CN202024998U - Protein suspension array system for detecting yellow fever antibodies - Google Patents
Protein suspension array system for detecting yellow fever antibodies Download PDFInfo
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- CN202024998U CN202024998U CN2010206842439U CN201020684243U CN202024998U CN 202024998 U CN202024998 U CN 202024998U CN 2010206842439 U CN2010206842439 U CN 2010206842439U CN 201020684243 U CN201020684243 U CN 201020684243U CN 202024998 U CN202024998 U CN 202024998U
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Abstract
The utility model discloses a protein suspension array system for detecting yellow fever antibodies, which comprises an array basal body, a liquid injection system, a suspension array detecting instrument and a computer, wherein related buffer solution and to-be-detected antibodies are arranged inside the array basal body; coding microspheres are arranged in the related buffer solution; coding microspheres are coated with capturing antigen; the liquid injection system sequentially adds second biotin-labeled detection antibodies and fluorescent signal testing samples to the array basal body; the suspension array detecting instrument comprises a microcapillary detection passage and detection lasers; the coding microspheres pass through the microcapillary detection passage in sequence; the detection lasers excite and recognize the colors of the coding microspheres and the colors of to-be-detected objects on the coding microspheres; in addition, the computer is connected with the suspension array detecting instrument, and records and analyzes the measurement results of the suspension array detecting instrument. A suspension array technology improves the accuracy and the repeatability of sample detection by utilizing the reaction of the microspheres in the solution and by utilizing the laser detection technique, is characterized by good repeatability and the like superior to a membrane chip, and is simple and convenient to operate.
Description
Technical field
The utility model relates to a kind of protein suspension chip system that detects yellow fever virus antibody.
Background technology
Yellow fever virus belongs to arboviruse B group, is RNA viruses, spherical in shape, about 20～60 nanometers.This virus is preserved down the vigor that still had in 10 years at subzero 70 ℃, and deactivation easily at room temperature lost activity through 48 hours under 0 ℃.Mosquito always obtains virus from various monkeys, bites by infected female mosquito (known have kind more than 20) then and propagates that to give people, people be the terminal host of virus, forms monkey-mosquito-people's chain type propagation, also may have people-mosquito-people's circulation.The acute infectious disease that yellow fever virus causes, the jaundice heat that occurs together often appears in patient, so name.Cardinal symptom has heating, headache, jaundice and hemorrhage etc.; The major lesions of yellow fever is at internal organ such as liver, kidney, the heart, stomach, intestines.Because liver function suffers damage, the synthetic minimizing of clotting factor, can cause upper gastrointestinal bleeding, oozing of blood and subcutaneous hemorrhage, also disseminated intravascular coagulation can take place, patients undergo symptom occurs after latent period on the 3rd～6, the similar flu of extremely light-duty patient's symptom has only 1～2 day heating, headache; Light-duty case is acute onset, tangible heating, headache are arranged, feels sick, nosebleed epistaxis, slight jaundice and albuminuria, recovery from illness after several days; The normal sudden onset of heavy case, the high heat headache of performance shiver with cold, backache, pantalgia, feel sick, vomiting, flushing, the hyperemia of eye binding film, leucocyte reduces in the peripheral blood, after continuing a few days, sx, and then heating occurs and begin that jaundice is arranged, companion's hemorrhagic tendency shows as the soft palate blutpunkte, nosebleed epistaxis, bleeding gums, albuminuria even anuria appear in the black watery blood of vomitting sometimes, relative infrequent pulse appears in 50% patient, Consciousness, liver function damage, the red matter of blood courage increases, prolonged prothrombin, transaminase raises, and hepatopathy hypoglycemia can occur when heavy, and shock appearred in 10～60% patients after 6～8 days, stupor so that dead.
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, add again after the washing enzyme labeling two anti-with immune complex in antibodies form 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, 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 the protein suspension chip method can detect the yellow warm antibody in the human serum, and its detection by quantitative ability how, still lacks model and evaluation.
The utility model content
The purpose of this utility model is to provide a kind of protein suspension chip system that detects yellow warm antibody, 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 No. 027 coding microball that wraps the antigen that is hunted down in the relevant damping fluid, adding liquid systems is communicated with chip basal body, anti-in chip basal body, to add biotin labeled two, fluorescence signal detects thing, the suspending chip detector comprises microcapillary sense channel and detection laser, the microcapillary sense channel is communicated with chip basal body, solution behind this microcapillary sense channel sucting reaction, 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 comprises that used microballoon dilution, the dilution described biotin labeled two of sample diluting liquid, the described coding microball of dilution that is used for described antibody dilution to be measured resists used antibody diluents, each reaction to wash the used microballoon cleaning fluid of coding microball, SA-PE dilution afterwards, detect damping fluid.
Further, described capture antigen is preferably yellow hot E3 albumen.
Further, described antibody to be measured is blood serum sample.
Further, described biotin labeled two anti-be preferably Biotin-goat-anti rabbit and/or Biotin-goat anti-human igg.
Further, described fluorescence signal detection thing is streptavidin-phycoerythrin.
Further, described chip basal body is 96 hole filter plate or microcentrifugal tubes.
Further, described detection laser comprises with exciting color in the described coding microball matrix, the sorting code number of recognition coding microballoon to determine the red laser of test item, and the color signal, tracer signal power that is used to excite and discern testing molecule is with the green laser of the content that detects described antibody to be measured.
Through a large amount of tests and deep research, the protein suspension chip preparation and the testing conditions thereof of yellow warm antibody have been done substantial improvement and innovation, it has following advantage:
1, the improvement of antigen coated amount
The package amount of yellow hot E3 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 4 μ g/ tests.
2, biotin labeled improvement
Usually, labelled antibody need use excessive biotin.Theoretically, in testing process, excessive biotin is fallen by suction filtration during cleaning because of the unmarked antibody of going up is not connected by the antibody in conjunction with capture antibody on the microballoon, can not react with the SA-PE that adds subsequently, does not influence detection.But in the actual detected process, false positive appears in the phenomenon that the detection signal that fell in may be too high, so after advising biotin labeling antibody, removes unnecessary biotin as far as possible.IgG (molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ L of 10mM biotin solution.
3, sensitivity of Jian Ceing and dynamic range
The protein suspension chip quantitative detecting method of the hot Richettsia antibody of blood serum sample Q of the present utility model and the sensitivity of suspending chip are titre 0.000007; And the suspension chip method dynamic detection range is 1: 10240～1: 40.
4, the specificity of method
The utility model is an envelope antigen by selecting yellow hot E3 albumen for use, is antibody to be measured with the anti-yellow warm antibody of rabbit, serves as to detect antibody with biotinylated goat-anti rabbit.This development test proves, exist the anti-St. Louis encephalitis NS1 serum of rabbit, mouse-anti west Buddhist nun sieve E serum, the hot Richettsia antibody of the anti-Q of rabbit, the anti-Rickettsia prowazeki of rabbit, the anti-horse beautiful eyes of rabbit antibody, the anti-Tula antibody of rabbit, rabbit anti-dengue serum, rabbit anteserum, rat blood serum, mice serum, BSA, casein, tryptone etc. to disturb under antibody or the albumen existence condition, this method has good specificity.
5, the detectability of sample
The utility model has been estimated the detectability of suspension chip method to human serum.By detection to human serum, tentative confirmation the practicality of this method yellow warm antibody in detecting human serum.
Description of drawings
Fig. 1 is the utility model structural representation;
Fig. 2 is that No. 027 microballoon that is coated with yellow hot E3 albumen detects yellow warm antibody synoptic diagram 1;
Fig. 3 is that No. 027 microballoon that is coated with yellow hot E3 albumen detects yellow warm antibody synoptic diagram 2;
Fig. 4 detects the anti-yellow warm antibody dose-response canonical plotting of rabbit for the protein suspension chip method.
Embodiment
As shown in Figures 1 to 4, a kind of protein suspension chip system that detects yellow warm antibody 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 coding microball No. 027 in the relevant damping fluid, be coated with yellow hot E3 albumen on the coding microball, be used for catching the antibody to be measured of blood serum sample to be detected, if yellow warm antibody is arranged in the sample, yellow hot E3 protein combination on yellow warm antibody and the coding microball, add again have biotin labeled two anti-, forming coding microball-yellow hot E3 albumen-yellow warm antibody-two resists-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-yellow hot E3 albumen-yellow warm antibody-two resists-biotin-streptavidin-phycoerythrin compound, by the suspending chip detector detection of phycoerythrin fluorescence signal is reached detection to yellow warm antibody in the blood serum sample, if there is not yellow warm antibody in the blood serum sample, the coding microball that is coated with yellow hot E3 albumen not can with add later biotin labeled two anti-, 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 resist 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 the ruddiness and the infrared light colour former of different proportion, and produce 100 kinds of different proportion colors, color numbers as 100 kinds of uniquenesses, every about 5.6 μ m of microballoon size, different research purposes such as immunoassay, nucleic acids research, enzyme analysis, acceptor and part discriminance analysis etc. be can comply with, and specific antibodies, nucleic acid probe and various acceptor probe demarcated according to different research purposes.The microballoon of label probe and determinand react in 96 orifice plates.After the reaction, utilize machine automatically reactant liquor to be picked up and by a microcapillary sense channel, only allow a microballoon to pass through sense channel at every turn.Being provided with twice laser in the sense channel, is red together, excites the color in the microballoon matrix, and the sorting code number of identification microballoon is to determine test item; Be green together, excite the color of reporter molecules, the tracer signal power is to detect the content of determinand.When the probe of sample to be tested and specific microballoon was attached together, the light that twice laser is excited all can be detected.And, then only have the exciting light in the microballoon to be detected if do not contain this subject matter in the sample.Microballoon kind and the quantity that is excited by machine and computing machine automatic statistical analysis twice laser again, thereby several test target things are arranged therein in the judgement sample to be tested, learn to have or not cause of disease to be measured to exist in the test sample book, or have several simultaneously to tens of kinds of cause of diseases.The suspending chip technology is owing to utilize microballoon to react in solution, overcome sheet film chip and when big Molecular Detection, be subjected to the influence to reaction kinetics such as surface tension, steric effect, utilize laser measuring technology simultaneously, improve the accuracy and the repeatability of sample detection greatly, had characteristics such as easy and simple to handle, the good reproducibility that is better than sheet film chip.
A kind of protein suspension chip system that detects yellow warm antibody of the utility model be described further by following embodiment, but the utility model is subjected to the qualification of this embodiment never in any form.
One, material
1. the relevant damping fluid of protein suspension chip:
(1) PBS damping fluid (pH7.4): NaCl 137mmol/L; KCl 2.7mmol/L; Na
2HPO
410mmol/L; KH
2PO
42mmol/L.With 800mL dissolved in distilled water 8gNaCl, 0.2gKCl, 1.44gNa
2HPO
4With 0.24g KH
2PO
4PH value to 7.4 with the HCl regulator solution adds water to 1L.121 ℃ of autoclavings 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 be in 250mL, pH6.2.
(4) the microballoon bag is cushioned liquid 0.05M MES, pH5.0:2.44g MES, and 5N NaOH 0.15mL, constant volume is in 250mL.
(5) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, 0.02%Tween-20,0.05% 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) SA-PE dilution: PBS (pH7.4), 1%BSA.
2. antigen and antibody
The employed antigen of the utility model is yellow hot E3 albumen, can be available from China Inst. of Quarantine Inspection Sciences.
The utility model uses and detects antibody is that rabbit resists yellow hot E3 IgG, can available from the modern times up to company, detecting antibody is biotinylation goat anti-rabbit igg and biotinylation goat anti-human igg, and described goat anti-rabbit igg and goat anti-human igg can be available from Beijing ancient cooking vessel state biotech companies.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is the anti-yellow hot IgG of rabbit, disturbed specimen or the sample of testing as the method specificity are other antibody or other protein of target detection beyond the region of objective existence, comprise the anti-St. Louis encephalitis NS1 serum of rabbit, mouse-anti west Buddhist nun sieve E serum, the hot Richettsia antibody of the anti-Q of rabbit, the anti-Rickettsia prowazeki of rabbit, the anti-horse beautiful eyes of rabbit antibody, the anti-Tula antibody of rabbit, rabbit anti-dengue serum, rabbit anteserum, rat blood serum, mice serum, BSA, casein, tryptose etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid-4 ℃ of preservations.
Anti-yellow hot IgG is the variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with rabbit to be analyzed, to draw the typical curve of sample detection dose-response, wherein several sample concentrations are lower than the susceptibility of detection, and enriched sample should make the binding site of coding microball be in state of saturation.
2, the processing of human serum sample
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, the yellow warm antibody of detection
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 yellow hot E3 proteantigen that mark can be caught yellow warm antibody, promptly utilizes yellow hot E3 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 yellow hot E3 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
6Promptly 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 2, bag is all dropped in its correct surveyed area by No. 027 microballoon of yellow hot E3 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 yellow warm antibody.
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.
Process verification experimental verification in the present utility model, find that the biotinylated antibody goat-anti of 2mg/mL rabbit detects yellow warm 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 the detection thing that detects yellow warm antibody in the human serum, testing result showed that biotinylation detects the detection effect that thing Biotin-goat anti-human igg can obtain the Supreme People's Procuratorate's measured value and low background to the biotinylated detection thing of 2mg/mL Biotin-goat anti-human igg 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 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) 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 yellow warm antibody is the corresponding detection substrate concentration of fluorescence intensity critical value (Cutoff).Wherein, the definition of Cutoff is to adopt blank sample (Blank) fluoroscopic examination signal MFI average to add 3 times of standard deviations (SD), and promptly the Cutoff value is=MFI (blank)+3 times standard deviation.Corresponding fluorescence intensity level then is judged to be the yellow warm antibody testing result positive if testing result is higher than LOD; Corresponding fluorescence intensity level then is judged to be yellow warm antibody testing result feminine gender if testing result is lower than LOD.
The using suspending chip detection method detects the anti-yellow hot IgG of rabbit, the anti-St. Louis encephalitis NS1 serum of rabbit, mouse-anti west Buddhist nun sieve E serum, the hot Richettsia antibody of the anti-Q of rabbit, the anti-Rickettsia prowazeki of rabbit, the anti-horse beautiful eyes of rabbit antibody, the anti-Tula antibody of rabbit, rabbit anti-dengue serum, rabbit anteserum, rat blood serum, mice serum, BSA, casein, tryptose etc. respectively, according to testing result criterion among the embodiment 3, only the anti-yellow hot IgG of rabbit is positive, and all the other interference antibody or albumen are all negative.Cross reaction or non-specific responding all do not take place in method for detecting suspension chip and other test antibody that the yellow warm antibody that the present invention sets up is described.
The foundation of embodiment 5, suspending chip detection by quantitative model
1, the anti-yellow hot IgG typical curve specimen preparation of rabbit
With sample diluting liquid the anti-yellow hot immune serum standard items of rabbit are become the series concentration sample with 4 times of multiple proportions gradient dilutions.
2, the drafting of dose-response typical curve
Detect above-mentioned series concentration sample according to the detection method among the embodiment 3, and draw dose-response typical curve (as shown in Figure 3) according to the suspension chip system testing result.Wherein.Wherein, X-axis is represented the titre of serum 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 titre of the yellow warm antibody of rabbit is respectively among Fig. 2: S1:1: 655360, and S2:1: 163840, S3:1: 40960, S4:1: 10240, S5:1: 2560, S6:1: 640, S7:1: 160, S8:1: 40, S9:1: 10.
Suspension chip system resists yellow warm antibody dose-response typical curve equation according to testing result match rabbit:
FI＝2.75079+(8515.88-2.75079)/[1+(Conc/0.00361469)
-1.22372]
1.02486
3, suspending chip detects sensitivity and the dynamic range of the anti-yellow hot IgG of rabbit
According to minimum detectability definition and dose-response typical curve equation, the utility model is that the sensitivity of the anti-yellow hot IgG of protein suspension chip method detection rabbit is: 0.000007 (Cutoff=6.3, MFI (blank)=5.25, standard deviation=1.05).
The present 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 the anti-yellow hot IgG concentration of rabbit, its corresponding MFI value also increases progressively thereupon, when the anti-yellow hot IgG titre of rabbit surpasses 1: 40, 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 behind the diluted sample again.
Therefore, can judge that according to minimum detectability and Supreme Procuratorate's rising limit the present invention is that the dynamic range of the anti-yellow hot IgG of suspending chip detection by quantitative rabbit is 1: 10240～1: 40.
Embodiment 6, human serum sample's detection
1, human serum sample's preparation
Human serum 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 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,, determine the concentration of yellow warm antibody in the human serum to be detected according to typical curve.
Through revision test repeatedly, biotinylation goat anti-human igg dilution ratio is selected dilution in 1: 2500 for use, the SA-PE dilution ratio is selected dilution in 1: 300 for use, through 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, be that C utoff value is 780, if the MFI value that detection obtains is greater than 780, promptly can be considered the yellow warm antibody positive may be infected by yellow fever virus, if the MFI value is less than 780, yellow warm antibody feminine gender be can be judged as, yellow fever virus or recessive carrier do not infected.
Claims (5)
1. protein suspension chip system that detects yellow warm antibody, it is characterized in that, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, coding microball is housed in the chip basal body No. 027, adding liquid systems is communicated with chip basal body, anti-in chip basal body, to add biotin labeled two, fluorescence signal detects thing, the suspending chip detector comprises microcapillary sense channel and detection laser, the microcapillary sense channel is communicated with chip basal body, solution behind this microcapillary sense channel sucting reaction, 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 is characterized in that described capture antigen is preferably yellow hot E3 albumen.
3. protein suspension chip as claimed in claim 1 is characterized in that, it is streptavidin-phycoerythrin that described fluorescence signal detects thing.
4. 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.
5. protein suspension chip as claimed in claim 1 system, it is characterized in that, described detection laser comprises with exciting color in the described coding microball matrix, the sorting code number of recognition coding microballoon to determine the red laser of test item, and the color signal, tracer signal power that is used to excite and discern testing molecule is with the green laser of the content that detects described antibody to be measured.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010206842439U CN202024998U (en) | 2010-12-28 | 2010-12-28 | Protein suspension array system for detecting yellow fever antibodies |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105738629A (en) * | 2015-12-28 | 2016-07-06 | 中华人民共和国上海出入境检验检疫局 | Indirect immuno-fluorescence detection method of yellow fever virus IgG antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105738629A (en) * | 2015-12-28 | 2016-07-06 | 中华人民共和国上海出入境检验检疫局 | Indirect immuno-fluorescence detection method of yellow fever virus IgG antibody |
| CN105738629B (en) * | 2015-12-28 | 2018-07-10 | 中华人民共和国上海出入境检验检疫局 | A kind of indirect immunofluorescene assay method of yellow fever virus IgG antibody |
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