CN101498720A - Protein suspending chip for quantitative detection of staphylococcal enterotoxin B and method for producing the same - Google Patents
Protein suspending chip for quantitative detection of staphylococcal enterotoxin B and method for producing the same Download PDFInfo
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- CN101498720A CN101498720A CNA2009100788184A CN200910078818A CN101498720A CN 101498720 A CN101498720 A CN 101498720A CN A2009100788184 A CNA2009100788184 A CN A2009100788184A CN 200910078818 A CN200910078818 A CN 200910078818A CN 101498720 A CN101498720 A CN 101498720A
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Abstract
The invention aims to provide a protein suspension chip capable of quantificationally detecting bacterial toxin and the preparation method thereof, and particularly relates to a protein suspension chip which is suitable for quantificationally detecting and analyzing staphylococcal enterotoxin. The method has high sensitivity and high particularity, good detection capacity and wide dynamic range and builds a novel detection modularity platform.
Description
Technical field
The present invention relates to protein suspending chip of a kind of detection by quantitative SEB and preparation method thereof.
Background technology
(staphylococcal enterotoxin B SEB) is the main paathogenic factor that causes food poisoning to Staphylococcus aureus enterotoxin B.The food poisoning morbidity is more anxious, outbreak in toxic on the feed usually vegetable food 1-6 hour, average 2-3 hour.
SEB Routine Test Lab diagnostic method has: 1) immunodiffusion: Oudin uni-directional diffusion technology can be used for the accurate quantitative measurement of staphylococcal enterotoxin, easily repeats, and not influenced by temperature, antiserum dilutability and action time etc.The susceptibility that detects various staphylococcal enterotoxin is about 10-20 μ g/mL.2) radioimmunology (RIA): detect enterotoxin A and B in the food, than responsive 100 times of SRID.3) reversed passive hemagglutination test (RPHA): can be with the toxin somatotype, and higher specificity and susceptibility are arranged.When measuring staphylococcal enterotoxin with the RPHA method of improvement, the concentration limit of purifying staphylococcal enterotoxin is 1-5ng/mL in the food.This law advantage is easy, quick, and need not concentrate and can detect low concentration toxin in the food.4) passive hemagglutination inhibition test (RPIA): with glutaraldehyde is bond, makes the passive hemagglutination inhibition test with the freeze-drying sensitized erythrocyte and measures SEB.Can measure the 0.01ng staphylococcal enterotoxin, but impure as the food leachate, often can disturb check result.The detection research of SEB is all significant with wartime at ordinary times.The present invention selects SEB to set up detection model as the representative of staphylococcal enterotoxin.
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.Report the earliest is that Horan in 1977 is used for immunology detection with this technology, and so far, 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.
This research selects Staphylococcus aureus enterotoxin B as the pathogen and the biological factor that threatens, preparation corresponding proteins matter suspending chip and being used for to real life field as milk powder, flour, starch, the fast detecting in the powdered sample such as treasure really, analyze the applicable cases of suspension chip method in the white powder analog sample detects, comprise susceptibility, specificity, stability and at different samples, have the applicability that detection method is descended in assorted bacterium interference.
Summary of the invention
The present invention relates to a kind of detection by quantitative staphylococcal enterotoxin, particularly protein suspension chip of Staphylococcus aureus enterotoxin B and preparation method thereof also relates to application and the detection method of protein suspending chip of the present invention in quantitatively, fast, directly detecting staphylococcal enterotoxin, especially Staphylococcus aureus enterotoxin B.
The inventor is by a large amount of and deep research, initiative develops that staphylococcal enterotoxin of the present invention detects protein suspending chip and detection method has the following advantages: the protein suspending chip of preparation can quicker, the described pathogen of detection by quantitative, high specificity; The protein suspending chip detection method is than ELISA method detection sensitivity height, and dynamic range is wideer; For the detection by quantitative of described pathogen is set up technology model, for example ricin (WA) is applied to other plant toxin etc. but also can further encourage described protein suspension chip.
The invention provides a kind of protein suspending chip of detection by quantitative staphylococcal enterotoxin, this suspending chip is made of: coding microball coated antibody, biotinylated antibody, streptavidin-phycoerythrin (SA-PE) and buffer solution, this microballoon specific antibody bag quilt of antistaphylohemolysin enterotoxin B (SEB), biotin labeled antibody is the SEB specific antibody.
The present invention also provides a kind of method for preparing described protein suspending chip, and this method comprises the following steps: (1) with the capture antibody bag microballoon that is encoded, and (2) biotin labeling detects antibody, (3) sample detection method.
The present invention also provides a kind of method of detection by quantitative staphylococcal enterotoxin, this method comprises the following steps: (1) usefulness antistaphylohemolysin enterotoxin antibody sandwich coding microball, (2) biotin labeling detects antistaphylohemolysin enterotoxin antibody, (3) sample detection method.
In the performance history of chip and method, the present invention has carried out a large amount of and complicated groping and studying to described protein suspending chip and preparation method thereof and detection method condition, and has obtained the improvement of following many aspects:
1, the improvement of antibody sandwich amount
The package amount of antibody need be optimized to detect effect, and the package amount of each antibody is from the 9-12 μ g beginning in this research, and the gradient test with 2 times, 4 times is chosen in 10-12 μ g and gets final product.
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
Immunological detection method---ELISA compares suspension chip method of the present invention with classical, and the result has good anastomose property, but suspension chip method is more highly sensitive than ELISA, and dynamic detection range is wideer.The highly sensitive classical standard in immunological method of suspension chip method-ELISA method also is confirmed from the testing result to multiple sample.
Prepare in the protein suspending chip in the present invention, antigen can be selected from natural purification, through the SEB of protein chromatographic system (Sapherose 4B, protein G post) purifying.Capture antibody is the SEB monoclonal antibody, also can be the anti-SEB of rabbit.Detecting antibody is the anti-SEB of Bio-rabbit, also can be the Bio-SEB monoclonal antibody.By detecting the contrast test of SEB, suspending chip of the present invention and ELISA method have good consistance (R
2Be 0.9785), be 60ng/mL but ELISA detects the minimal detectable concentration of SEB, the range of linearity is 100-800ng/mL.Suspending chip of the present invention in detection than ELISA method highly sensitive 300 times, dynamic range is wideer.
Warp and the comparative study of corresponding ELISE method, with preliminary assessment and examinations such as artificial contamination's sample detection, laboratory standard test, blind sample tests, many pathogen of suspending chip quantitative detecting method that this research is set up has higher susceptibility and wideer dynamic detection range than ELISA method, realize the detection by quantitative of pathogen, the detection of target analytes is not subjected to the influence of background interference such as other pathogen existence, can be used for the detection of environment such as " white powders " and food samples, the sensitivity of detection is subjected to the influence of different sample substrates with dynamic range and changes to some extent.The foundation of method for detecting suspension chip is for the fast detecting of ricin (WA), and the detection of the bio-terrorism factor in the suspicious environmental sample is had very important significance.
Detection by quantitative when we have developed the coding microball suspending chip and can realize ricin (WA).Investigated method sensitivity, specificity, multiple detectability, detect the applicability of powdered sample etc., and compare with the ELISA method, the sensitivity of suspension chip method is higher than corresponding ELISA method respectively.The test result of artificial contamination's sample and blind sample has also been proved the practicality of coding microball suspending chip quantitative detecting method.
Many pathogen of suspending chip detection by quantitative model that this research is set up reaches accurate, quick, easy, multi-functional, many index, high flux, standardized requirement and lays a good foundation for further solving detection technique.
Description of drawings:
Fig. 1: with No. 43 microballoon bags by SEB antibody test synoptic diagram;
Fig. 2: the coding microball suspending chip detects the canonical plotting of SEB;
Fig. 3: the ELISA method detects the SEB canonical plotting;
Fig. 4: microsphere chip and ELISA method detect the anastomose property of SEB;
Fig. 5: SEB dose-response performance graph in the powdered milk sample.
Embodiment
Protein suspending chip of the detection by quantitative staphylococcal enterotoxin that the present invention relates to and preparation method thereof will the invention will be further described with following embodiment, but the present invention is subjected to the restriction of this embodiment never in any form.
One, material
The present invention adopts following damping fluid to prepare suspending chip and carries out the detection of object:
(1) 0.03M PB damping fluid (pH7.2): 2.83g Na
2HPO
4, 1.36g KH
2PO
4Be settled to 1L.
(2) PB:0.01M phosphate buffer, pH7.2.Form with the dilution of 0.03M PB damping fluid.
(3)PBS(pH7.4)：NaCl137mmol/L；KCl2.7mmol/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 20min, 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 NaOH1.5mL, constant volume be in 250mL, pH6.2.
(6) the microballoon bag is cushioned liquid 0.05M MES, pH5.0:2.44g MES, and 5N NaOH0.15mL, 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.
Two, the preparation of testing sample
1, the preparation of sample
The target detection matter sample is SEB (SEB).Disturbed specimen or the sample of testing as the method specificity are other bacterium or other protein of target detection beyond the region of objective existence, comprise BONT, Recombinant HIV P24 antigen, BSA, casein, tryptone, avian influenza virus HA albumen, NH albumen etc.With above-mentioned sample to be analyzed all be dissolved in sample diluting liquid PB (0.01M, pH7.2) in the solution, 4 ℃ of preservations.The storing solution concentration of SEB is 1mg/mL.Toxin and recombinant protein quality sample are used preceding dilution facing.The concentration range of SEB is 10pg/mL-5 μ g/mL.In the comparative experiments, same sample is used for the detection of ELISA and suspending chip.
Bacterium to be analyzed is diluted to 10 times of different gradients with PB, staphylococcal enterotoxin is diluted to 4 times of different gradients with PB, 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 preparation of sample is added in simulation
Respectively powder such as 0.5g milk powder, cornstarch, wheat flour, instant fruit treasure are joined in the 5mL sample diluting liquid (PB damping fluid), with anthrax spore, plague bacillus, SARS-CoVN albumen, ricin (WA) and the SEB of variable concentrations wherein one or more, be incorporated in the powdered sample, through the abundant mixing of VOTEX, leave standstill more than the 2h, target analytes and simulation white powder 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 (1000rpm, 2min) after, supernatant carries out the detection of suspension chip method as sample to be checked.
Three, antigen-antibody
Natural purification, through SEB, the anti-SEB of Bio-rabbit of protein chromatographic system (Sapherose 4B, Protein G post) purifying through caprylic acid-saturated ammonium sulfate method of purification antibody purification IgG.
The anti-SEB of rabbit is through caprylic acid-saturated ammonium sulfate method of purification purifying.The choose any one kind of them antibody (SEB antibody) of microballoon (as 043) mark capturing SEB of coding.
The activation of A, coding microball
Get 100 μ L coding microballs in the 1.5mL centrifuge tube, 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.Add the microballoon activation damping fluid of 100 μ L, then elder generation adds the EDC (50mg/mL) of the fresh configuration of 10 μ L, and then adds the Sulfo-NHS (50mg/mL) of the fresh configuration of 10 μ L, jolts 20min 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 corresponding antibody 5-50 μ g joins activation, be settled to 500 μ L with PBS (pH7.4), room temperature jolts 2 hours.14000g is centrifugal, careful sucking-off and abandoning supernatant.PBS (pH7.4) with 500 μ L washes once, and 14000g is centrifugal, careful sucking-off and abandoning supernatant.Add the sealing damping fluid suspended coding microballoon of 250 μ L, jolt 30min 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.
The biotin labeling of embodiment 2, detection antibody
Antibody to be marked comprises the anti-SEB antibody of rabbit, goat-anti SEB antibody, SEB monoclonal antibody, at the detection antibody of every kind of analyte mark biotin have at least two kinds alternative.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 30min (or 2 hours) on ice in room temperature, packing after the post desalination excessively, and-20 ℃ are frozen standby.
The detection of embodiment 3, suspending chip
Adopt double-antibody sandwich immunology detection pattern, 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, and the washing lotion washing is also used the vacuum pump suction filtration;
2) add 50 μ L test sample, the room temperature lucifuge jolts 30min behind the mixing, washing lotion 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 30min behind the mixing, washing lotion washing and vacuum pump suction filtration;
4) SA-PE of adding 50 μ L, the room temperature lucifuge jolts 10min 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 VOTEX;
6) read FMI numerical value and analyze data with suspension chip system.
Above-mentioned testing process can be finished the detection of object in 96 samples at 30 minutes.
The suspending chip detection method of embodiment 4, SEB
The suspending chip detection system of optimizing can successfully be used for SEB and detect.Bag is all dropped in its correct surveyed area by No. 043 microballoon of SEB antibody, sees shown in Figure 1.The antibody of selecting after the method optimization is to being combined as: (the antibody sandwich amount is 10 μ g/1.25 * 10 to the microballoon bag by the monoclonal antibody of SEB
6Individual coding microball), detection antibody is 1: 50 the anti-SEB of Bio-rabbit (label concentration is 1mg/mL).
Embodiment 5, suspension chip method detect the typical curve of SEB
The typical curve of coding microball suspending chip detection SEB comprises the point (Fig. 2) of 6 orders of magnitude.Detect SEB since 4 times of serial dilutions of 7.5 μ g/mL to 10pg/mL.The detection dynamic range is 0.02-1653.4ng/mL, and LOD is 203pg/mL.
Embodiment 6, suspending chip of the present invention and ELISA method detect the comparison of SEB
SEB is diluted to the variable concentrations gradient with the diluted sample damping fluid,, compares suspending chip and the situation that meets of ELI SA method and the susceptibility of method with suspending chip and the parallel detection of ELISA method.The mean value of getting three ELISA detections is seen Fig. 3 as detected value drawing standard curve.The detected value of zero content detection sample is a blank value.The ratio that the LOD value of ELI SA method is got OD value and blank value is 2.1 o'clock a detectable concentration.Suspending chip and ELISA method testing result anastomose property are relatively seen Fig. 4, and solid line represents two kinds of methods respectively to the connecting line of same sample detected value among the figure, and dotted line is the Trendline of interpolation.The comparative result of two kinds of detection method LOD values sees Table 1.
1, ELISA method
In the ELISA method of setting up, every kind of combinations of pairs that detects used capture antibody of thing and detection antibody as far as possible with the used antibody of corresponding suspension chip method to consistent.After the different capture antibodies that detect thing dilute with the PB damping fluid respectively, the every hole of elisa plate package amount 200ng-1 μ g.Test sample corresponds respectively to suspending chip single-factor detection of analytes sample.
All the other testing processes are with first's corresponding contents.
2, the detection by quantitative of suspending chip
Dilute test sample in the SEB storing solution, detect the back and on the typical curve of correspondence, read its content; Sample detects for three times, gets detection signal mean value and calculates its standard deviation, the coefficient of variation, the repeatability that examination detects.
3, suspending chip and ELISA method detection SEB susceptibility and sensing range are relatively
Table 1: suspension chip method and ELISA method detect performance relatively
By inspection SEB, relatively the detection performance of suspending chip and ELISA method can be reached a conclusion, and detects similar sample, and in the finite concentration scope, suspension chip method and ELISA method have good correlativity.Suspending chip has higher detection sensitivity and wideer dynamic range than ELISA method, but to different detection things, superior performance degree difference specifically sees Table 1.Suspending chip of the present invention and ELISA method have good consistance (R
2Be 0.9785).The minimal detectable concentration that ELISA detects SEB is 60ng/mL, and the range of linearity is 100-800ng/mL.Highly sensitive 300 times than ELISA method of suspension chip methods, dynamic range is wideer.
The specificity of embodiment 7, suspension chip method
1, suspension chip method is to the detection of different bacterium and protein
Test by the suspension chip method specificity, the result shows, the MFI value of MFI values such as ricin (WA), SARS-CoV N albumen, colon bacillus, proteus, avian influenza virus HA albumen, avian influenza virus NH albumen, casein, BSA, BONT, tryptone and blank does not have the difference of conspicuousness, illustrate above bacterium, virus, toxin and other protein all not with generation cross reaction of target detection thing or nonspecific reaction.
2, suspending chip detection by quantitative SEB
Prepare the SEB sample of variable concentrations successively, after detecting with suspension chip method, apply mechanically the dose-response typical curve, draw the detection by quantitative concentration of SEB in the sample.Each sample detection 3 times is got the mean value (MFI) of its fluoroscopic examination signal, carries out the calculating of corresponding detectable concentration and standard deviation, the results are shown in Table 2.SEB five concentration levels from 0.46ng/mL to 468.75ng/mL all can quantitatively detect average coefficient of variation 6.788% in the sample.
Table 2: suspending chip detection by quantitative SEB result
Embodiment 8, suspending chip of the present invention detect the SEB in the sample
SEB detection by quantitative in the milk powder artificial contamination sample
SEB is quantitatively added in the powdered milk sample, with 4.25 * 10
6Cfu/mL beginning 4 times of gradients are carried out serial dilution, draw dose-response curve (Fig. 5).The dynamic detection range of SEB is about 1-62500ng/mL in the powdered milk sample.Carry out detection by quantitative to adding the SEB sample in the milk powder,, see Table 3 with the ratio calculating detection efficiency of detected value with the interpolation value.
Table 3: the simulation powdered milk sample adds the detection efficiency of SEB
Conclusion
Warp and the comparative study of corresponding ELISE method, with preliminary assessment and examinations such as artificial contamination's sample detection, laboratory standard test, blind sample tests, many pathogen of suspending chip quantitative detecting method that this research is set up has higher susceptibility and wideer dynamic detection range than ELISA method, realize the detection by quantitative of pathogen, the detection of target analytes is not subjected to the influence of background interference such as other pathogen existence, can be used for the detection of environment such as " white powders " and food samples, the sensitivity of detection is subjected to the influence of different sample substrates with dynamic range and changes to some extent.The sensitivity of suspension chip method detection SEB is respectively 2.2ng/mL in milk powder, and respectively good dose-response relationship is arranged in the 1-62500ng/mL concentration range.
The foundation of method for detecting suspension chip is for the fast detecting of SEB, and the detection of the bio-terrorism factor in the suspicious environmental sample is had very important significance.
Many pathogen of suspending chip detection by quantitative model that this research is set up reaches accurate, quick, easy, multi-functional, many index, high flux, standardized requirement and lays a good foundation for further solving detection technique.
Claims (10)
1, a kind of protein suspension chip of detection by quantitative SEB, it is characterized in that, described suspending chip is made of target polystyrene coding microball in the band fluorescein, this microballoon antibody sandwich that can catch the respective objects molecule, the described antibody of biotin labeling, the fluorescence phycoerythrin of Avidinization.
2, protein suspending chip as claimed in claim 1 is characterized in that, described bag be can be monoclonal antibody by the antibody of microballoon, how anti-ly also can be.
3, protein suspending chip as claimed in claim 1 is characterized in that, the required antibody sandwich amount of suspending chip is every hole 20-40ng/2500-5000 microballoon/test.
4, protein suspending chip as claimed in claim 2 is characterized in that, described biotinylated detection antibody be selected from the anti-SEB monoclonal antibody of biotinylated rabbit or many anti-in one or more.
5, protein suspending chip as claimed in claim 2, it is characterized in that, the SEB antibody of selected microballoon mark is capture antibody, is selected from biotin labeled SEB antibody as detecting antibody, and the suspending chip that is used for the double-antibody sandwich pattern detects staphylococcal enterotoxin.
6, a kind of preparation method of described protein suspending chip, it is characterized in that, this method comprise the following steps: (1) activation coding microball, (2) with the capture antibody bag be encoded microballoon and the counting, (3) detect antibody with biotin labeling, the washing of (4) cleaning fluid.
7, preparation method as claimed in claim 6, wherein said bag is selected from by the antibody of microballoon: one or more among the anti-SEB of rabbit, SEB monoclonal antibody, the goat-anti SEB.
8, preparation method as claimed in claim 6, wherein said biotinylated detection antibody and consumption thereof can be selected from: one or more among the anti-SEB of 6 μ g-40 μ g rabbits, SEB monoclonal antibody, the goat-anti SEB.
9, preparation method as claimed in claim 6, the biotin in the wherein said method is the biotin of carboxyl activity.
10, a kind of method of detection by quantitative staphylococcal enterotoxin, it is characterized in that, this method comprises the following steps: (1) usefulness antistaphylohemolysin enterotoxin antibody sandwich coding microball, (2) biotin labeling detects antistaphylohemolysin enterotoxin antibody, (3) sample detection, wherein said described bag is selected from by the antibody of microballoon: one or more among the anti-SEB of rabbit, SEB monoclonal antibody, the goat-anti SEB.
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|---|---|---|---|
| CNA2009100788184A CN101498720A (en) | 2009-03-04 | 2009-03-04 | Protein suspending chip for quantitative detection of staphylococcal enterotoxin B and method for producing the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102455356A (en) * | 2010-10-29 | 2012-05-16 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chip detection method for staphylococcal enterotoxin and fumonisin |
| CN103376319A (en) * | 2013-08-22 | 2013-10-30 | 南京师范大学 | High sensitivity fungaltoxin multi-detection method by using photonic crystal micro-sphere liquid-phase chip chemiluminiscence method |
| CN107402297A (en) * | 2017-06-04 | 2017-11-28 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Enterotoxin B detection kit and detection method based on immune bio-barcode |
| CN110563839A (en) * | 2019-08-19 | 2019-12-13 | 西北农林科技大学 | Staphylococcus aureus enterotoxin B nano antibody B1, application and kit |
-
2009
- 2009-03-04 CN CNA2009100788184A patent/CN101498720A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102455356A (en) * | 2010-10-29 | 2012-05-16 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chip detection method for staphylococcal enterotoxin and fumonisin |
| CN103376319A (en) * | 2013-08-22 | 2013-10-30 | 南京师范大学 | High sensitivity fungaltoxin multi-detection method by using photonic crystal micro-sphere liquid-phase chip chemiluminiscence method |
| CN103376319B (en) * | 2013-08-22 | 2015-08-05 | 南京师范大学 | The method of photon crystal micro-ball liquid-phase chip chemoluminescence method high sensitivity Multiple detection mycotoxin |
| CN107402297A (en) * | 2017-06-04 | 2017-11-28 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Enterotoxin B detection kit and detection method based on immune bio-barcode |
| CN110563839A (en) * | 2019-08-19 | 2019-12-13 | 西北农林科技大学 | Staphylococcus aureus enterotoxin B nano antibody B1, application and kit |
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