CN101533022A - New method and product for detecting influenza antibody in serum sample - Google Patents
New method and product for detecting influenza antibody in serum sample Download PDFInfo
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- CN101533022A CN101533022A CN200910083002A CN200910083002A CN101533022A CN 101533022 A CN101533022 A CN 101533022A CN 200910083002 A CN200910083002 A CN 200910083002A CN 200910083002 A CN200910083002 A CN 200910083002A CN 101533022 A CN101533022 A CN 101533022A
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Abstract
The invention relates to a new quantitative detection method and a product for detecting influenza antibody in a serum sample. The method of the invention has good detection capability, high sensitivity, strong specificity and wide dynamic range. The invention establishes an open detection modularised platform of pathogenic bacteria antibody protein suspension chip detection with the influenza antibody as a representative.
Description
Technical field
The present invention relates to the quantitative detecting method of influenza antibodies in a kind of new detection serum sample and product and preparation method thereof.
Background technology
Influenza virus; be called for short influenza virus; be that a kind of mankind of causing and animal suffer from grippal RNA viruses; on taxonomy; influenza virus belongs to Orthomyxoviridae family; it can cause acute upper respiratory infection, and propagates rapidly by air, and regular meeting has periodically and is very popular all over the world.Influenza virus can cause more serious symptom the more weak old man of immunity or the patient of child and some immune disorders, as pneumonia or cardiopulmonary depletion etc.Influenza virus constitutes by three layers, and internal layer is a virus nucleocapsid, contains nucleoprotein (NP), P albumen and PNA.NP is soluble antigen (a S antigen), has type specificity, and antigenicity is stable.P albumen (P1, P2, P3) may be rna transcription and duplicate required polymerase, the middle level is a virus envelope, be made of one deck lipoid and skim albumen, skin is the radial projection that two kinds of different sugar albumen constitute, i.e. agglutinin of blood (H) and neuraminidase (N).H can cause red cell agglutination, is the instrument that virus is adsorbed in the sensitive cells surface, and N then can the hydrolysis mucus protein, and the N-n acetylneuraminic acid n of hydrolysis cell surface receptor specificity glycoprotein end is that virus replication is finished and broken away from the instrument that cell shows.H and N all have variability, so have only the special antigenicity of strain, its antibody has protective effect.
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 at cell factor at present, whether can detect the plague antibodies in the human serum, and its detection by quantitative ability how, still lacks model and evaluation.
Summary of the invention
The present invention relates to the protein suspension chip of influenza antibodies in a kind of new detection serum, this chip comprises: coding microball, as wrapping by the influenza NP proteantigen of microballoon antigen, biotin labeled sheep anti-mouse igg and goat anti-human igg are as detecting antibody, streptavidin-phycoerythrin and suitable buffer solution, described coding microball can be selected the arbitrary number coding microball.
The invention provides the preparation method of the protein suspension chip of above-mentioned detection influenza antibodies, it is characterized in that, in relevant buffer solution, coding microball with influenza NP albumen bag by, be that sheep anti-mouse igg and goat anti-human igg, usefulness streptavidin-phycoerythrin (SA-PE) are as signal detection with biotin labeled detection antibody.
The invention provides a kind of protein suspension chip quantitative detecting method of influenza antibodies, 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 influenza NP proteantigen, 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 influenza antibodies in the serum, 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 be according to the dynamic detection range of dose-response curve decision method, the unknown concentration sample can go out the concentration of test sample according to the dose-response Equation for Calculating.
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.
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 influenza antibodies, and it has following advantage:
1, the improvement of antigen coated amount
The package amount of influenza NP 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, influenza NP proteantigen package amount of the present invention is 3 μ g/1.25 * 10
6Individual microballoon, i.e. 6-40ng/2500-5000 microballoon/test, and general ELISA to test the package amount of required antigen be 5 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 influenza antibodies of the present invention and the sensitivity of suspending chip are 5.6813ng/ml; And the suspension chip method dynamic detection range is 48.828～50000ng/ml.
4, the specificity of method
The present invention is an envelope antigen by the NP albumen of selecting influenza for use, is antibody to be measured with mouse-anti influenza NP IgG, and resisting with two of biotinylated sheep anti mouse serves as to detect antibody.This development test proves, exists rabbit antitubercle serum, rabbit anti-avian influenza H5N1 serum, the anti-F 1 antibody of plague bacterium polyclonal antibody of rabbit, the anti-SARS 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 the detectability of suspension chip method to human serum.By the detection of human serum, tentative confirmation the practicality of this method influenza antibodies in detecting human serum.
Description of drawings:
Fig. 1: No. 031 microballoon bag is by influenza NP Protein Detection influenza antibodies synoptic diagram;
Fig. 2: the protein suspension chip method detects mouse-anti influenza NP IgG dose-response typical curve.
Embodiment
Protein suspension chip preparation method, detection and the quantivative approach of the detection tuberculosis 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
4And 0.24gKH
2PO
4PH 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 NaH
2PO
4: 3g NaH
2PO
4, 5N NaOH1.5mL, 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 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 used herein is influenza NP albumen.
Antibody to be measured used herein is mouse-anti influenza NP IgG, and detecting antibody is biotinylation sheep anti-mouse igg and biotinylation goat anti-human igg, sheep anti-mouse igg and goat anti-human igg.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is mouse-anti influenza NP 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 approach IgG, 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 mouse-anti influenza NP IgG is 0.5mg/mL.
Is variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with mouse-anti influenza NP 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 1:10 dilution, 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 suspension chip of embodiment 1, detection influenza antibodies
1, the capture antigen bag microballoon that is encoded
No. 031 coding microball that the present invention adopts is available from U.S. BIO-RAD company, and coding microball is used for the influenza NP proteantigen that mark can be caught influenza antibodies, promptly utilizes influenza NP 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 influenza NP proteantigen 4～50 μ g join 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.
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, 5 μ g, 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g is encoded to No. 031 microballoon by 100 μ L respectively.Effect compares after testing, with 10 μ g/1.25 * 10
6Individual microballoon is that 20-40ng/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. 031 microballoon of influenza NP 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 detection of influenza antibodies.
2, the optimization of biotinylated antibody
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, 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 inventor is through verification experimental verification, find that the biotinylated sheep anti-mouse antibody of 2mg/mL detects influenza antibodies in the mouse serum with the 1:1000 dilution as detecting antibody, testing result shows that biotinylation detects the detection effect that antibody Bio-sheep anti-mouse antibody can obtain the Supreme People's Procuratorate's measured value and low background; As detecting influenza antibodies in the human serum, testing result shows 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 goat anti-human antibody of 2mg/mL with the 1:2500 dilution.
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 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 influenza antibodies 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 influenza antibodies testing result positive if testing result is higher than Cutoff; If being lower than the corresponding fluorescence intensity level of Cutoff, testing result then is judged to be knot influenza health check-up survey feminine gender as a result.
Embodiment 4, suspending chip are tested the specificity of influenza NP proteantigen
The using suspending chip detection method detects mouse-anti influenza NP IgG, rabbit approach IgG, the anti-SARSIgG 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 hairy rat anti influenza NP 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 method for detecting suspension chip and other test antibody that the influenza antibodies that the present invention sets up is described.
Embodiment 5, to the suspending chip detection by quantitative of influenza antibodies in the blood serum sample
1, mouse-anti influenza NP IgG typical curve specimen preparation
With sample diluting liquid mouse-anti influenza NP IgG standard items are become series concentration sample with 4 times of multiple proportions gradient dilutions to 0.763ng/mL by 0.05 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 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 mouse-anti influenza NP is respectively among Fig. 2: S1:0.763, S2:3.052, S3:12.207, S4:48.828, S5:195.312, S6:781.25, S7:3125.0, S8:12500.0, S9:50000.0.
Suspension chip system according to testing result match mouse-anti influenza NP IgG dose-response typical curve equation is:
FI＝0.670815+(389620-0.670815)/[1+(Conc/89.5047)
-0.175674]
10
3, suspending chip method of the present invention detects sensitivity and the dynamic range of mouse-anti influenza NP IgG in the serum
According to minimum detectability definition and dose-response typical curve equation, the present invention is that the sensitivity of protein suspension chip method detection mouse-anti influenza NP IgG is: 5.6813ng/mL (Cutoff=25.9319, MFI (blank)=22.75, standard deviation=1.0606).
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 mouse-anti influenza NP antibody concentration, its corresponding MFI value also increases progressively thereupon, when mouse-anti influenza NP antibody concentration surpasses 50000ng/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, need the sample of high concentration to detect again, but the restriction that is subjected to sample concentration can only reach 50000ng/mL fails to determine highest detection and limits, but but preliminary judgement the present invention is the dynamic range of suspending chip detection by quantitative mouse-anti influenza NP IgG is 48.828～50000ng/mL.
Embodiment 6, human serum sample's detection
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) go into the SA-PE of 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 influenza antibodies in the human serum to be detected according to typical curve.
Through revision test repeatedly, biotinylation goat anti-human igg dilution ratio is preferably diluted with 1:2500, the SA-PE dilution ratio preferably with the 1:300 dilution, is checked that by the Bio-plex detection system fluorescent value of income earner's serum sample is brought curvilinear equation FI=0.670815+ (389620-0.670815)/[1+ (Conc/89.5047) into
-0.175674]
10Can obtain the concentration of influenza antibodies in the human serum.
Claims (7)
1, a kind of indirect immunology quantitative detecting method that adopts protein suspension chip to detect influenza antibodies 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 an influenza NP proteantigen by the capture antigen of microballoon,
(2) go into adding to the hole and contain the working solution that wraps the antigen encoding microballoon that is hunted down, clean 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 adopting biotin labeled goat anti-human igg as detecting antibody, and itself and the combination composition indirect method detection architecture of influenza NP albumen.
3, the method for claim 1 is characterized in that, described bag is 2-10 μ g/1.25 * 10 by the capture antigen influenza NP proteantigen consumption of microballoon
6Individual coding microball or 6～40ng/2500-5000 microballoon/test.
4, 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.
5, 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.
6, the protein suspension chip method of influenza antibodies 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 is characterized in that the positive detection sample of adding is mouse-anti influenza NP IgG according to the concentration of influenza antibodies in dose-response curve and the equation judgement unknown sample.
7, a kind of protein suspension chip that detects influenza antibodies in the blood serum sample, this chip comprises: coding microball, is influenza NP proteantigen as bag by microballoon antigen, biotin labeled sheep anti-mouse igg and goat anti-human igg are as detecting antibody, and streptavidin-phycoerythrin is as signal detection and suitable buffer solution.
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| CN200910083002A CN101533022A (en) | 2009-04-28 | 2009-04-28 | New method and product for detecting influenza antibody in serum sample |
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|---|---|---|---|
| CN200910083002A CN101533022A (en) | 2009-04-28 | 2009-04-28 | New method and product for detecting influenza antibody in serum sample |
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ID=41103750
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915836A (en) * | 2010-07-23 | 2010-12-15 | 中国检验检疫科学研究院 | Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof |
-
2009
- 2009-04-28 CN CN200910083002A patent/CN101533022A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915836A (en) * | 2010-07-23 | 2010-12-15 | 中国检验检疫科学研究院 | Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof |
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Application publication date: 20090916 |