CN202002930U - Protein suspension chip system for detecting SARS (Severe Acute Respiratory Syndromes) antibody in serum sample - Google Patents

Abstract

The utility model discloses a protein suspension chip system for detecting SARS (Severe Acute Respiratory Syndromes) antibody in serum sample, which comprises a chip basal body, a liquid adding system, a suspension chip detector and a computer, related buffer solution and an antibody to be detected are arranged in the chip basal body; a coding microballoon is arranged in the related buffer solution, envelope captured antigen is arranged on the coding microballoon, and the liquid adding system is used for sequentially adding a second antibody marked by biotin and fluorescent signal detection objects into the chip basal body; the suspension chip detector comprises a microcapillary detection channel and detection laser, the coding microballoon sequentially passes through the microcapillary detection channel, and the detection laser excites and identifies the color of the coding microballoon and the color of objects to be detected on the coding microballoon; the computer is connected with the suspension chip detector and records and analyzes the measurement result of the suspension chip detector. The protein suspension chip technology utilizes the reaction of the microballoon in the solution and utilizes the laser detection technology to increase the accuracy and the repeatability of sample detection, and the protein suspension chip system has characteristics of simple operation, good repeatability and the like which are superior to that of the piece film chip.

Description

A kind of protein suspension chip system that detects SARS antibody in the serum sample

Technical field

The utility model relates to a kind of protein suspension chip system that detects SARS antibody in the serum sample.

Background technology

The serious acute respiratory syndrome (SevereAcute Respiratory Syndrome, SARS, severe acute respiratory syndrome) of wreaking havoc the whole world in year spring winter to 2003 in 2002 is exactly a coronaviridae, a kind of in the coronavirus genus.Coronavirus is one of main pathogen of adult's common cold, and the childhood infection rate is higher, mainly is the infection of the upper respiratory tract, generally seldom involves lower respiratory tract.In addition, also can cause baby and neonate's acute gastroenteritis, cardinal symptom is water sample stool, heating, vomiting, can draw 10 every day surplus time, severe patient even the blood watery stool occurs also causes nervous system syndrome under the rare occasion.SARS-CoV is the sub-thread positive chain RNA virus, belongs to the nido virales, coronaviridae, coronavirus genus.According to serotype, coronavirus genus mainly is divided into 3 Group, comprises multiple mammal coronavirus and bird infectious bronchitis virus.SARS-CoV is first coronavirus that causes human serious disease.SARS-CoV primary structure albumen has: S albumen (spike glycoprotein), M albumen (transmembrane protein), E albumen (envelope protein), N albumen (NP).N albumen is wherein the most stable structural proteins, also is virus protein the abundantest in the course of infection, and confirmation is desirable diagnostic antigen.

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 suspension chip method can detect the SARS 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 SARS antibody in the serum, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, relevant buffer solution and antibody to be measured are housed in the chip basal body, be provided with coding microball No. 044 in the relevant damping fluid, be coated with capture antigen on the coding microball, adding liquid systems adds biotin labeled two anti-successively in chip basal body, 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 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 SARS-CoV N albumen.

Further, described antibody to be measured is blood serum sample.

Further, described biotin labeled two anti-be preferably Biotin-goat anti-rabbit igg 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 SARS antibody have been done substantial improvement and innovation, it has following advantage:

1, the improvement of antigen coated amount

The package amount of SARS-CoV N 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 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, the specificity of method

The utility model is an envelope antigen by selecting SARS-CoV N albumen for use, is antibody to be measured with the anti-SARS antibody of rabbit, serves as to detect antibody with biotinylated goat-anti rabbit.This development test proves, exists mouse-anti influenza NP IgG, rabbit antitubercle serum, rabbit approach antibody, the anti-pestis F 1 IgG of rabbit 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 protein suspension chip method to human serum.By detection to human serum, tentative confirmation the practicality of this method SARS antibody in detecting human serum.

Description of drawings:

Fig. 1 is the utility model structural representation;

Fig. 2 is that No. 044 microballoon bag is by SARS-CoV N Protein Detection SARS antibody synoptic diagram;

Fig. 3 detects the anti-SARS antibody dosage of rabbit-reaction normal curve map for the protein suspension chip method.

Fig. 4 detects the anti-SARS antibody of rabbit canonical plotting for the ELISA method;

Fig. 5 is the correlativity that protein suspension chip and ELISA method detect the anti-SARS antibody of rabbit.

Embodiment

Extremely shown in Figure 5 as Fig. 1, a kind of protein suspension chip system that detects SARS 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 SARS-CoV N albumen on the coding microball, be used for catching the antibody to be measured of blood serum sample to be detected, if SARS antibody is arranged in the sample, SARS-CoVN protein combination on SARS antibody and the coding microball, add again have biotin labeled two anti-, forming coding microball-SARS-CoV N albumen-SARS antibody-two resists-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-SARS-CoV N albumen-SARS antibody-two resists-biotin-streptavidin-phycoerythrin compound, reach SARS detection of antibodies in the blood serum sample by of the detection of suspending chip detector the phycoerythrin fluorescence signal, if there is not SARS antibody in the blood serum sample, the coding microball that is coated with SARS-CoV N 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 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 SARS antibody in the serum sample of the utility model be described further by following embodiment, but the utility model is subjected to the qualification of this embodiment never in any form.

One, material

1. the relevant damping fluid of protein suspension chip:

(1) PBS damping fluid (pH7.4): NaCl 137mmol/L; KCl 2.7mmol/L; 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 5N NaOH0.15mL, constant volume is in 250mL.

(5) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, and 0.02%TWEEN, 0.05% azide, pH 7.4.

(6) microballoon confining liquid PBS-BN:PBS, 1%BSA, 0.05% azide, pH7.4.

(7) detect damping fluid: PBS, 1%BSA, pH7.4.

(8) antibody diluent PBS, pH7.4.

(9) microballoon dilution: PBS, 1%BSA, pH7.4.

(10) sample diluting liquid PVX:PBS, 0.5%PVA, 0.8%PVP;

(11) SA-PE dilution: PBS (pH7.4), 1%BSA.

2. antigen and antibody

The employed antigen of the utility model is that SARS-CoV N proteantigen for example is reorganization SARS-CoV total length nucleoprotein-N32 (aa1-422) and reorganization SARS-CoV nucleoprotein fragment N13 (aa221-422), all through nickel affinity column purifying, can be available from China Inst. of Quarantine Inspection Sciences.

The utility model uses detection antibody to be the anti-SARS IgG of rabbit, can be available from microorganism epidemic research institute of military medical courses in general institute, it is biotinylation goat anti-rabbit igg and biotinylation goat anti-human igg that detection two resists, and described goat anti-rabbit igg and goat anti-rabbit 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-SARS 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 rabbit antitubercle serum, rabbit anti influenza NP IgG, rabbit anti-avian influenza H5N1 serum, the anti-pestis F 1 IgG of rabbit, BSA, casein, tryptone etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid-4 ℃ of preservations.The storing solution concentration of the anti-SARS N of the anti-rabbit of rabbit IgG is 0.036mg/mL.In the comparative experiments, same sample is used for the detection of ELISA and suspending chip.

Anti-SARS 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, detection SARS antibody

1, the capture antigen bag microballoon that is encoded

No. 044 coding microball that the utility model adopts is available from U.S. Bio-Rad company, and coding microball is used for the SARS-CoV N proteantigen that mark can be caught SARS antibody, promptly utilizes SARS-CoV N 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 SARS-CoV N proteantigen 0.01～90 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, protein suspension 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 1, bag is all dropped in its correct surveyed area by No. 044 microballoon of SARS-CoV N proteantigen, and obtain high s/n ratio result (the MFI value is much larger than 2000), illustrate that the protein suspension chip detection system of optimizing can successfully be used for the SARS 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 SARS 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 SARS 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-goat anti-human antibody with dilution in 1: 2500.

Embodiment 3, protein suspension chip specimen preparation, 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) 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 SARS 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 SARS antibody test positive as a result if testing result is higher than LOD; Corresponding fluorescence intensity level then is judged to be SARS antibody test feminine gender as a result if testing result is lower than LOD.

Embodiment 4, protein suspension chip are to SARS-CoV N proteantigen specific detection

Using the protein suspension chip detection method detects rabbit approach IgG, mouse-anti influenza NP IgG, the anti-SARS IgG of rabbit, rabbit anti-avian influenza H5N1 serum, the anti-pestis F 1 IgG of rabbit, BSA, casein, tryptone etc. respectively, according to testing result criterion among the embodiment 3, only the anti-SARS 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 protein suspension chip detection method and other test antibody that the SARS antibody that the present invention sets up is described.

The foundation of embodiment 5, protein suspension chip detection by quantitative model

1, the anti-SARS IgG of rabbit typical curve specimen preparation

With sample diluting liquid the anti-SARS IgG of rabbit standard items are become series concentration sample with 4 times of multiple proportions gradient dilutions to 0.55ng/mL by 0.036mg/mL.

2, the drafting of dose-response typical curve

Detect above-mentioned series concentration sample according to the detection method among the embodiment 3, and draw dose-response typical curve (as shown in Figure 3) according to protein suspension chip system testing result.Wherein, X-axis is represented the concentration (ng/mL) of antibody, the fluorescent value (MFI) that on behalf of the suspending chip instrument, Y-axis detect.The testing result mean value that each is data represented 3 times, coordinate axis is set with logarithm-logarithmic relationship, the concentration (ng/mL) of the anti-SARS antibody of rabbit is respectively S1:0.549 among Fig. 3, S2:2.197, S3:8.789, S4:35.156, S5:140.625, S6:562.5, S7:2250.0, S8:9000.0.

The protein suspension chip system is according to the anti-SARS IgG of testing result match rabbit dose-response typical curve equation:

FI＝0.774363+(15544.5-0.774363)/[1+(Conc/2884.69) ^-7.77675] ^0.0999461

3, protein suspension 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 the anti-SARS IgG of protein suspension chip method detection rabbit is: 105.57pg/mL (Cutoff=6.31066, MFI (blank)=5.25, standard deviation=0.35355).

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 the anti-SARS IgG of rabbit concentration, its corresponding MFI value also increases progressively thereupon, when the anti-SARS IgG of rabbit concentration reaches 0.025mg/mL, 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 utility model is that the dynamic range of the anti-SARS IgG of protein suspension chip detection by quantitative rabbit is 4.883～25000ng/mL.

Embodiment 6, protein suspension chip method and ELISA method detect the comparison of the anti-SARS IgG of rabbit

1, biotin-avidin ELISA (BAS-ELISA) detection method

Biotin-avidin ELISA adopts and comprises the following steps:

1) adds the SARS-CoV N albumen 5-50 μ g of 50 μ L with the dilution of PBS damping fluid, 4 ℃ of static spending the night to the every hole of common ELISA microwell plate;

2) add confining liquid, 37 ℃ of sealings 2 hours;

3) washing lotion is washed 3 times, pats dry;

4) add test sample, every hole 50 μ L, room temperature was placed 40 minutes; Washing lotion is washed 3 times, pats dry;

5) add an amount of biotinylation and detect antibody, every hole 50 μ L, room temperature was placed 30 minutes; Washing lotion is washed 3 times, pats dry;

6) add an amount of SA/HRP (horseradish enzyme labeling Streptavidin), 50 μ L/ holes, room temperature was placed 3 minutes; Washing lotion is washed 3 times, pats dry;

7) add TMB colour developing liquid (solvable type single component tmb substrate solution) colour developing, 50 μ L/ holes, room temperature was placed 10 minutes;

8) use 2M H ₂SO ₄Cessation reaction;

9) use the microplate reader survey and read A450nm numerical value.

2, the protein suspension chip detection method of sample

Adopt indirect method to survey the immunology detection pattern of antibody, total overall reaction is all carried out on 96 hole filter plates in the testing process.

1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washing lotion washing and with vacuum pump suction filtration 2 times;

2) add 50 μ L test sample, the room temperature lucifuge jolts 30 minutes behind the mixing, with cleaning fluid washing and suction filtration 3 times;

3) add 50 μ L debita spissitudos with the biotinylated antibody after the antibody diluent dilution, the room temperature lucifuge jolts 30 minutes behind the mixing, washing lotion washing and vacuum pump suction filtration 3 times;

4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10 minutes behind the mixing.With cleaning fluid washing and vacuum pump suction filtration 3 times;

5) the detection damping fluid of adding 125 μ L is through the resuspended mixing of spiral;

6) read MFI numerical value and analyze data with the Bio-Plex suspension chip system.

3, the comparison of the sensitivity of two kinds of detection methods and dynamic range and correlativity

The anti-SARS antibody of the mutually same group rabbit sample of preparation uses protein suspension chip simultaneously with sample diluting liquid 4 doubling dilutions and the ELISA method detects.Draw the typical curve (horizontal ordinate is a sample concentration among the figure, and ordinate is an absorbance, and coordinate axis is taken the logarithm-logarithmic relationship) that the ELISA method detects the anti-SARS antibody of rabbit, and compare with the protein suspension chip methods and results.

According to two kinds of method standard curves: protein suspension chip method sensitivity as shown in Figure 3 is 402.9pg/mL, linear detection range 146.48-2400000pg/mL; ELISA method sensitivity as shown in Figure 4 is 600ng/mL, linear detection range 0.6-38.4 μ g/mL.Can reach a conclusion: detect the anti-SARS antibody of identical rabbit sample, the protein suspension chip method has higher detection sensitivity than ELISA method, and is promptly high 1000 times; And has wideer dynamic detection range, promptly high 3 orders of magnitude.

In addition, two kinds of method testing results are compared as shown in Figure 4 in 0.6-38.4 μ g/mL scope, can judge that protein suspension chip method and ELISA method have good correlativity, related coefficient is 0.9518.

Embodiment 7, human serum sample's detection

1, human serum sample's preparation

Human serum is used for suspending 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 SARS antibody in the human serum to be detected according to typical curve.

Through revision test repeatedly, biotinylation goat anti-human antibody 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 3123.355, being scaled concentration value is 366ng/mL, if the MFI value that detection obtains is greater than 3123.355, be that SARS antibody concentration in the serum surpasses 366ng/mL and can be considered the SARS antibody positive and may be infected by SARS virus, if the MFI value is less than 3123.355, be that SARS antibody concentration in the serum is lower than 9.6ng/mL and can be judged as the SARS negative antibody, do not infect SARS virus or recessive carrier.

Claims (8)

1. protein suspension chip system that detects SARS antibody in the serum sample, it is characterized in that, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, relevant buffer solution and antibody to be measured are housed in the chip basal body, be provided with coding microball No. 044 in the relevant damping fluid, be coated with capture antigen on the coding microball, adding liquid systems adds biotin labeled two anti-successively in chip basal body, 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 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.

3. protein suspension chip as claimed in claim 2 system is characterized in that described capture antigen is preferably SARS-CoVN 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-ly are preferably Biotin-goat anti-rabbit igg and/or Biotin-goat anti-human igg.

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 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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