CN101545905A - Methods for preparing, quantifying and detecting protein suspension chip of ricin - Google Patents
Methods for preparing, quantifying and detecting protein suspension chip of ricin Download PDFInfo
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- CN101545905A CN101545905A CN200910080254A CN200910080254A CN101545905A CN 101545905 A CN101545905 A CN 101545905A CN 200910080254 A CN200910080254 A CN 200910080254A CN 200910080254 A CN200910080254 A CN 200910080254A CN 101545905 A CN101545905 A CN 101545905A
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Abstract
The invention relates to a protein suspension chip of ricin and methods for preparing, quantifying and detecting the same. The detecting method comprises the following steps: adding a working solution and a sample to be tested into a hole; adding a detection antibody; adding SA-PE; adding a detection buffer solution and mixing the mixture evenly; and reading numerical values, analyzing data and judging the detection result. The quantifying method comprises the following steps: adding a positive test sample or a standard product for dilution through gradient multiple proportions, detecting a series of diluted samples in a suspension chip system, and reading a corresponding fluorescence value (MFI); making a dosage-reaction curve; and fitting the dosage-reaction curve and an equation. The suspension chip comprises a coded microsphere, a ricin capturing antibody coated on the microsphere, a biotin-marked ricin detection antibody, a streptavidin-phycoerythrin and the buffer solution. The methods have the advantages of good detectability, high sensitivity, strong specificity and wide dynamic range.
Description
Technical field
The present invention relates to the protein suspending chip preparation and the quantitative detecting method of a kind of ricin (WA) (ricin).
Background technology
Ricin (WA) (ricin toxin is hereinafter to be referred as ricin) is a kind of highly toxic glycoprotein that contains in the castor bean, and by the heterodimer that A, B polypeptied chain are formed by disulfide bond, the A chain is the effect chain, has RNA N-glycosidase activity, can make the ribosomes inactivation; The B chain is a marriage chain, contains two galactose binding sites, and mediation A chain enters performance toxic action in the cell.Ricin (WA) all can cause the people through respiratory tract suction, alimentary canal absorption and intramuscular injection and poison.People's per os lethal quantity 0.15-0.2g, intravenous injection lethal quantity 20mg, mouse peritoneal LD50 are about 3.0 μ g/kg.Ricin (WA) toxicity is big, stable in properties, and it is wider to originate, and extraction cost is cheap.Along with a plurality of terroristic organizations and extremist development with use the message of ricin (WA) to enter medium in succession, this biotoxin fatal, that toxicity is extremely strong is classified as the bio-terrorism factor that most possibly is used as the attack of terrorism by states such as the U.S..Aspect the quick detection of ricin (WA), country such as the U.S., European Union attaches great importance to always, has set up for laboratory and the on-the-spot method of using: as enzyme linked immunosorbent assay (ELISA), chemical analysis technology, biosensor technology and colloidal gold immunochromatographimethod method etc.; ELISA and colloidal gold immunochromatographimethod method and the supporting diagnostic reagent thereof that detects ricin (WA) also set up in China Taiwan.The fast quantification reagent of setting up ricin (WA) has important practical significance.Simultaneously, the present invention selects ricin (WA) to set up detection model as the representative of phytotoxin.
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 for example ricin (WA) of phytotoxin, 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 protein suspending chip that the purpose of this invention is to provide a kind of ricin (WA) (ricin) also provides the quantitative detecting method based on the ricin (WA) of this chip simultaneously.
Technical scheme of the present invention is specific as follows:
A kind of detection ricin (WA) protein suspending chip, this suspending chip comprises: coding microball (for example No. 027), bag is detected antibody (for example ricin (WA) monoclonal antibody RAC), streptavidin-phycoerythrin (SA-PE) and relevant buffer solution by the capture antibody of the ricin (WA) of microballoon (for example rabbit antiricin A antibody), biotin labeled ricin (WA).
A kind of protein suspending chip detection method of ricin (WA), 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 ricin (WA), this method comprises the following steps: that positive detection sample that (1) adds or standard items are through 4 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 ricin (WA), 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
Ricin (WA) 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=0.9934).Simultaneously, it is highly sensitive more than 4 times that suspension chip method sensitivity (29.1ng/mL) is higher than ELISA method (119ng/mL); And suspension chip method dynamic detection range (29.1-100852.1ng/mL) is higher than 2 orders of magnitude of ELISA method dynamic detection range (119-58500ng/mL).
4, the specificity of method
The present invention is a capture antibody with rabbit antiricin A antibody (RTA2-G) respectively by selecting for use the ricin (WA) toxin for detecting target antigen, detects antibody with biotinylated ricin (WA) monoclonal antibody Bio-RAC.This development test proves, this method has good specificity under the interference antigen existence conditions such as botulinum toxin (BONT) Recombinant HIV P24 antigen, bovine serum albumin(BSA) (BSA), casein, tryptone, avian influenza virus HA albumen, avian influenza virus NH albumen 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 ricin (WA).
Description of drawings:
Fig. 1: ricin (WA) detects with No. 027 microballoon bag antibody rabbit antiricin IgG detection synoptic diagram that is hunted down;
Fig. 2: protein suspension chip method detects ricin (WA) dose-response canonical plotting;
Fig. 3: the ELISA method detects the dose-response canonical plotting of ricin (WA);
Fig. 4: protein suspension chip method and ELISA method detect the correlativity of ricin (WA).
Embodiment
Protein suspending chip preparation method, detection and the quantivative approach of the ricin (WA) 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 be in 250mL, pH6.2.
(6) the microballoon bag is cushioned liquid 0.05M MES, pH5.0:2.44g MES, and 5N NaOH 0.15mL, constant volume is in 250mL.
(7) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, 0.02% TWEEN, 0.05% azide, pH7.4.
(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. antigen and antibody
Ricin (WA) antigen used herein is natural purification, obtains through protein chromatographic system (Sapherose 4B, Protein G post) purifying.
Capture antibody used herein is a rabbit antiricin A antibody, adopts caprylic acid-saturated ammonium sulfate method of purification to extract by this laboratory and obtains; Detecting antibody is biotinylation ricin (WA) monoclonal antibody RCA, available from U.S. Sigma company.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
The present invention with ricin (WA) as the target analysis matter sample.Disturbed specimen or the sample of testing as the method specificity are other toxin or the antigen protein of target detection beyond the region of objective existence, comprise BONT, Recombinant HIV P24 antigen, BSA, casein, tryptone, avian influenza virus HA albumen, avian influenza virus NH albumen etc.Above-mentioned sample to be analyzed all is dissolved in the sample diluting liquid liquid 4 ℃ of preservations.The storing solution concentration of ricin (WA) is 1mg/mL.In the comparative experiments, same sample is used for the detection of ELISA and suspending chip.
Is variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with ricin (WA) 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, 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 ricin (WA) 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 ricin (WA) virus
1, the capture antibody bag microballoon that is encoded
No. 027 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 ricin (WA), promptly utilizes rabbit antiricin A 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 the anti-castor-oil plant A of capture antibody rabbit 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; 1/400mm
2) under simple microscope, count.According to formula (each big lattice number * 10
4* extension rate * volume (mL)) calculates microballoon quantity.
2, detect the antibody labeling biotin
The mark of A, biotin
Preparing concentration respectively is the antibody-solutions to be marked of 10mM biotin (Biotin) solution and 2mg/mL, the biotin that has calculated volume is joined in the antibody-solutions to be marked, jolt 30 minutes in room temperature (or 2 hours) on ice, packing after the post desalination excessively ,-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:
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 H2SO4 cessation reaction at last.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. 027 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. 027 microballoon of rabbit antiricin A 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 ricin (WA) 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 1:50 dilution, and testing result shows that biotinylation detects the detection effect that antibody Bio-RCA 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 ricin (WA) 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 ricin (WA) testing result positive if testing result is higher than LOD; Corresponding fluorescence intensity level then is judged to be ricin (WA) testing result feminine gender if testing result is lower than LOD.
Embodiment 4, suspending chip are to the specific detection of ricin (WA) antigen
The using suspending chip detection method detects BONT, Recombinant HIV P24 antigen, BSA, casein, tryptone, avian influenza virus HA albumen, avian influenza virus NH albumen etc. respectively, according to testing result criterion among the embodiment 3, only ricin (WA) 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 ricin (WA) antigen protein method for detecting suspension chip and other test antigen that the present invention sets up.
The foundation of embodiment 5, suspending chip detection by quantitative model
1, ricin (WA) antigen typical curve specimen preparation
With sample diluting liquid the ricin (WA) standard items are become series concentration sample with 4 times of multiple proportions gradient dilutions to 3.56ng/mL by 234 μ g/mL.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 (ng/mL) of antigen, 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 ricin (WA) dose-response typical curve equation:
FI＝22.9039+(10162.2-22.9039)/[1+(Conc/23169.6)
-1.70809]
0.558067
3, suspending chip detects the sensitivity and the dynamic range of ricin (WA) antigen
According to minimum detectability definition and dose-response typical curve equation, the present invention is that the sensitivity of protein suspension chip method detection ricin (WA) antigen is: and 29.1ng/mL (Cutoff=40.28, Blank=26.0, SD=4.76).
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 ricin (WA) concentration, its corresponding MFI value also increases progressively thereupon, when ricin (WA) concentration surpasses 100852.1ng/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 present invention is that the dynamic range of suspending chip detection by quantitative ricin (WA) antigen is 29.1～100852.1ng/mL.
Embodiment 6, protein suspension chip method and ELISA method detect the comparison of ricin (WA)
1, double-antibody sandwich 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 ricin (WA) 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 ricin (WA) antigen, 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 29.1ng/mL, linear detection range 29.1～100852.1ng/mL; ELISA method (as shown in Figure 3) sensitivity is 119ng/mL, linear detection range 119-58500ng/mL.Can reach a conclusion: detect identical ricin (WA) antigen samples, protein suspension chip method has higher detection sensitivity than ELISA method, and is promptly high 4 times; And has wideer dynamic detection range, promptly high 2 orders of magnitude.
In addition, two kinds of method testing results are compared (as shown in Figure 4) can judge that protein suspension chip method and ELISA method have good correlativity in the 119-58500ng/mL scope, related coefficient is 0.9934.
The detection of embodiment 7, artificial contamination's " white powder " sample
1, the preparation of artificial contamination's " white powder " sample
The ricin (WA) 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 ricin (WA) pollution fast, accurately and efficiently.
Claims (12)
1, a kind of method that adopts protein suspending chip to detect ricin (WA) 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 antiricin A antibody, adopt biotin labeled antiricin A 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 rabbit antiricin A 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 marker detection antibody ricin (WA) monoclonal antibody RCA in the described method is the biotin of carboxyl activity.
5, the method for claim 1 is characterized in that, the biotinylated detection antibody of 2mg/mL Bio-RCA detects as detecting antibody with 1:50-1:1000 dilution.
6, a kind of protein suspension chip method of detection by quantitative ricin (WA) 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 4 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 ricin (WA) 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 rabbit antiricin A antibody, adopt biotin labeled ricin (WA) monoclonal antibody RCA 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 rabbit antiricin A 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 marker detection antibody ricin (WA) monoclonal antibody RCA in the described method is the biotin of carboxyl activity.
10, method as claimed in claim 6 is characterized in that, the biotinylated detection antibody of 2mg/mL Bio-RCA detects as detecting antibody with 1:50-1:1000 dilution.
11, a kind of protein suspending chip that detects ricin (WA) is characterized in that: comprising: coding microball, bag is detected antibody, streptavidin-phycoerythrin and described buffer solution by the capture antibody of the ricin (WA) of microballoon, biotin labeled ricin (WA).
12, the described protein suspending chip of claim 11 is characterized in that: being used to wrap by the capture antibody of microballoon is antiricin A antibody, adopt biotin labeled antiricin antibody as detection antibody, and detection architecture is formed in its combination.
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| CN200910080254A CN101545905A (en) | 2009-03-17 | 2009-03-17 | Methods for preparing, quantifying and detecting protein suspension chip of ricin |
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| CN200910080254A CN101545905A (en) | 2009-03-17 | 2009-03-17 | Methods for preparing, quantifying and detecting protein suspension chip of ricin |
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| CN102288769A (en) * | 2011-05-10 | 2011-12-21 | 中国检验检疫科学研究院 | Liquid-phase chip for testing Bt cry1 Ac protein and application of same |
| CN102411056A (en) * | 2011-10-11 | 2012-04-11 | 山东莱博生物科技有限公司 | Kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101936988A (en) * | 2010-07-23 | 2011-01-05 | 中国检验检疫科学研究院 | Protein suspension chip for detecting west nile antibody in serum sample and preparation method and using method thereof |
| CN102288769A (en) * | 2011-05-10 | 2011-12-21 | 中国检验检疫科学研究院 | Liquid-phase chip for testing Bt cry1 Ac protein and application of same |
| CN102411056A (en) * | 2011-10-11 | 2012-04-11 | 山东莱博生物科技有限公司 | Kit for quantitatively detecting GFAP concentration in human serum by polystyrene microsphere |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
| WO2020147735A1 (en) * | 2019-01-17 | 2020-07-23 | 北京市疾病预防控制中心 | Gold/quantum dot nanoprobe for detecting active ricin in complex matrix and use thereof |
| US11391711B2 (en) | 2019-01-17 | 2022-07-19 | Beijing Center For Disease Prevention And Control | Gold/quantum dot nanoprobe for detecting active ricin in complex matrix and application thereof |
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Application publication date: 20090930 |