CN101915836A - Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof - Google Patents
Protein suspension chip for detecting dengue antibody in serum sample and preparation method and using method thereof Download PDFInfo
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Abstract
The invention relates to a protein suspension chip for detecting a dengue antibody in a serum sample and a preparation method and a using method thereof. The methods of the invention have high detection capability, high sensitivity, strong specificity and wide dynamic range and establish an open detection modelization platform for viral antibody, represented by the dengue antibody, protein suspension chip detection.
Description
Technical field
The present invention relates to a kind of protein suspension chip that detects antibody of dengue fever virus in the serum sample and preparation method thereof and using method.
Background technology
Dengue fever is that dengue fever virus causes, complies with mosquito-borne a kind of acute infectious disease.Clinical symptoms is that onset is hurried, high heat, and whole-body muscle, marrow and arthralgia, extremely tired, part is suffered from can fash, hemorrhagic tendency and enlargement of lymph nodes.Dengue fever virus belongs to group B arbovirus, is included into Alphaherpesvirinae (togaviridae) yellow fever virus now and belongs to (flavivirus).Virion be dumbbell shaped (700 * 20～40nm), it is bar-shaped or spherical that (diameter is 20～50nm).Nucleus pulposus is a sub-thread wire RNA (ribonucleic acid) (RNA).Virion is similar to japanese encephalitis virus, and outermost layer is the coating that two kinds of glycoprotein are formed, and coating contains type and group specific antigen, can identify its type with neutralization test.Dengue virus can be divided into 4 serotypes, but with other group B arbovirus such as japanese encephalitis virus cross-immune reaction.
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 dengue antibody in the human serum, and its detection by quantitative ability how, still lacks model and evaluation.
Summary of the invention
The object of the present invention is to provide a kind of protein suspension chip that detects dengue antibody in the serum, this chip comprises: coding microball, dengue fever E2 proteantigen, standard items, sample to be checked, biotin labeled two anti-, streptavidin-phycoerythrin, and relevant buffer solution, suspension chip system detects fluorescent value.
The invention provides the preparation method of the protein suspension chip of dengue antibody in the above-mentioned detection serum sample, be specially: coding microball is the microballoon that has carboxyl, with the dengue fever E2 albumen bag microballoon that is encoded, mouse-anti dengue fever IgG is standard items, the human serum sample is as sample to be checked, biotin labeled detection thing is biotinylated SPA (Biotin-SPA) and biotinylation sheep anti-mouse igg, detect thing with streptavidin-phycoerythrin (SA-PE) as fluorescence signal, above reagent is all with relevant buffer solution dilution, the preferred No. 031 carboxyl coding microball of described coding microball.
The invention provides a kind of indirect immunological detection method that adopts above-mentioned protein suspension chip to detect dengue antibody in the serum sample, respond in testing process and can carry out on 96 micropore filter plates or in microcentrifugal tube, comprising the following step: (1) wraps the microballoon that is encoded with dengue fever E2 proteantigen as capture antigen; (2) add in every hole or the pipe to contain and wrap the antigen that is hunted down, promptly the working solution of the coding microball of dengue fever E2 proteantigen bag quilt cleans with cleaning fluid; (3) add the test serum sample, hatch the back and clean; (4) add with biotinylated detection thing, hatch the back and clean; (5) add streptavidin-phycoerythrin (SA-PE), hatch the back and clean, mixing after (6) adding detection damping fluid, (7) read MFI numerical value (being average fluorescent strength) with suspension chip system and analyze the data judging testing result negative or positive.
In this method, adopt biotin labeled SPA as detecting antibody, and itself and the combination composition indirect method detection architecture of dengue fever E2 albumen; The capture antigen dengue fever E2 proteantigen consumption that wraps the microballoon that is encoded is 0.1～100 μ g/1.25 * 10
6Individual coding microball or 0.02～400ng/2500～5000 microballoon/test; The biotin of marker detection antibody SPA is the biotin of carboxyl activity, and adopts the biotinylated detection antibody of 2mg/mL Bio-SPA to detect as detecting antibody with dilution in 1: 2500.
The present invention also provides a kind of method that adopts dengue antibody in the above-mentioned protein suspension chip detection by quantitative serum sample, this method comprises the following steps: positive detection sample that (1) adds or standard items through 4 times of gradient doubling dilutions, and (2) are detected series of diluted samples and read corresponding fluorescent value (MFI) in suspension chip system; (3) the dose-response typical curve of the corresponding MFI value of making sample concentration, (4) with analysis software match dose-response curve and equation, (5) 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.It finishes detection in following suspension chip system, the microballoon of label probe and determinand react in 96 orifice plates, after the reaction, utilizes machine automatically reactant liquor to be picked up and by a microcapillary sense channel, only allows a microballoon to pass through sense channel at every turn.Being provided with twice laser in the sense channel, is red together, excites the color in the microballoon matrix, and the sorting code number of identification microballoon is to determine test item; Be green together, excite the color of reporter molecules, the tracer signal power is to detect the content of determinand.When the probe of sample to be tested and specific microballoon was attached together, the light that twice laser is excited all can be detected.And, then only have the exciting light in the microballoon to be detected if do not contain this subject matter in the sample.Microballoon kind and the quantity that is excited by machine and computing machine automatic statistical analysis twice laser again, thereby several test target things are arranged therein in the judgement sample to be tested, learn to have or not cause of disease to be measured to exist in the test sample book, or have several simultaneously to tens of kinds of cause of diseases.The suspending chip technology is owing to utilize microballoon to react in solution, overcome sheet film chip and when big Molecular Detection, be subjected to the influence to reaction kinetics such as surface tension, steric effect, utilize laser measuring technology simultaneously, improve the accuracy and the repeatability of sample detection greatly, had characteristics such as easy and simple to handle, the good reproducibility that is better than sheet film chip.
The inventor has done substantial improvement and innovation through a large amount of and deep research to the protein suspension chip preparation and the testing conditions thereof of dengue antibody, and it has following advantage:
1, the improvement of antigen coated amount
Whether the package amount of dengue fever E2 albumen is suitable 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, dengue fever E2 proteantigen package amount of the present invention is 0.1～100 μ g/1.25 * 10
6Individual microballoon, i.e. 0.02～400ng μ g/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 dengue antibody of the present invention and the sensitivity of suspending chip are 18.3ng/mL; And the suspension chip method dynamic detection range is 4.14～265ng/mL.
4, the specificity of method
The present invention is an envelope antigen by the E2 albumen of selecting dengue fever for use, is antibody to be measured with mouse-anti dengue fever IgG, serves as to detect thing with biotinylation-SPA.This development test proves, this method has good specificity under the interference antibody existence conditions such as rabbit antitularense serum, rabbit anti-avian influenza H5 serum, the anti-Xi Niluo antibody of rabbit existing.
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 dengue antibody in detecting human serum.
Description of drawings:
Fig. 1 is that No. 031 microballoon bag is by dengue fever E2 Protein Detection dengue antibody synoptic diagram;
Fig. 2 detects mouse-anti dengue fever IgG dose-response typical curve for the protein suspension chip method.
Embodiment
Protein suspension chip preparation method, detection and the quantivative approach of the detection dengue antibody that the present invention relates to are described further by following embodiment, but the present invention is subjected to the qualification of this embodiment never in any form.
One, material
1. the relevant damping fluid of protein suspension chip:
(1) PBS damping fluid (pH7.4): NaCl 137mmol/L; KCl 2.7mmol/L; Na
2HPO
410mmol/L; KH
2PO
42mmol/L.With 800mL dissolved in distilled water 8gNaCl, 0.2gKCl, 1.44g Na
2HPO
4With 0.24g KH
2PO
4PH value to 7.4 with the HCl regulator solution adds water to 1L.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 NaOH 1.5mL, constant volume are in 250mL, and pH 6.2.
(4) the microballoon bag is cushioned liquid 0.05M MES, pH 5.0:2.44g MES, and 5N NaOH0.15mL, constant volume is in 250mL.
(5) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, and 0.02%TWEEN, 0.05% azide, pH 7.4.
(6) microballoon confining liquid PBS-BN:PBS, 1%BSA, 0.05% azide, pH7.4.
(7) detect damping fluid: PBS, 1%BSA, pH7.4.
(8) antibody diluent PBS, pH7.4.。
(9) microballoon dilution: PBS, 1%BSA, pH7.4.
(10) sample diluting liquid PVX:PBS, 0.5%PVA, 0.8%PVP;
(11) biotinylation detect thing dilution: PBS-TBN (PBS, 0.1%BSA, 0.02%TWEEN-20,0.05%NaN3, pH7.4).
(12) SA-PE dilution: PBS (pH7.4), 1%BSA.
2. antigen and antibody
Antigen dengue fever E2 albumen used herein, it can be available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C.
Antibody to be measured used herein is mouse-anti dengue fever E2IgG, it can be available from Microbiology and Epidemic Disease Inst., Academy of Military-Medical Sciences (C, detecting antibody is biotinylation sheep anti-mouse igg and biotinylation SPA, sheep anti-mouse igg and SPA, and 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 mouse-anti dengue fever 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 antitularense serum, rabbit anti-avian influenza H5 serum, the anti-Xi Niluo antibody 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 dengue fever E2IgG is 1.06 μ g/mL.
Is variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with mouse-anti dengue fever 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 suspension chip method as sample to be checked.
The preparation of the protein suspension chip of embodiment 1, detection dengue antibody
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 dengue fever E2 proteantigen that mark can be caught dengue antibody, promptly utilizes dengue fever E2 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 dengue fever E2 proteantigen 1～50 μ g joins activation, be settled to 500 μ L with the PBS damping fluid, room temperature jolts 2 hours.14000 * g is centrifugal, careful sucking-off and abandoning supernatant.PBS damping fluid with 500 μ L is washed once, and 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.Add the sealing damping fluid suspended coding microballoon of 250 μ L, jolt 30 minutes in room temperature, 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.The microballoon that adds 500 μ L is preserved liquid washing coding microball, and 16000 * g is centrifugal, careful sucking-off and abandoning supernatant.Preserve liquid suspended coding microballoon with the microballoon of 150 μ L at last, keep in Dark Place standby in 4 ℃.
C, bag are by the counting of microballoon
Draw an amount of microballoon, after the dilution, with blood counting chamber (0.10mm; 1/400mm
2) under simple microscope, count.According to formula (each big lattice number * 10
4* extension rate * volume (mL)) calculates microballoon quantity.
2, detect the 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, 35 μ g, 40 μ g, 45 μ g, 50 μ g is encoded to No. 031 microballoon by 100 μ L respectively.Effect compares after testing, with 1～50 μ g/1.25 * 10
6Promptly 0.2～200ng/2500～5000 microballoon/bag is best by effect for individual coding microball, 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 dengue fever E2 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 dengue antibody.
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 dengue antibody in the mouse serum with dilution in 1: 1000 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 dengue 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 the biotinylated SPA of 2mg/mL 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 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 dengue 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 dengue antibody 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 dengue fever health check-up survey feminine gender as a result.
The using suspending chip detection method detects mouse-anti dengue fever E2IgG, rabbit approach IgG, the anti-SARS IgG of rabbit, the anti-fowl dengue fever of rabbit 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-dengue E2IgG 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 dengue antibody that the present invention sets up is described.
1, mouse-anti dengue fever E2IgG typical curve specimen preparation
With sample diluting liquid mouse-anti dengue fever E2IgG standard items are become series concentration sample with 4 times of multiple proportions gradient dilutions to 0.004ng/mL by 1.06 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 dengue fever E2 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, S9:265, S10:1060.Suspension chip system according to testing result match mouse-anti dengue fever E2IgG dose-response typical curve equation is:
FI＝-2.98016+(8993.39+2.98016)/[(1+(Conc/13386.5)
-1.32899]
1.84993
3, suspending chip method of the present invention detects sensitivity and the dynamic range of mouse-anti dengue fever E2IgG 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 dengue fever E2IgG is: 18.3ng/mL (Cutoff=65, MFI (blank)=55.75, standard deviation=3.0606).
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 dengue fever E2 antibody concentration, its corresponding MFI value also increases progressively thereupon, when mouse-anti dengue fever E2 antibody concentration surpasses 265ng/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 265ng/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 dengue fever E2IgG is 4.14～265ng/mL.
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 dengue 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 792, if the MFI value that detection obtains is greater than 792, it is the dengue antibody excessive concentration in the serum, can be considered the dengue antibody positive may be by dengue virus infection, if the MFI value is less than 792, promptly the low normal value of the dengue antibody concentration in the serum can be judged as the dengue antibody feminine gender, does not infect the recessive carrier of dengue fever virus or dengue fever virus.
Claims (10)
1. protein suspension chip that detects dengue antibody in the serum sample, it is characterized in that, this chip comprises: coding microball, is dengue fever E2 proteantigen as bag by microballoon antigen, biotin labeled sheep anti-mouse igg and/or SPA are as detecting antibody, and streptavidin-phycoerythrin is as signal detection and relevant buffer solution.
2. preparation method who detects the protein suspension chip of dengue antibody, it is characterized in that, this preparation method is specially: in relevant buffer solution, coding microball is that sheep anti-mouse igg and/or SPA, adding streptavidin-phycoerythrin are as signal detection with dengue fever E2 proteantigen bag quilt, with biotin labeled detection antibody.
3. the preparation method of the protein suspension chip of detection dengue antibody as claimed in claim 2 is characterized in that, described coding microball is preferably coding microball No. 031.
4. an indirect immunological detection method that adopts above-mentioned protein suspension chip to detect dengue antibody in the serum sample is characterized in that this method comprises the following steps:
1) dengue fever E2 proteantigen is wrapped the microballoon that is encoded as capture antigen;
2) adding contains the working solution that wraps the antigen encoding microballoon that is hunted down in detecting carrier, cleans with cleaning fluid;
3) add the test serum sample, hatch the back and clean;
4) add with biotin labeled detection antibody, hatch the back and clean;
5) add streptavidin-phycoerythrin, hatch the back and clean;
6) adding mixes after detecting damping fluid;
7) read average fluorescent strength numerical value and analyze the data judging testing result with suspension chip system.
5. detection method as claimed in claim 4 is characterized in that, adopts biotin labeled SPA as detecting antibody, and itself and the combination composition indirect method detection architecture of dengue fever E2 albumen.
6. detection method as claimed in claim 4 is characterized in that, bag is 0.1～100 μ g/1.25 * 10 by the dengue fever E2 proteantigen consumption of described coding microball in the described step 1)
6Individual coding microball or 0.02～400ng/2500～5000 microballoon/test.
7. a method that adopts dengue antibody in the protein suspension chip detection by quantitative 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;
2) series of diluted samples is detected in suspension chip system read corresponding fluorescent value;
3) dose-response curve of the corresponding fluorescent value of making sample concentration;
4) with analysis software match dose-response curve and equation;
5) judge dynamic detection range according to dose-response curve, according to the concentration of dengue antibody in the dose-response equation judgement sample.
8. method as claimed in claim 7 is characterized in that, the gradient doubling dilution is 4 times in the described step 1).
9. method as claimed in claim 7 is characterized in that, the positive detection sample that adds in the described step 1) is mouse-anti dengue fever E2IgG.
10. method as claimed in claim 7 is characterized in that, described step 2) in adopt the biotinylated detection antibody of 2mg/mL Bio-sheep anti-mouse antibody to detect as detecting antibody with dilution in 1: 1000.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102393454A (en) * | 2011-08-24 | 2012-03-28 | 中国检验检疫科学研究院 | Colloidal gold test strip for detecting dengue virus antibody and preparation method and application thereof |
| CN109444414A (en) * | 2018-09-10 | 2019-03-08 | 珠海国际旅行卫生保健中心 | Using the method and corresponding composition, kit of AlphaLISA detection dengue virus |
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