CN203133084U - Protein suspension array system for detecting tick-borne encephalitis antibody in serum sample - Google Patents
Protein suspension array system for detecting tick-borne encephalitis antibody in serum sample Download PDFInfo
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- CN203133084U CN203133084U CN 201020269920 CN201020269920U CN203133084U CN 203133084 U CN203133084 U CN 203133084U CN 201020269920 CN201020269920 CN 201020269920 CN 201020269920 U CN201020269920 U CN 201020269920U CN 203133084 U CN203133084 U CN 203133084U
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Abstract
The utility model discloses a protein suspension array system for detecting a tick-borne encephalitis antibody in a serum sample. The system comprises an array matrix, a liquid adding system, a suspension array detector and a computer, wherein a related buffer solution and an antibody to be detected are filled in the array matrix; encoding microspheres are arranged in the related buffer solution and are enveloped by captured antigens; the liquid adding system adds a biotin labelled second antibody and/or SPA and a fluorescence signal detecting object to the array matrix in sequence; the suspension array detector comprises a microcapillary detection channel and detection laser; the encoding microspheres pass through the microcapillary detection channel in sequence; the detection laser excites and recognizes colors of the encoding microspheres and colors of objects to be detected on the encoding microspheres; and the computer is connected with the suspension array detector and records and analyzes the measuring results of the suspension array detector. The suspension array technology utilizes the microspheres to react in the solution, and by utilizing the laser detection technology, the system improves the accuracy and repeatability of sample detection and has better characteristics of simplicity and convenience in operation, good repeatability and the like than membrane arrays.
Description
Technical field
The utility model relates to a kind of protein suspension chip system that detects tick encephalitis antibody in the serum sample.
Background technology
Tick encephalitis (TBE), it is common viral central nervous system (CNS) disease, hundreds of routine inpatients are arranged every year, virulence factor is tick-brone encephalitis virus (TBEV), a kind of by tick-borne flavivirus, be feature with happen suddenly high heat, meningeal irritation sign, the disturbance of consciousness and paralysis etc. clinically, cerebrospinal fluid has ANOMALOUS VARIATIONS, and sequelae is often arranged.
Utilizing antigen and antibody specific reaction to detect antigen or antibody in the immunological method enzyme linked immunological experiment (ELISA) mainly contains by double antigens sandwich and surveys antibody, double-antibody sandwich survey antigen, competition law, indirect method etc.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, add the anti-antibody with immune complex in of enzyme labeling two after the washing again and be combined formation enzyme labeling two anti-antibodies-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, utilize the microsphere of band coding 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 method evaluation and the optimization of laboratory detection method at present, to shorten detection time, the detection cost of reduction method.But whether suspension chip method can detect the tick encephalitis antibody in the human serum, and its quantitative detectability 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 tick encephalitis 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 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 sample diluting liquid 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 coding microball is preferably coding microball No. 027.
Further, described capture antigen is preferably tick encephalitis E-D3 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 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 for exciting and identifying testing molecule is with the green laser of the content that detects described antibody to be measured.
Through a large amount of tests and deep research, protein suspension chip preparation and the testing conditions thereof of tick encephalitis antibody have been done substantial improvement and innovation, it has following advantage:
1, the improvement of antigen coated amount
The package amount of tick encephalitis E-D3 proteantigen 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 wrap very lowly by the required antigen coated amount of suspending chip, antigen coated amount of the present utility model is 0.01～90 μ g/1.25 * 10
6Individual coding microball, i.e. 0.002～360ng/2500～5000 microballoon/test, and the package amount of traditional immunology ELISA method is 1 μ g/ test.
2, biotin labeled improvement
Usually, labelled antibody need use excessive biotin.Theoretically, in testing process, excessive biotin is fallen by suction filtration during cleaning because the unmarked antibody of going up is not connected by the antibody in conjunction with capture antibody on the microballoon, can not react with the SA-PE that adds subsequently, does not influence detection.But in the actual detected process, false positive appears in the phenomenon that the detection signal that fell in may be too high, so after advising biotin labeling antibody, removes unnecessary biotin as far as possible.IgG (molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ L of 10mM biotin solution.
3, the specificity of method
The utility model is envelope antigen by selecting tick encephalitis E-D3 albumen for use, is antibody to be measured with rabbit tick-borne encephalitis resisting antibody, serves as to detect antibody with biotinylated goat-anti rabbit.This development test proves, exists mouse-anti dengue fever IgG, rabbit anti-avian influenza H5 serum, the anti-soil of rabbit to draw FopA polyclonal antibody, rabbit anti-dengue NS3 antibody etc. to disturb under the condition of antibody existence, and this method has good specificity.
4, the detectability of sample
The utility model has been estimated the detectability of suspension chip method to human serum.By the detection to human serum, tentative confirmation the practicality of this method tick encephalitis antibody in detecting human serum.
Description of drawings:
Fig. 1 is the utility model structural representation;
Fig. 2 is that No. 027 microballoon bag is by tick encephalitis E-D3 Protein Detection tick encephalitis antibody synoptic diagram;
Fig. 3 detects rabbit tick-borne encephalitis resisting antibody dosage-reaction normal curve map for the protein suspension chip method.
Embodiment
As shown in Figure 1 to Figure 3, a kind of protein suspension chip system that detects tick encephalitis 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 tick encephalitis E-D3 albumen on the coding microball, be used for catching the antibody to be measured of blood serum sample to be detected, if tick encephalitis antibody is arranged in the sample, tick encephalitis E-D3 protein combination on tick encephalitis antibody and the coding microball, add again and have biotin labeled two anti-and/or SPA, forming coding microball-tick encephalitis E-D3 albumen-tick encephalitis antibody-two resists/the SPA-biotin composite, add streptavidin-phycoerythrin at last, streptavidin is combined with biotin, forming coding microball-tick encephalitis E-D3 albumen-tick encephalitis antibody-two resists/SPA-biotin-streptavidin-phycoerythrin compound, reach tick encephalitis detection of antibodies in the blood serum sample by the detection of suspending chip detector to the phycoerythrin fluorescence signal, if there is not tick encephalitis antibody in the blood serum sample, the coding microball that is coated with tick encephalitis E-D3 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, all with microballoon cleaning fluid washing microballoon, making did not have the material of combination to remove system in conjunction with afterwards.
The ultimate principle of suspending chip is to utilize the microballoon of polystyrene (polystyrene) made, coat 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 excites all can be detected.And if do not contain this subject matter in the sample, then only have the exciting light in the microballoon to be detected.Microballoon kind and the quantity that excites 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 to tens of kinds of cause of diseases simultaneously.The suspending chip technology is owing to utilize microballoon to react in solution, having overcome sheet film chip is subjected to surface tension, steric effect etc. to the influence of reaction kinetics when big Molecular Detection, utilize laser measuring technology simultaneously, improve 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 tick encephalitis antibody in the serum sample of the utility model be described further by following embodiment, but the utility model is subjected to the restriction of this embodiment never in any form.
One, material
1. the relevant damping fluid of protein suspension chip:
(1) PBS damping fluid (pH7.4): NaCl 137mmol/L; KCl 2.7mmol/L; Na
2HPO
410mmol/L; KH
2PO
42mmol/L.With 800mL dissolved in distilled water 8gNaCl, 0.2gKCl, 1.44g Na
2HPO
4With 0.24g KH
2PO
4PH value to 7.4 with the HCl regulator solution adds water to 1L.After the packing at 15psi (1.05kg/cm
2) high pressure steam 20 minutes, or filtration sterilization, be stored in room temperature.
(2) microballoon cleaning fluid: PBS (pH7.4), 0.05%TWEEN-20.
(3) microballoon activation damping fluid 100mM NaH
2PO
4: 3g NaH
2PO
4, 5N NaOH 1.5mL, constant volume are in 250mL, and pH 6.2.
(4) the microballoon bag is cushioned liquid 0.05M MES, pH 5.0:2.44g MES, and 5NNaOH0.15mL, 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 employed antigen of the utility model is tick encephalitis E-D3 albumen, can be available from China Inst. of Quarantine Inspection Sciences.
The utility model uses detection antibody to be rabbit tick-borne encephalitis resisting IgG, can be available from China Inst. of Quarantine Inspection Sciences, detect two and anti-ly be biotinylation goat anti-rabbit igg and biotinylation SPA, described goat anti-rabbit igg can be available from Beijing ancient cooking vessel state biotech company, SPA can be available from Sigma company.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is rabbit tick-borne encephalitis resisting 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 mouse-anti dengue fever IgG, rabbit anti-avian influenza H5 serum, rabbit anti-dengue NS3 antibody, BSA, casein, tryptone etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid-4 ℃ of preservations.The storing solution titre of rabbit tick-borne encephalitis resisting IgG is 0.01.In the comparative experiments, same sample is used for the detection of ELISA and suspending chip.
Be variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with rabbit tick-borne encephalitis resisting 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 sample dilution dilution in 1: 10, after for example human serum sample 5 μ L add sample dilution 50 μ L mixings, is carried out the detection of suspension chip method as sample to be checked.
The preparation of the protein suspending chip of embodiment 1, detection tick encephalitis antibody
1, the capture antigen bag microballoon that is encoded
No. 027 coding microball that the utility model adopts is available from U.S. Bio-Rad company, and coding microball is used for the tick encephalitis E-D3 proteantigen that mark can be caught tick encephalitis antibody, namely utilizes tick encephalitis E-D3 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 capture antigen and be in the coding microball after tick encephalitis E-D3 proteantigen 1～50 μ g joins activation, be settled to 500 μ L with the PBS damping fluid, room temperature jolts 2 hours.14000 * g is centrifugal, careful sucking-off and abandoning supernatant.PBS damping fluid with 500 μ L is washed once, and 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.Add the sealing damping fluid suspended coding microballoon of 250 μ L, jolt 30 minutes in room temperature, 14000 * g is centrifugal, careful sucking-off and abandoning supernatant.The microballoon that adds 500 μ L is preserved liquid washing coding microball, and 16000 * g is centrifugal, careful sucking-off and abandoning supernatant.Preserve liquid suspended coding microballoon with the microballoon of 150 μ L at last, keep in Dark Place standby in 4 ℃.
C, bag are by the counting of microballoon
Draw an amount of microballoon, after the dilution, with blood counting chamber (0.10mm; 1/400mm
2) under simple microscope, count.According to formula (each big lattice number * 10
4* extension rate * volume (mL)) calculates microballoon quantity.
2, detect the substance markers biotin
The mark of A, biotin
Preparing concentration respectively is the antibody-solutions to be marked of 10mM biotin solution and 2mg/mL, and the biotin that has calculated volume is joined in the thing solution to be marked, jolts 30 minutes in room temperature (or 2 hours) on ice, packing after the post desalination excessively, and-20 ℃ are frozen standby.
The consumption of B, antibody
IgG (molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ l of 10mM biotin solution.
The optimization of embodiment 2, suspending chip preparation method condition
1, the selection of microballoon envelope antigen package amount
Amount bag with 1 μ g, 2.5 μ g, 5 μ g, 7.5 μ g, 10 μ g, 12.5 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g is encoded to No. 027 microballoon by 100 μ L respectively.Effect compares after testing, with 1～30 μ g/1.25 * 10
6Namely 0.2～120ng/2500～5000 microballoon/test pack 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. 027 microballoon of tick encephalitis E proteantigen, and obtain high s/n ratio result (the MFI value is much larger than 2000), illustrate that the suspending chip detection system of optimizing can successfully be used for the tick encephalitis detection of antibodies.
2, biotinylation detects the optimization of thing
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 quality testing survey 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 antibody goat-anti of 2mg/mL rabbit detects tick encephalitis antibody in the rabbit anteserum with dilution in 1: 500 as detecting thing, testing result shows that biotinylation detects the detection effect that thing Bio-goat anti-rabbit antibody can obtain the Supreme People's Procuratorate's measured value and low background; As detecting tick encephalitis detection of antibodies thing in the human serum, testing result showed that biotinylation detects the detection effect that thing Biotin-SPA can obtain the Supreme People's Procuratorate's measured value and low background to the biotinylated detection thing of 2mg/mL Biotin-SPA with dilution in 1: 2500.
1, testing sample preparation
With sample diluting liquid sample to be tested is 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 at 96 hole filter plates, and testing process is as follows:
1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washs and use the vacuum pump suction filtration with cleaning fluid;
2) add 50 μ L test sample, the room temperature lucifuge jolts 30 minutes behind the mixing, with cleaning fluid washing and suction filtration;
3) biotinylation with after the antibody diluent dilution that adds 50 μ L debita spissitudos detects thing, and the room temperature lucifuge jolts 30 minutes behind the mixing, washing lotion washing and vacuum pump suction filtration;
4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10 minutes behind the mixing.Washing lotion washing and vacuum pump suction filtration;
5) the detection damping fluid of adding 125 μ L is through the resuspended mixing of spiral;
6) read MFI numerical value and analyze data with suspension chip system.
3, testing result is judged
According to experimental study result and correlative study report, the minimum detectability (LOD value) that the utility model definition protein suspension chip method detects tick encephalitis 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 namely the Cutoff value is=MFI (blank)+3 times standard deviation.Corresponding fluorescence intensity level then is judged to be the tick encephalitis antibody test positive as a result if testing result is higher than LOD; Corresponding fluorescence intensity level then is judged to be tick encephalitis antibody test feminine gender as a result if testing result is lower than LOD.
The using suspending chip detection method draws FopA polyclonal antibody, rabbit anti-dengue NS3 antibody, BSA, casein, tryptone etc. to detect to rabbit tick-borne encephalitis resisting IgG, mouse-anti dengue fever IgG, rabbit anti-avian influenza H5 serum, the anti-soil of rabbit respectively, according to testing result criterion among the embodiment 3, only rabbit tick-borne encephalitis resisting 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 tick encephalitis antibody that the utility model is set up is described.
The foundation of embodiment 5, the quantitative detection model of suspending chip
1, rabbit tick-borne encephalitis resisting IgG typical curve specimen preparation
Be 0.01 with 4 times multiple proportions gradient dilutions to titre to be 5 * 10 with rabbit tick-borne encephalitis resisting IgG standard items by titre with sample diluting liquid
-7Become the series concentration sample.
2, the drafting of dose-response typical curve
Detect above-mentioned series concentration sample according to the detection method among the embodiment 3, and draw dose-response typical curve (as shown in Figure 2) according to the suspension chip system testing result.Wherein, X-axis represents titre, and Y-axis represents the fluorescent value (MFI) that the suspending chip instrument detects.Each data represents 3 times testing result mean value, and coordinate axis is set with logarithm-logarithmic relationship, and the titre of rabbit tick-borne encephalitis resisting serum is respectively among Fig. 2: S1:8 * 10
-6, S2:3.2 * 10
-5, S3:1.28 * 10
-4, S4:5.12 * 10
-4, S5:2.048 * 10
-3, S6:8.19 * 10
-3, S7:2.5 * 10
-3, S8:0.01.
Adopt indirect method to survey the immunology detection pattern of antibody, total overall reaction is all carried out at 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.
Suspension chip system is according to testing result match rabbit tick-borne encephalitis resisting IgG dose-response typical curve equation:
FI＝-2.07898+(13723.6+2.07898)/[1+(Conc/0.141595)
-1.37795]
0.715313
3, suspending chip detects sensitivity and the dynamic range of rabbit tick-borne encephalitis resisting IgG
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 tick-borne encephalitis resisting IgG is: 0.000009 (Cutoff=24.1818, MFI (blank)=21, standard deviation=1.0606).
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 tick-borne encephalitis resisting IgG concentration, its corresponding MFI value also increases progressively thereupon, when rabbit tick-borne encephalitis resisting IgG titre degree surpasses 0.01, the immune response of the antigen-antibody state that reaches capacity, the MFI value begins to enter plateau, substrate concentration to be checked in the interpret sample is too high, need will detect after the sample dilution again.
Therefore, can judge that according to minimum detectability and Supreme Procuratorate's rising limit the utility model is that the dynamic range that suspending chip quantitatively detects rabbit tick-borne encephalitis resisting IgG is 0.000008～0.01.
1, human serum sample's preparation
Human serum is used for protein suspension chip with sample diluting liquid dilution (human serum sample 5 μ L add sample dilution 50 μ L mixings) in 1: 10 back to be detected.
2, human serum sample's detection
1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washs and use the vacuum pump suction filtration with cleaning fluid;
2) add 50 μ L human serum sample to be detected, the room temperature lucifuge jolts 30 minutes behind the mixing, with cleaning fluid washing and suction filtration;
3) add 50 μ L debita spissitudos with the biotinylation SPA after the antibody diluent dilution, the room temperature lucifuge jolts 30 minutes behind the mixing, washing lotion washing and vacuum pump suction filtration;
4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10 minutes behind the mixing.Washing lotion washing and vacuum pump suction filtration;
5) the detection damping fluid of adding 125 μ L is through the resuspended mixing of spiral;
6) read MFI numerical value with suspension chip system, according to typical curve, determine the concentration of tick encephalitis antibody in the human serum to be detected.
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 the detection to normal human serum, 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, it is the Cutoff value, if the MFI value that detection obtains is greater than the Cutoff value, it is the tick encephalitis excessive concentration in the serum, can be considered the tick encephalitis antibody positive may be infected by tick-brone encephalitis virus, if the MFI value is less than the Cutoff value, namely the low normal value of the tick encephalitis antibody concentration in the serum can be judged as the tick encephalitis negative antibody, does not infect tick-brone encephalitis virus or recessive carrier.
Claims (8)
1. protein suspension chip system that detects tick encephalitis antibody in the serum, it is characterized in that, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, chip basal body be used for carrying antibody to be measured and in relevant buffer solution is housed, be provided with coding microball in the relevant damping fluid, coding microball is No. 027 coding microball, 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 sample diluting liquid 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 tick encephalitis E-D3 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 Biotin-goat-anti rabbit and/or Biotin-SPA.
6. protein suspension chip as claimed in claim 2 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 for exciting and identifying testing molecule is with the green laser of the content that detects described antibody to be measured.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2562841C2 (en) * | 2013-12-30 | 2015-09-10 | ЗАО "МБС-Технология" | Method of preparing tick tissues for biological analysis |
| CN105785025A (en) * | 2014-12-18 | 2016-07-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Kit for detecting tick-borne encephalitis virus infected serum |
-
2010
- 2010-07-23 CN CN 201020269920 patent/CN203133084U/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2562841C2 (en) * | 2013-12-30 | 2015-09-10 | ЗАО "МБС-Технология" | Method of preparing tick tissues for biological analysis |
| CN105785025A (en) * | 2014-12-18 | 2016-07-20 | 中国人民解放军军事医学科学院微生物流行病研究所 | Kit for detecting tick-borne encephalitis virus infected serum |
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