CN101504415A - Preparation for protein suspending chip of bacillus anthracis spore and its quantitative determination method - Google Patents

Abstract

The invention relates to a preparation method, a detection method and a quantitative method for a suspension chip of a bacillus anthracis spore protein. The methods have the advantages of good detection capability, high sensitivity, strong specificity and wide dynamic range, and establish an open detection modeling platform for bacterial spores represented by bacillus anthracis.

Description

A kind of protein suspending chip preparation and quantitative detecting method of bacillus anthracis spore

Technical field

The present invention relates to the preparation method and the quantitative detecting method thereof of a kind of Bacillus anthracis (Bacillus anthracis) gemma (endospore) protein suspending chip.

Background technology

Bacillus anthracis (Bacillus anthracis) is the pathogen that causes Amphixenosis-anthrax, can form the aerobic bacteria of gemma for Gram-positive, gemma can pass through respiratory tract, alimentary canal, the skin contact infection mankind, infection symptoms appearred in back 7 days in contact generally, and the most common with localized anthrax.Suck a large amount of anthrax spores (greater than 8000) and can cause the induction type anthrax, also claim pulmonary anthrax.Because anthrax spore has the extremely strong resistibility of environment to external world, cause and pollute sustainable existence, militarily be listed in is one of No.1 biological warfare agent always.Once produced and stored in some countries, and became the attack of terrorism now again and select agent for use, the mankind were caused new bigger threat as weapon.The antigen of Bacillus anthracis comprises somatic antigen and gemma antigen.The existence of Bacillus anthracis, propagation do not need specific condition equipment and environment, just can breed in soil, manually cultivate in a large number and to make it to become gemma very easy.Because gemma unique biological proterties and harm are significant to the fast quantification detection of anthrax spore.

For the purpose of public health and national security, research and development is quick, the qualitative and quantitative analysis infectious disease pathogens are targets of the affiliated technical field midium or long term endeavouring to pursue.Especially for anthrax-bacilus,, can realize that detection by quantitative is most important as a kind of pathogen of deadly infectious disease.The laboratory conventional sense method that is used for anthrax-bacilus at present has the molecular biology method of bacteriological detection method, serology detection method and detection nucleic acid.But there are many defectives in existing detection techniques such as bacteriological detection method, serology detection method (for example indirect hemagglutination method) and nucleic acid detection method.For the bacteriological detection method, microbe growth is consuming time longer, is not suitable for the fast detecting to anthrax-bacilus.For nucleic acid detection method, although adopted round pcr anthrax-bacilus to be detected and analyzes the specificity height, be not easy contaminated, but the method for existing round pcr is loaded down with trivial details, particularly these class methods comprise extraction and the complicated operations step of DNA/RNA, and can not be used for the detection of non-nucleic acid substances such as protein, so nucleic acid detection method has limitation and inconvenience.The immunological detection method biosensor technology of newly-developed, more with the antibody test report as Fibre Optical Sensor, electrochemical sensor, up-converting phosphor biosensor, nano-sensor etc., the report that antigen directly detects is less.The present invention selects anthrax spore to set up the protein suspending chip detection model as the representative of sporiferous bacterium and gemma thereof.

Suspending chip (suspension array) also claim liquid-phase chip (liquid array, liquidchip), 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 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 5.5 μ m of microballoon size, different research purposes such as immunoassay, nucleic acids research, enzyme analysis, acceptor and part discriminance analysis etc. be can comply with, and specific antibodies, nucleic acid probe and various acceptor probe demarcated according to different research purposes.The microballoon of label probe and determinand react in 96 orifice plates.After the reaction, utilize machine automatically reactant liquor to be picked up and by a microcapillary sense channel, only allow a microballoon to pass through sense channel at every turn.Being provided with twice laser in the sense channel, is red together, excites the color in the microballoon matrix, and the sorting code number of identification microballoon is to determine test item; Be green together, excite the color of reporter molecules, the tracer signal power is to detect the content of determinand.When the probe of sample to be tested and specific microballoon was attached together, the light that twice laser is excited all can be detected.And, then only have the exciting light in the microballoon to be detected if do not contain this subject matter in the sample.Microballoon kind and the quantity that is excited by machine and computing machine automatic statistical analysis twice laser again, thereby several test target things are arranged therein in the judgement sample to be tested, learn to have or not cause of disease to be measured to exist in the test sample book, or have several simultaneously to tens of kinds of cause of diseases.The suspending chip technology is owing to utilize microballoon to react in solution, overcome sheet film chip and when big Molecular Detection, be subjected to the influence to reaction kinetics such as surface tension, steric effect, utilize laser measuring technology simultaneously, improve the accuracy and the repeatability of sample detection greatly, had characteristics such as easy and simple to handle, the good reproducibility that is better than sheet film chip.

The 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.Whether but whether suspending chip can detect anthrax and gemma thereof, be fit to directly detect fast from samples such as powder, liquid, and its detection by quantitative ability how, still lacks model and evaluation.

Summary of the invention

The purpose of this invention is to provide the protein suspending chip of a kind of Bacillus anthracis (Bacillus anthracis) gemma, the quantitative detecting method based on the bacillus anthracis spore of this chip also is provided simultaneously.

Technical solution of the present invention is as follows:

A kind of detection anthrax spore protein suspending chip, this suspending chip comprises: coding microball (for example No. 025), bag is detected antibody (for example anti-sterne antibody of rabbit), streptavidin-phycoerythrin (SA-PE) and relevant buffer solution by the capture antibody of the described anthrax spore of microballoon (for example goat-anti sterne (anthrax vaccine strain) antibody), biotin labeled anthrax spore.

A kind of protein suspending chip detection method of anthrax spore, total overall reaction can carry out also can carrying out in microcentrifugal tube on 96 hole filter plates in the testing process.Comprise the following steps: that (1) every hole adding contains the working solution that wraps the antibody coding microballoon that is hunted down, cleans with cleaning fluid; (2) add test sample, hatch the back and clean; (3) adding detects antibody with biotinylation, hatches the back and cleans; (4) add streptavidin-phycoerythrin (SA-PE), hatch the back and clean, mixing after (5) adding detection damping fluid, (6) read FMI numerical value (average fluorescent strength) with suspension chip system and analyze the data judging testing result negative or positive.

A kind of protein suspending chip quantitative detecting method of anthrax spore, this method comprises the following steps: that the anthrax spore suspension of (1) adding is through 10 times of gradient multiple proportions serial dilutions, (2) the dose-response typical curve of the corresponding FMI value of making sample concentration, (3) with analysis software match dose-response curve and equation, (4) the unknown concentration sample can be according to dose-response curve and equation judgement sample detectable concentration, decidable method detectability and dynamic detection range.

The inventor has done substantial optimization through a large amount of and deep research to the protein suspending chip preparation and the testing conditions thereof of anthrax spore, and it has following advantage:

1, the improvement of antibody sandwich amount

The package amount of antibody need be optimized to detect effect, and the required antibody sandwich amount of suspending chip is very low, and optimizing the result is 10 μ g/1.25 * 10 ⁶Individual microballoon, i.e. 20-40ng/2500-5000 microballoon/test, and the package amount of traditional immunology ELISA method is the 200ng/ test.

2, biotin labeled improvement

The used biotin of labelled antibody is excessive, and the computing formula of reference is arranged in the excessive value kit.Theoretically, in testing process, excessive biotin is fallen by suction filtration during cleaning because of the unmarked antibody of going up is not connected by the antigen in conjunction with capture antibody on the microballoon, can not react with the SA-PE that adds subsequently, does not influence detection.But in the actual detected process, the phenomenon that the detection signal that fell in may reduce, so after advising biotin labeling antibody, remove unnecessary biotin as far as possible.

3, sensitivity of Jian Ceing and dynamic range

Anthrax spore protein suspending chip quantitative detecting method of the present invention is compared with the immunology ELISA detection method of classics, and the result has good anastomose property (coefficient R 2=0.9564).Simultaneously, suspension chip method sensitivity (103cfu/mL) is higher than 1 order of magnitude of ELISA method (104cfu/mL) sensitivity; And suspension chip method dynamic detection range (103-107cfu/mL) is higher than 1 order of magnitude of ELISA method dynamic detection range (104-107cfu/mL).

4, the specificity of method

The present invention is to detect target antigen by selecting anthrax spore for use, is capture antibody with goat-anti sterne antibody respectively, serves as to detect antibody with the anti-sterne antibody of rabbit.This development test proves, this method has good specificity under the quiet and secluded conditions of interference such as false knot pyrenomycetes, enterocolitis bacterium, yersinia pestis, Bacillus cereus, bacillus subtilis, bacillus megaterium, deep shape bacillus, Escherichia coli, Salmonella typhimurtum, enterobacter cloacae, Fu Shi citrobacter, staphylococcus aureus, proteus vulgaris existing.

5, the detectability of sample

The present invention has estimated the detectability of suspension chip method to environment such as " white powders " and food samples.By simulations such as milk powder, wheat flour, cornstarch, the precious powder of mixed type fruit being added the detection of sample, tentative confirmation the practicality of this method in " white powder " sample that detects suspicious pollution anthrax spore.

Description of drawings:

Fig. 1: anthrax spore detects with No. 025 microballoon bag by anthrax spore antibody test synoptic diagram;

Fig. 2: protein suspension chip method detects the anthrax spore canonical plotting;

Fig. 3: the ELISA method detects the anthrax spore canonical plotting;

Fig. 4: protein suspending chip and ELISA method detect the correlativity of anthrax spore.

Embodiment

Protein suspending chip preparation method, detection and the quantivative approach of the anthrax spore that the present invention relates to are described further by following embodiment, but the present invention is subjected to the qualification of this embodiment never in any form.

One, material

1. the relevant damping fluid of protein suspending chip:

(1) 0.03M PB damping fluid (pH7.2): 2.83g Na ₂HPO ₄, 1.36g KH ₂PO ₄Be settled to 1L.

(2) 0.01M PB damping fluid (pH7.2): form by the dilution of 0.03M PB damping fluid.

(3) 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.After the packing at 15psi (1.05kg/cm ²) high pressure steam 20 minutes, or filtration sterilization, be stored in room temperature.

(4) microballoon cleaning fluid: PBS (pH7.4), 0.05% TWEEN-20.

(5) 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.

(6) the microballoon bag is cushioned liquid 0.05M MES, pH 5.0:2.44g MES, and 5N NaOH 0.15mL, constant volume is in 250mL.

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

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

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

(10) antibody diluent: 0.01mmol/L PB (pH7.2).

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

(12) sample diluting liquid: 0.01M PB, pH7.2.

(13) biotinylated antibody dilution: PBS-TBN (PBS, 0.1%BSA, 0.02%TWEEN-20,0.05%NaN ₃, pH7.4).

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

2. bacterial strain

Bacillus anthracis used herein is a vaccine strain Sterne bacterial strain.

3. antigen and antibody

Anthrax spore antigen used herein is produced gemma by vaccine strain Sterne bacterial strain.

Capture antibody used herein is a goat-anti Sterne antibody, and detecting antibody is the anti-Sterne antibody of biotinylation rabbit, for this research department prepares with caprylic acid-saturated ammonium sulfate method of purification purifying.

Two, the preparation of testing sample

1, the preparation of target analysis matter sample

Target analytes is an anthrax spore, but relates to deadly infectious disease bio-safety problem, with Bacillus anthracis vaccine strain Sterne gemma as the target analysis matter sample.Bacillus anthracis vaccine strain (Sterne) is inoculated in gemma nutrient culture media DSM, cultivated for 2～3 weeks for 37 ℃, the husky yellow spore staining of peacock green, microscopy is observed, and gathers in the crops gemma when above when gemma accounts for thalline several 95%, sterilization PB damping fluid (0.01M, Ph7.2) flushing is 2 times, after PB is resuspended, and 10 times of dilutions, and determine the gemma copy number with dull and stereotyped viable bacteria counting method, preserve for 4 ℃ and be equipped with inspection.Dilution back bacteria suspension concentration range is

10 ⁷Cfu/mL.

Disturbed specimen or the sample of testing as the method specificity are other bacteriums of target detection beyond the region of objective existence, comprise the false knot pyrenomycetes, the enterocolitis bacterium, yersinia pestis, Bacillus cereus, bacillus subtilis, bacillus megaterium, deep shape bacillus, Escherichia coli, Salmonella typhimurtum, enterobacter cloacae, the Fu Shi citrobacter, staphylococcus aureus, proteus vulgaris etc. are inoculated in nutrient agar, 37 ℃ 24 hours, the homogeneity of visual inspection lawn, scrape and get bacterium colony, with aseptic PB damping fluid dilution bacterium liquid, Maxwell standard turbidimetry is tentatively counted, and preserves for 4 ℃ to be equipped with inspection.Bacteria suspension concentration is about

10 ⁷Cfu/mL.

In the comparative experiments, identical anthrax spore sample is used for the detection of ELISA and suspending chip.

2, simulating pollution sample

Respectively powder such as 0.5g milk powder, cornstarch, wheat flour, instant fruit treasure are joined in the 5mL sample diluting liquid, the anthrax spore of variable concentrations is incorporated in the powdered sample, through the shake well mixing, leave standstill more than the 2h, target analytes and simulation white powder to be measured are fully adsorbed.Again with absorbent cotton, thin filter paper, thick filter paper, 0.45 μ m filter membrane filter paper filtering or low-speed centrifugal (2000rpm, 1min) after, supernatant carries out the detection of suspension chip method as sample to be checked.

The preparation of the protein suspending chip of embodiment 1, detection anthrax spore

1, the capture antibody bag microballoon that is encoded

No. 025 coding microball that the present invention adopts is available from U.S. BIO-RAD company, and coding microball is used for the antibody that mark can be caught anthrax spore, promptly utilizes goat-anti Sterne antibody sandwich 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 14000g is centrifugal, careful sucking-off and abandoning supernatant.The microballoon cleaning buffer solution that adds 100 μ L suspends, and concussion and ultrasonic back 14000g are centrifugal, careful sucking-off and abandoning supernatant.The microballoon activation damping fluid that adds 100 μ L, the EDC (50mg/mL) that then adds the fresh configuration of 10 μ L earlier, (biotin-LC-hydrazide) or the biotin of carboxyl activity (are the Sulfo-NHS-biotin to add the amino active biotin of the 50mg/mL of the fresh configuration of 10 μ L again, the biotin of SH-activity), jolt 20 minutes in room temperature.Add the PBS (pH7.4) of 150 μ L, after the concussion, 14000g is centrifugal, careful sucking-off and abandoning supernatant.PBS (pH7.4) the suspended coding microballoon that adds 100 μ L.

B, use the antibody sandwich coding microball

Get in the coding microball after capture antibody goat-anti Sterne antibody 4-50 μ g joins activation, be settled to 500 μ L with the PBS damping fluid, room temperature jolts 2 hours.14000g is centrifugal, careful sucking-off and abandoning supernatant.PBS damping fluid with 500 μ L is washed once, and 14000g 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, 14000g is centrifugal, careful sucking-off and abandoning supernatant.The microballoon that adds 500 μ L is preserved liquid washing coding microball, and 16000g 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; 1/400mm2) under simple microscope, count.According to formula (each big lattice number * 10 ⁴* extension rate * volume (mL)) calculates microballoon quantity.

2, detect the antibody labeling biotin

The mark of A, biotin

Preparing concentration respectively is the antibody-solutions to be marked of 10mM biotin solution and 2mg/mL, and the biotin that has calculated volume is joined in the antibody-solutions to be marked, jolts 30 minutes in room temperature (or 2 hours) on ice, packing after the post desalination excessively, and-20 ℃ are frozen standby.

B, antibody consumption calculate

IgG (immunoglobulin (Ig), molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ l of 10mM biotin solution.

Computation process is as follows:

$1\mathrm{ml\; lgG}\&times;\frac{2\mathrm{<figure-callout\; id="1"\; label="mg\; lgG"\; filenames="A200910080259E00182.png"\; state="\{\{state\}\}">mg\; lgG</figure-callout>}}{1\mathrm{ml\; lgG}}\&times;\frac{1\mathrm{mmol\; lgG}}{\mathrm{150,000}\mathrm{mg\; lgG}}\&times;\frac{20\mathrm{mmol\; Biotin}}{1\mathrm{mmol\; lgG}}=0.000266\mathrm{mmol\; Biotin}$

$0.000266\mathrm{mmol\; Biotin}\&times;\frac{\mathrm{1,000,000}\mathrm{\&mu;l}}{L}\&times;\frac{L}{10\mathrm{mmol}}=26.6\mathrm{\&mu;l\; Biotin\; Reagent}$

The optimized choice of C, biotinylated antibody

With the ELISA method biotinylated antibody is carried out quality control, method is as follows:

1) adds 50 μ L, 4 ℃ of static spending the night with the every hole of antigen coated ELISA 96 orifice plates;

2) add confining liquid (the PBS solution that contains 5% BSA) at 37 ℃ of sealing 1h;

3) washing lotion (PBS that contains 0.5% Tween20) is washed 3 times, pats dry;

4) add biotinylated antibody, every hole 50 μ L, room temperature is placed 30min;

5) 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 is placed 30min;

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

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

9) use 2M H at last ₂The SO4 cessation reaction.Use the microplate reader survey and read A450nm numerical value.

The result judges: very light blue if negative control hole occurs, positive hole color is very dark, and then biotinylated antibody can use; Otherwise, need carry out the biotinylation mark of new round antibody again.

The optimization of embodiment 2, suspending chip preparation method condition

1, the selection of microballoon coated antibody and antibody sandwich amount

Amount bag with 4 μ g, 8 μ g, 10 μ g, 16 μ g, 24 μ g, 40 μ g, 48 μ g is encoded to No. 025 microballoon by 100 μ L respectively.Effect compares after testing, with 10 μ g/1.25 * 10 ⁶Individual microballoon is that 20-40ng/2500-5000 microballoon/test pack is best by effect, and microscopically counting back lucifuge stored refrigerated is stand-by.As shown in Figure 1, bag is all dropped in its correct surveyed area by No. 025 microballoon of goat-anti Sterne antibody, 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 anthrax spore and detect.

2, the optimization of biotinylated antibody

The present invention uses amino active biotin respectively, and (biotin-LC-hydrazide) biotin with the carboxyl activity (is the Sulfo-NHS-biotin, the biotin of SH-activity) marker detection antibody, detect effect relatively through the Quality Control process, it is better that the biotin labeling of SH-activity detects the antibody test effect in this experiment, and the method that detects effect adopts the conventional method of this area or the described method of product description of installation manufacturer.

The inventor finds that through verification experimental verification the biotinylated antibody of 2mg/mL detects as detecting antibody with the 1:200 dilution, and testing result shows that biotinylation detects the detection effect that the anti-Sterne antibody of antibody Bio-rabbit can obtain the Supreme People's Procuratorate's measured value and low background.

Embodiment 3, suspending 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 suspending chip detection.

2, the detection of sample and result judge

The testing process total overall reaction is all carried out on 96 hole filter plates, and testing process is as follows:

1) every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washs and use the vacuum pump suction filtration with cleaning fluid;

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

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

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

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

6) read FMI numerical value and analyze data with suspension chip system.

3, testing result is judged

According to experimental study result and correlative study report, the present invention defines protein suspension chip method and detects the detection substrate concentration of the minimum detectability (LOD value) of anthrax spore for fluorescence intensity critical value (Cutoff) correspondence.Wherein, the definition of Cutoff is to adopt blank sample (Blank) fluoroscopic examination signal MFI average to add 3 times of standard deviations (SD), and promptly the Cutoff value is=MFI _Blank+ 3 * SD.Corresponding fluorescence intensity level then is judged to be the anthrax spore testing result positive if testing result is higher than LOD; Corresponding fluorescence intensity level then is judged to be anthrax spore testing result feminine gender if testing result is lower than LOD.

Embodiment 4, suspending chip are to the specific detection of anthrax spore antigen

The using suspending chip detection method is respectively to bacterial concentration 10 ⁵-10 ⁷Cfu/mL false knot pyrenomycetes, enterocolitis bacterium, yersinia pestis, Bacillus cereus, bacillus subtilis, bacillus megaterium, deep shape bacillus, Escherichia coli, Salmonella typhimurtum, enterobacter cloacae, Fu Shi citrobacter, staphylococcus aureus, common deformed rod etc. detect, according to testing result criterion among the embodiment 3, only anthrax spore antigen is positive, and all the other disturb antigen all negative.Illustrate that cross reaction or non-specific responding all do not take place for anthrax spore antigen method for detecting suspension chip and other bacteria tested that the present invention sets up.

The foundation of embodiment 5, suspending chip detection by quantitative model

1, anthrax spore antigen typical curve specimen preparation

With sample diluting liquid with the anthrax spore bacteria suspension by 1.84 * 10 ⁷Cfu/mL is with 10 times of multiple proportions gradient dilution to 1.84 * 10 ¹Cfu/mL becomes the series concentration sample.Simultaneously, same sample is used for the detection of ELISA method.

2, the drafting of dose-response typical curve

Detect above-mentioned series concentration sample according to the detection method among the embodiment 3, and draw dose-response typical curve (as shown in Figure 2) according to the suspension chip system testing result.Wherein, X-axis is represented the concentration (cfu/mL) of anthrax spore, 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.

Suspension chip system is according to testing result match anthrax spore dose-response typical curve equation:

FI＝3.73169+(229.979-3.73169)/[1+(Conc/96161.2) ^-1.13863] ^0.893333

3, suspending chip detects the sensitivity and the dynamic range of anthrax spore

According to minimum detectability definition and dose-response typical curve equation, the present invention is that the sensitivity of protein suspension chip method detection anthrax spore is: 10 ³Cfu/mL (Cutoff=12.86, Blank=6.5, SD=2.12).

It is to make the binding site of coding microball be in the detection substrate concentration of state of saturation that the present invention defines Supreme Procuratorate's rising limit.According to the rising of typical curve along with anthrax spore concentration, its corresponding MFI value also increases progressively thereupon, when anthrax spore concentration surpasses 10 ⁷During cfu/mL, the immune response of the antigen-antibody state that reaches capacity, the MFI value begins to enter plateau, and the substrate concentration to be checked in the interpret sample is too high, need 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 suspending chip detection by quantitative anthrax spore is 10 ³～10 ⁷Cfu/mL.

Embodiment 6, protein suspension chip method and ELISA method detect the comparison of anthrax spore

1, ELISA detection method

As to this, adopt the double-antibody sandwich elisa that comprises the following steps to detect:

1) adds the capture antibody 5-50 μ g of 50 μ L with the dilution of PB 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 A _450nmNumerical value.

2, the method for detecting suspension chip of sample

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 FMI 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 mutually same group anthrax spore sample while using suspending chip and the ELISA method of preparation detects among the embodiment 5.Draw the typical curve (horizontal ordinate is a sample concentration among Fig. 2 and 3, and ordinate is an absorbance, and coordinate axis is taken the logarithm-logarithmic relationship) that the ELISA method detects anthrax spore, and with embodiment 5 in the suspension chip method result compare.

According to two kinds of method standard curves: suspension chip method (as shown in Figure 2) sensitivity is 10 ³Cfu/mL, dynamic detection range 10 ³～10 ⁷Cfu/mL; ELISA method (as shown in Figure 3) sensitivity is 10 ⁴Cfu/mL, linear detection range 10 ⁴～10 ⁷Cfu/mL.Can reach a conclusion: detect identical anthrax spore sample, protein suspension chip method has higher detection sensitivity than ELISA method, is high 1 order of magnitude; And has wideer dynamic detection range, promptly high 1 order of magnitude.

In addition, with two kinds of method testing results 10 ⁴～10 ⁷Comparing (as shown in Figure 4) in the cfu/mL scope can judge that protein suspension chip method and ELISA method have good correlativity, and related coefficient is 0.9564.

The detection of embodiment 7, artificial contamination's " white powder " sample

1, the preparation of artificial contamination's " white powder " sample

The anthrax spore of certain content or blank (sample diluting liquid) are mixed powdered samples such as milk powder, starch, flour, instant fruit treasure respectively prepare artificial contamination's sample according to variable concentrations.The 0.5g powder is joined in the 5mL diluted sample damping fluid, and fully mixing left standstill 2 hours, allowed testing sample and white powder fully adsorb.

2, the detection of artificial contamination's " white powder " sample

Above-mentioned contaminated " white powder " sample is handled, select cotton, thin filter paper, thick filter paper, 0.45 μ m filter membrane, 0.22 μ m membrane filtration for use, and 2000rpm, 5 minutes and 1000rpm, 4 minutes methods such as low-speed centrifugal handle the artificial powder solution that adds behind the sample, carries out the suspending chip analyzing and testing according to embodiment after collecting filtrate or supernatant.

Table 1: suspending chip is to artificial contamination's " white powder " test result of samples

In 18 samples that detected, testing result (as shown in table 1) is all correct, has proved that tentatively protein suspension chip method can be applied to the actual detected work of " white powder " sample of anthrax spore pollution fast, accurately and efficiently.

Claims (12)

1, a kind of method of utilizing protein suspending chip to detect bacillus anthracis spore is characterized in that total overall reaction can be carried out in the testing process in 96 hole filter plates or microcentrifugal tube, and this method comprises the following steps:

(1) adding contains the working solution that wraps the antibody coding microballoon that is hunted down to the hole, cleans with cleaning fluid;

(2) add testing sample, hatch the back and clean;

(3) add with biotinylated detection antibody, hatch the back and clean;

(4) add SA-PE, hatch the back and clean;

(5) mixing after the adding detection damping fluid;

(6) read average fluorescent strength (FMI) numerical value and analyze the data judging testing result with suspension chip system.

2, the method for claim 1 is characterized in that, being used to wrap by the capture antibody of microballoon is anti-anthrax spore antibody, adopt biotin labeled anti-anthrax spore antibody as detection antibody, and detection architecture is formed in its combination.

3, the method for claim 1 is characterized in that, described bag is 10 μ g/1.25 * 10 by the capture antibody goat-anti Sterne antibody consumption of microballoon ⁶Individual coding microball or 20-40ng/2500-5000 microballoon/test.

4, the method for claim 1 is characterized in that, the biotin of the anti-Sterne antibody of marker detection antibody rabbit in the described method is the biotin of carboxyl activity.

5, the method for claim 1 is characterized in that, the anti-Sterne antibody of the biotinylated detection antibody of 2mg/mL Bio-rabbit detects as detecting antibody with the 1:50-1:5000 dilution.

6, a kind of protein suspension chip method of detection by quantitative bacillus anthracis spore is characterized in that, this method comprises the following steps:

(1) positive detection sample of Jia Ruing or standard items are through the gradient doubling dilution, and described gradient doubling dilution is 10 times;

(2) series of diluted samples is detected in suspension chip system read corresponding fluorescent value (MFI);

(3) dose-response curve of the corresponding MFI value of making sample concentration;

(4) with analysis software match dose-response curve and equation;

(5) the unknown concentration sample can be judged anthrax spore concentration in the unknown sample according to dose-response curve and equation.

7, method as claimed in claim 6 is characterized in that, being used to wrap by the capture antibody of microballoon is goat-anti Sterne antibody, adopt the anti-Sterne antibody of biotin labeled rabbit as detection antibody, and detection architecture is formed in its combination.

8, method as claimed in claim 6 is characterized in that, described bag is 10 μ g/1.25 * 10 by the capture antibody goat-anti Sterne antibody consumption of microballoon ⁶Individual coding microball or 20-40ng/2500-5000 microballoon/test.

9, method as claimed in claim 6 is characterized in that, the biotin of the anti-Sterne antibody of marker detection antibody rabbit in the described method is the biotin of carboxyl activity.

10, method as claimed in claim 6 is characterized in that, the anti-Sterne antibody of the biotinylated detection antibody of 2mg/mL Bio-rabbit detects as detecting antibody with the 1:50-1:5000 dilution.

11, a kind of protein suspending chip that detects bacillus anthracis spore, it is characterized in that: comprising: coding microball, bag is detected antibody, streptavidin-phycoerythrin and described buffer solution by the capture antibody of the described anthrax spore of microballoon, biotin labeled anthrax spore.

12, the described protein suspending chip of claim 11 is characterized in that: being used to wrap by the capture antibody of microballoon is goat-anti Sterne antibody, adopt the anti-Sterne antibody of biotin labeled rabbit as detection antibody, and detection architecture is formed in its combination.

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