball, be used for catching the antibody to be measured of blood serum sample to be detected, if avian influenza antibody is arranged in the sample, bird flu protein combination on avian influenza antibody and the coding microball, add again and have biotin labeled two anti-and/or SPA, forming coding microball-bird flu H5 albumen-avian influenza antibody-two resists/the SPA-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-bird flu H5 albumen-avian influenza antibody-two resists/SPA-biotin-streptavidin-phycoerythrin compound, by the suspending chip detector detection of phycoerythrin fluorescence signal is reached detection to avian influenza antibody in the blood serum sample, if there is not avian influenza antibody in the blood serum sample, the coding microball that is coated with bird flu H5 albumen not can with the biotin labeled two anti-and/or SPA that add later, streptavidin-phycoerythrin combination, do not have when detecting by the suspending chip detecting instrument at last fluorescence signal or should signal very a little less than.
In the said chip system, coding microball is at the microballoon dilution, antibody to be measured is in sample diluting liquid, biotin labeled two anti-and/or SPA are in antibody diluent, fluorescence signal detects thing in the SA-PE dilution, the coding microball compound was in detecting damping fluid when the suspending chip detector detected, and per step, making did not have the material of combination to remove system in conjunction with all washing microballoon with cleaning fluid afterwards.
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.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.
A kind of protein suspension chip system that detects avian influenza antibody in the serum sample of the utility model be described further by following embodiment, but the utility model 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; Na2HPO4 10mmol/L; KH2PO4 2mmol/L.With 800mL dissolved in distilled water 8gNaCl, 0.2gKCl, 1.44g Na2HPO4 and 0.24g KH2PO4.PH value to 7.4 with the HCl regulator solution adds water to 1L.15psi (1.05kg/cm2) high pressure steam 20 minutes, or filtration sterilization was stored in room temperature after the packing.
(2) microballoon cleaning fluid: PBS (pH7.4), 0.05%TWEEN-20.
(3) microballoon activation damping fluid 100mM NaH2PO4:3g NaH2PO4,5N 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) SA-PE dilution: PBS (pH7.4), 1%BSA.
2. antigen and antibody
The utility model uses antigen recombinant fowl influenza virus H5 albumen, and it can be available from the Harbin veterinary institute.
The utility model uses antibody to be measured to be rabbit anti-avian influenza H5N1 serum, it can be available from CDC virosis prevention and control research institute, detecting antibody is biotinylation goat anti-rabbit igg and biotinylation SPA, goat anti-rabbit igg and SPA, it can be glad through biological company limited of section available from Beijing.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is rabbit anti-avian influenza H5N1 IgG, 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, the anti-SARS IgG of rabbit, rabbit anti-avian influenza H5N1 serum, the anti-plague IgG of rabbit, BSA, casein, tryptone etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid liquid 4 ℃ of preservations.The storing solution concentration of anti-avian influenza H5N1 IgG is 0.2mg/mL.In the comparative experiments, same sample is used for the detection of ELISA and protein suspension chip.
Is variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with rabbit anti-avian influenza H5N1 IgG 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 protein suspension chip method as sample to be checked.
The preparation of the protein suspension chip of embodiment 1, detection avian influenza antibody
1, the capture antigen bag microballoon that is encoded
No. 032 coding microball that the utility model adopts is available from U.S. Bio-Rad company, and coding microball is used for the bird flu H5 proteantigen that mark can be caught avian influenza antibody, promptly utilizes bird flu H5 albumen bag by microballoon.
The activation of A, coding microball
032 coding microball is in the 1.5mL centrifuge tube to get 100 μ L (1.25 * 106), 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 032 coding microball after capture antigen bird flu H5 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; 1/400mm2) under simple microscope, count.Calculate microballoon quantity according to formula (each big lattice number * 104 * extension rate * volume (mL)).
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.
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, protein suspension chip preparation method condition
1, the selection of microballoon envelope antigen package amount
Amount bag with 1 μ g, 5 μ g, 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g, 45 μ g, 50 μ g, 100 μ g is encoded to No. 032 microballoon by 100 μ L respectively.Effect is that 0.2～200ng/2500～5000 microballoon/bag is best by effect with 1～50 μ g/1.25 * 106 coding microball relatively after testing, 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. 032 microballoon of bird flu H5 proteantigen, and obtain high s/n ratio result (the MFI value is much larger than 2000), illustrate that the protein suspension chip detection system of optimizing can successfully be used for the detection of avian influenza antibody.
2, the optimization of biotinylated antibody
The utility model is used 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, it is better that the Sulfo-NHS-biotin labeling 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 utility model people is through verification experimental verification, find that the biotinylated goat anti-rabbit antibody of 2mg/mL detects avian influenza antibody in the rabbit anteserum with dilution in 1: 250 as detecting antibody, 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 avian influenza antibody in the human serum, testing result showed that biotinylation detects the detection effect that antibody Bio-SPA can obtain the Supreme People's Procuratorate's measured value and low background to 2mg/mL biotinylation SPA with dilution in 1: 2500.
The preparation of embodiment 3, sample, 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 the protein suspension chip system.
3, testing result is judged
According to experimental study result and correlative study report, the minimum detectability (LOD value) that the utility model definition protein suspension chip method detects avian influenza antibody is the corresponding detection substrate concentration of fluorescence intensity critical value (Cutoff).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 avian influenza antibody testing result positive if testing result is higher than Cutoff; Corresponding fluorescence intensity level then is judged to be avian influenza antibody testing result feminine gender if testing result is lower than Cutoff.
Using the protein suspension chip detection method detects rabbit approach IgG, mouse-anti influenza NP IgG, the anti-SARS IgG of rabbit, rabbit anti-avian influenza H5N1 serum, the anti-plague IgG of rabbit, BSA, casein, tryptone etc. respectively, according to testing result criterion among the embodiment 3, only rabbit anti-avian influenza H5N1 IgG 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 avian influenza antibody that the utility model is set up is described.
1, rabbit anti-avian influenza H5N1 IgG typical curve specimen preparation
With sample diluting liquid rabbit anti-avian influenza H5N1 IgG standard items are become series concentration sample with 4 times of multiple proportions gradient dilutions to 191p g/mL by 20 μ g/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 3) according to protein 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, and the concentration (ng/mL) of rabbit anti-avian influenza H5N1 serum is respectively and does 4 doubling dilution scopes and be among Fig. 2: S1:0.004, S2:0.0162, S3:0.647, S4:0.259, S5:1.04, S6:4.14, S7:16.56, S8:66.25, S9:265, S10:1060.
The protein suspension chip system according to testing result match rabbit anti-avian influenza H5N1 serum dosage-reaction normal curvilinear equation is:
FI＝-1.31918+(13360.5+1.31918)/[1+(Conc/3.93328)
-0.957367]
0.953689
3, the utility model protein suspension chip method detects sensitivity and the dynamic range of rabbit anti-avian influenza H5N1IgG in the serum
According to minimum detectability definition and dose-response typical curve equation, the utility model is that the sensitivity of protein suspension chip method detection rabbit anti-avian influenza H5N1IgG is: 0.0015ng/mL (Cutoff=8.6213, MFI (blank)=6.5, standard deviation=0.7071).
The utility model definition Supreme Procuratorate rising limit is to make the binding site of coding microball be in the detection substrate concentration of state of saturation.According to the rising of typical curve along with rabbit anti-avian influenza H5N1IgG concentration, its corresponding MFI value also increases progressively thereupon, when rabbit anti-avian influenza H5N1IgG concentration surpasses 0.05mg/mL, the immune response of the antigen-antibody state that do not reach capacity, the MFI value does not also enter plateau, substrate concentration to be checked in the interpret sample does not also reach the concentration that makes the MFI value saturated, needs the sample of high concentration to detect again.Therefore, can judge that according to minimum detectability and Supreme Procuratorate's rising limit the present invention is that the dynamic range of protein suspension chip detection by quantitative rabbit anti-avian influenza H5N1 IgG is 48.828～50000ng/mL.
1, biotin-avidin ELISA (BAS-ELISA) detection method
As a comparison, biotin-avidin ELISA adopts and comprises the following steps:
1) adds the bird flu H5 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 protein suspension chip detection method 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 mutually same group rabbit anti-avian influenza H5N1 blood serum sample of preparation uses protein suspension chip simultaneously with sample diluting liquid 10 doubling dilutions and the ELISA method detects.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 rabbit anti-avian influenza H5N1 serum, and compare with the protein suspension chip methods and results.
According to two kinds of method standard curves: the sensitivity as shown in Figure 2 of protein suspension chip method is 4.767 * 10-6 (serum titer), linear detection range 2 * 10-7～2 * 10-2; The sensitivity as shown in Figure 3 of ELISA method is 105 (serum titers), linear detection range 10-5～10-2.Can reach a conclusion: detect identical rabbit anti-avian influenza H5N1 blood serum sample, 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 2 orders of magnitude.
In addition, as shown in Figure 4, two kinds of method testing results are compared in 10-5～10-2 scope can judge that protein suspension chip method and ELISA method have good correlativity, related coefficient is 0.9863.
1, human serum sample's preparation
The human serum sample 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 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 avian influenza antibody in the human serum to be detected 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 2286, being scaled concentration value is 0.68ng/mL, if the MFI value that detection obtains is greater than 2286, it is the avian influenza antibody excessive concentration in the serum, can be considered the avian influenza antibody positive may be by avian flu virus infection, if the MFI value is less than 2286, be that dengue antibody concentration in the serum is lower than 0.68ng/mL and can be judged as the avian influenza antibody feminine gender, do not infect the recessive carrier of avian influenza virus or avian influenza virus.
Claims (8)
1. protein suspension chip system that detects avian influenza antibody in the serum sample, it is characterized in that, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, relevant buffer solution and antibody to be measured are housed in the chip basal body, be provided with coding microball No. 032 in the relevant damping fluid, be coated with capture antigen on the coding microball, add liquid systems and in chip basal body, add biotin labeled two anti-and/or SPA successively, fluorescence signal detects thing, the suspending chip detector comprises microcapillary sense channel and detection laser, reacted solution is drawn in the microcapillary sense channel, so that each coding microball is successively by the microcapillary sense channel, detection laser excites and the color of recognition coding microballoon and the fluorescence signal of last determinand thereof, computing machine links to each other with the suspending chip detector, and record, analyze the measurement result of suspending chip detector.
2. protein suspension chip as claimed in claim 1 system, it is characterized in that described relevant buffer solution washs the used microballoon cleaning fluid of coding microball, SA-PE dilution after comprising the used microballoon dilution of the sample diluting liquid that is used for described antibody dilution to be measured, the described coding microball of dilution, dilution described biotin labeled two are anti-and/or SPA is used antibody diluent, each reaction, detects damping fluid.
3. protein suspension chip as claimed in claim 2 system is characterized in that described capture antigen is preferably bird flu H5 albumen.
4. protein suspension chip as claimed in claim 2 system is characterized in that described antibody to be measured is blood serum sample.
5. protein suspension chip as claimed in claim 2 system is characterized in that, described biotin labeled two anti-and/or SPA are preferably Biotin-goat-anti rabbit and/or Biotin-SPA.
6. protein suspension chip as claimed in claim 2 system is characterized in that, it is streptavidin-phycoerythrin that described fluorescence signal detects thing.
7. protein suspension chip as claimed in claim 1 system is characterized in that described chip basal body is 96 hole filter plate or microcentrifugal tubes.
8. protein suspension chip as claimed in claim 1 system, it is characterized in that, described detection laser comprises with exciting color in the described coding microball matrix, the sorting code number of recognition coding microballoon to determine the red laser of test item, and the color signal, tracer signal power that is used to excite and discern testing molecule is with the green laser of the content that detects described antibody to be measured.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010206284428U CN201903543U (en) | 2010-11-24 | 2010-11-24 | Protein suspension array system for detecting avian influenza antibody in blood serums |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010206284428U CN201903543U (en) | 2010-11-24 | 2010-11-24 | Protein suspension array system for detecting avian influenza antibody in blood serums |
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| CN201903543U true CN201903543U (en) | 2011-07-20 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114720684A (en) * | 2022-04-01 | 2022-07-08 | 武汉爱博泰克生物科技有限公司 | Interleukin cytokine multi-flow type fluorescence detection kit, detection method and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114720684A (en) * | 2022-04-01 | 2022-07-08 | 武汉爱博泰克生物科技有限公司 | Interleukin cytokine multi-flow type fluorescence detection kit, detection method and application |
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