CN201984071U - Protein suspension array system for quickly screening important fever pathogeny antibodies - Google Patents
Protein suspension array system for quickly screening important fever pathogeny antibodies Download PDFInfo
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- CN201984071U CN201984071U CN2010206284752U CN201020628475U CN201984071U CN 201984071 U CN201984071 U CN 201984071U CN 2010206284752 U CN2010206284752 U CN 2010206284752U CN 201020628475 U CN201020628475 U CN 201020628475U CN 201984071 U CN201984071 U CN 201984071U
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Abstract
The utility model discloses a protein suspension array system for quickly screening important fever pathogeny antibodies, which comprises an array base body, a liquid injection system, a suspension array detector and a computer, wherein the array base body is filled with related buffer solution and to-be-detected antibodies; a plurality of coding microspheres are arranged in the related buffer solution; the coding microspheres are coated with corresponding capture antigens; a second antibody labeled by biotin and a fluorescent signal detection object are added in the array base body through the liquid injection system; the suspension array detector comprises a microcapillary detection passage and detection lasers; the coding microspheres passes through the microcapillary detection passage sequentially; the detection lasers are used for exciting and recognizing colors on the coding microspheres and colors of the to-be-detected objects on the coding microspheres; and the computer is connected with the suspension array detector, and is used for recording and analyzing the measurement result of the suspension array detector. The suspension array technique utilizing the microspheres reacting in the solution is adopted, and the laser detection technique is adopted, thereby improving the accuracy and the repeatability of the sample detection; and the system has the characteristics of simplicity and convenience in operation, good repeatability, and the like.
Description
Technical field
The utility model relates to the protein suspension chip system that detects several important heating pathogen detection of antibodies such as tuberculosis antibody, influenza antibodies, avian influenza antibody, plague antibodies, these several antibody of SARS antibody simultaneously.
Background technology
Global SARS in 2003 is popular, countries in Asia again and again outburst bird flu, dengue fever, the pernicious malaria in middle remote border, the encephalitis of rising one after another, influenza, enter the plague of active period, the West Nile fever of the U.S., the Ebola hemorrhagic fever in Africa etc. again, cause the extensive fear of the common people to infectious disease.The World Health Organization (WHO) pointed out that the whole world per hour had 1500 people to die from infectious disease in 2002 in the analysis report about infectious disease.And generate heat is one of the most common the most outstanding clinical symptoms of infectious disease.The present known material of heating that can cause has many kinds, but maximally related with heating is biogenic, as Much's bacillus, influenza virus, avian flu, plague bacillus, SARS, malicious bacillus anthracis, dengue virus, west nile virus, Chlamydia pneumoniae, mycoplasma infection etc.Since each the infected all be one flow fast, the unmanageable infection sources, implement fast detecting, differentiate in time, fast that cause of disease is that the infectious disease prevention and control are early found, early warning early, basis and the prerequisite early disposed.The Fast Detection Technique of cause of disease development at present is very fast, has developed different method for quick respectively at different biological markers.As detect the technology such as all kinds of PCR, in situ hybridization, DNA chip, NASBA of nucleic acid, and detect the ELISA, immunochromatography technique, protein chip technology of antigen such as protein, antibody materials etc., all have characteristics separately.Need rely on the gene magnification and the electrophoresis process of instrument at the Fast Detection Technique such as PCR of cause of disease nucleic acid, also rely on technical level of operators, be difficult to satisfy the on-the-spot easy rapid screening needs in port.General method mainly detects single cause of disease or infectious disease of planting, in the face of a fever patient, and examination when being difficult to realize multi-pathogenesis.Rapid screening is the key of EARLY RECOGNITION infectious disease, the Fast Detection Technique level of infectious disease not only concerns the people's life and health, quarantine level credibility and the reputation in the world that is also concerning China, affect the international image of the movable and country of the external personnel transfer of China, also affect and to control importing into of infectious disease and spread out of and avoid the foreign affairs dispute.Therefore, the raising of Fast Detection Technique is infectious disease prevention and control important link, it is very urgent to strengthen the Fast Detection Technique applied basic research, have very important significance: be the needs that improve China's health quarantine epidemic prevention overall work level and quality, safeguard the needs of China's public health security again.
Suspending chip (suspension array) also claim liquid-phase chip (liquid array, liquid chip), it is the biochip technology of new generation that the seventies in 20th century, U.S. Luminex company developed, the microsphere that utilizes the band coding is as carrier, flow cytometer is measured biomolecule such as nucleic acid, protein on a large scale as detection platform.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, be provided with twice laser in the sense channel, be red together, excite the color in the microballoon matrix, 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.
Compare with technology in the past, the biochip technology of current development can be realized the purpose of the disposable joint-detection of several heating pathogens, the suspending chip technology that produces still is a kind of of biochip at present, it is except can detecting multiple cause of disease, also have than ELISA and react liquid phase environment more fully, and have time saving and energy saving, high specificity, highly sensitive, and AT advantage.Therefore it has or not the monitoring of carrying zymad that important Research Significance is arranged to China inward and outward personnel's fast detecting to develop and develop a kind of important heating pathogen fast screening system.
The utility model content
The purpose of this utility model is to provide for people a kind of protein suspension chip system of important heating pathogen antibody rapid screening, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, relevant buffer solution and antibody to be measured are housed in the chip basal body, be provided with No. 027 in the relevant damping fluid, No. 031, No. 032, No. 033, No. 043, No. 044 coding microball, be coated with corresponding capture antigen on the various coding microballs respectively, adding liquid systems adds biotin labeled two anti-successively in chip basal body, fluorescence signal detects thing, the suspending chip detector comprises microcapillary sense channel and detection laser, reacted solution is drawn in the microcapillary sense channel, so that each coding microball is successively by the microcapillary sense channel, detection laser excites and the color of recognition coding microballoon and the fluorescence signal of last determinand thereof, computing machine links to each other with the suspending chip detector, and record, analyze the measurement result of suspending chip detector.
Further, described relevant buffer solution comprises that used microballoon dilution, the dilution described biotin labeled two of sample diluting liquid, the described coding microball of dilution that is used for described antibody dilution to be measured resists used antibody diluents, each reaction to wash the used microballoon cleaning fluid of coding microball, SA-PE dilution afterwards, detect damping fluid.
Further, the 38KD albumen of Much's bacillus and 16KD proteantigen wrap by described No. 027 coding microball as capture antigen, influenza NP proteantigen wraps by described No. 031 coding microball as capture antigen, LAM antigen wraps by described No. 033 coding microball as capture antigen, bird flu H5 antigen wraps by described No. 032 coding microball as capture antigen, F 1 antibody of plague bacterium wraps by described No. 043 coding microball as capture antigen, and SARS-CoV N proteantigen wraps by described No. 044 coding microball as capture antigen.
Further, described antibody to be measured is blood serum sample.
Further, described biotin labeled two anti-be preferably Biotin-goat-anti rabbit and/or Biotin-goat anti-human igg.
Further, described fluorescence signal detection thing is streptavidin-phycoerythrin.
Further, described chip basal body is 96 hole filter plate or microcentrifugal tubes.
Further, described detection laser comprises with exciting color in the described coding microball matrix, the sorting code number of recognition coding microballoon to determine the red laser of test item, and the color signal, tracer signal power that is used to excite and discern testing molecule is with the green laser of the content that detects described antibody to be measured.
Through a large amount of tests and deep research, protein suspension chip preparation and testing conditions thereof to synchronous examination mycobacterium tuberculosis antibody, Antibody of Influenza, avian influenza virus antibody, yersinia pestis antibody, SARS virus antibody have been done substantial improvement and innovation, and it has following advantage:
1, the improvement of antigen coated amount
The package amount of antigen is the key that successfully detects, the utility model is carried out substantive optimization to bag by the antigen coated amount of microballoon makes the detection effect of serum sample very good, and wrap very lowly by the required antigen coated amount of suspending chip, antigen coated amount of the present utility model is 0.01～250 μ g/1.25 * 10
6Individual microballoon, i.e. 0.004～1000ng/5000 microballoon/test, and the package amount of traditional immunology ELISA method is 1 μ g/ test.
2, biotin labeled improvement
Usually, labelled antibody need use excessive biotin.Theoretically, in testing process, excessive biotin is fallen by suction filtration during cleaning because of the unmarked antibody of going up is not connected by the antibody in conjunction with capture antibody on the microballoon, can not react with the SA-PE that adds subsequently, does not influence detection.But in the actual detected process, false positive appears in the phenomenon that the detection signal that fell in may be too high, so after advising biotin labeling antibody, removes unnecessary biotin as far as possible.IgG (molecular weight 150,000) 1mL solution with mark 2mg/mL is example, needs to add the about 27 μ L of 10mM biotin solution.
3, the sensitivity of Jian Ceing
The sensitivity that the protein suspension chip system of a kind of important heating pathogen antibody of the utility model rapid screening detects tuberculosis antibody is 1.5815ng/mL; Detecting influenza antibodies sensitivity is 968.8ng/mL; Detecting avian influenza virus antibody sensitivity is 69.4ng/mL; Detecting the sensitivity of yersinia pestis antibody is 6.29475ng/mL; Detecting the sensitivity of SARS virus antibody is 15.5265ng/mL.
4, the specificity of method
38KD albumen, 16KD albumen, the LAM antigen of the utility model by selecting Much's bacillus for use, influenza NP albumen, bird flu H5 antigen, F 1 antibody of plague bacterium, SARS-CoV N albumen are envelope antigen, being antibody to be measured with rabbit approach antibody, mouse-anti influenza NP IgG, mouse-anti H 5 N 1 avian influenza serum, the anti-plague IgG of rabbit, the anti-SARS IgG of rabbit respectively, is capture antibody with biotinylated goat anti-rabbit igg/sheep anti-mouse igg.This development test proves: the antigentic specificity that each is corresponding and corresponding antibodies with other antibody generation cross reaction, do not illustrate that this method has good specificity.
5, the detectability of sample
The utility model has been estimated the detectability of protein suspension chip method to human serum.By detection to human serum, tentative confirmation the practicality of this method mycobacterium tuberculosis antibody, Antibody of Influenza, avian influenza virus antibody, yersinia pestis antibody, SARS virus antibody in detecting human serum.
Description of drawings
Fig. 1 is the utility model structural representation;
Fig. 2 is multiple pathogenic autoantibody synoptic diagram for the protein suspension chip system detects;
Fig. 3 detects rabbit approach antibody dosage-reaction normal curve map for the protein suspension chip system;
Fig. 4 detects mouse-anti influenza NP dose-response canonical plotting for the protein suspension chip system;
Fig. 5 detects mouse-anti H 5 N 1 avian influenza antibody dosage-reaction normal curve map for the protein suspension chip system;
Fig. 6 detects the anti-pestis F 1 antibody dosage of rabbit-reaction normal curve map for the protein suspension chip system;
Fig. 7 detects the anti-SarsN13 antibody dosage of rabbit-reaction normal curve map for the protein suspension chip system.
Embodiment
Extremely shown in Figure 7 as Fig. 1, the protein suspension chip system of a kind of important heating pathogen antibody of the utility model rapid screening, comprise chip basal body 1, add liquid systems 2, suspending chip detector 3 and computing machine 4, wherein, chip basal body 1 is 96 hole filter plate or microcentrifugal tubes, relevant buffer solution and antibody to be measured are housed in it, be provided with some kinds of coding microballs in the relevant damping fluid, be coated with corresponding capture antigen on the various coding microballs, coding microball comprises coding microball No. 027, No. 031 coding microball, No. 032 coding microball, No. 033 coding microball, No. 043 coding microball, No. 044 coding microball, wherein, the 38KD albumen of Much's bacillus and 16KD proteantigen wrap by No. 27 carboxyl microballoons as capture antigen, influenza NP proteantigen wraps by No. 31 carboxyl microballoons as capture antigen, LAM antigen wraps by No. 33 coding microballs as capture antigen, bird flu H5 antigen wraps by No. 32 coding microballs as capture antigen, F 1 antibody of plague bacterium wraps by No. 43 coding microballs as capture antigen, and SARS-CoV N proteantigen wraps by No. 44 coding microballs as capture antigen.
The 38KD albumen of Much's bacillus and 16KD albumen, LAM antigen, influenza NP albumen, bird flu H5 antigen, F 1 antibody of plague bacterium, SARS-CoV N albumen are used for catching the antibody to be measured of blood serum sample to be detected.For example: if tuberculosis antibody is arranged in the sample, the 38KD albumen of the Much's bacillus on tuberculosis antibody and the coding microball and 16KD protein combination, add again have biotin labeled two anti-, the 38KD albumen and the 16KD albumen-tuberculosis antibody-two that form coding microball-Much's bacillus resist-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, form the 38KD albumen of coding microball-Much's bacillus and 16KD albumen-tuberculosis antibody-two anti--biotin is multiple-streptavidin-phycoerythrin compound, by the suspending chip detector detection of phycoerythrin fluorescence signal is reached detection to tuberculosis antibody in the blood serum sample, if there is not tuberculosis antibody in the blood serum sample, the coding microball that is coated with the 38KD albumen of Much's bacillus and 16KD albumen not can with add later biotin labeled two anti-, streptavidin-phycoerythrin combination, do not have when detecting by the suspending chip detecting instrument at last fluorescence signal or should signal very a little less than.
In like manner: if influenza antibodies is arranged in the sample, influenza NP protein combination on influenza antibodies and the coding microball, add again have biotin labeled two anti-, forming coding microball-influenza NP albumen-influenza antibodies-two resists-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-influenza NP albumen-influenza antibodies-two resists-biotin-streptavidin-phycoerythrin compound, by the suspending chip detector detection of phycoerythrin fluorescence signal is reached detection to influenza antibodies in the blood serum sample, if there is not Tula antibody in the blood serum sample, the coding microball that is coated with influenza NP albumen not can with add later biotin labeled two anti-, streptavidin-phycoerythrin combination, do not have when detecting by the suspending chip detecting instrument at last fluorescence signal or should signal very a little less than.
If avian influenza antibody is arranged in the sample, bird flu H5 protein combination on avian influenza antibody and the coding microball, add again have biotin labeled two anti-, forming coding microball-bird flu H5 albumen-avian influenza antibody-two resists-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-bird flu H5 albumen-avian influenza antibody-two resists-biotin-streptavidin-phycoerythrin compound, by the suspending chip detector detection of phycoerythrin fluorescence signal is reached detection to avian influenza antibody in the blood serum sample, if there is not avian influenza antibody in the blood serum sample, the coding microball that is coated with bird flu H5 albumen not can with add later biotin labeled two anti-, streptavidin-phycoerythrin combination, do not have when detecting by the suspending chip detecting instrument at last fluorescence signal or should signal very a little less than.
If plague antibodies is arranged in the sample, pestis F 1 protein combination on plague antibodies and the coding microball, add again have biotin labeled two anti-, forming coding microball-pestis F 1 albumen-plague antibodies-two resists-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-pestis F 1 albumen-plague antibodies-two resists-biotin-streptavidin-phycoerythrin compound, by the suspending chip detector detection of phycoerythrin fluorescence signal is reached detection to dengue antibody in the blood serum sample, if there is not plague antibodies in the blood serum sample, the coding microball that is coated with pestis F 1 albumen can not combine with the biotin labeled two anti--streptavidin-phycoerythrin that add later, do not have when detecting by the suspending chip detecting instrument at last fluorescence signal or should signal very a little less than.
If SARS antibody is arranged in the sample, SARS-CoV N protein combination on SARS-CoV N antibody and the coding microball, add again have biotin labeled two anti-, forming coding microball-SARS-CoV N albumen-SARS antibody-two resists-biotin composite, add streptavidin-phycoerythrin at last, streptavidin combines with biotin, forming coding microball-SARS-CoV N albumen-SARS antibody-two resists/SPA-biotin-streptavidin-phycoerythrin compound, reach SARS detection of antibodies in the blood serum sample by of the detection of suspending chip detector the phycoerythrin fluorescence signal, if there is not SARS antibody in the blood serum sample, the coding microball that is coated with SARS-CoV N albumen not can with add later biotin labeled two anti-, streptavidin-phycoerythrin combination, do not have when detecting by the suspending chip detecting instrument at last fluorescence signal or should signal very a little less than.
In the said chip system, coding microball is at the microballoon dilution, antibody to be measured is in sample diluting liquid, biotin labeled two resist in antibody diluent, fluorescence signal detects thing in the SA-PE dilution, the coding microball compound was in detecting damping fluid when the suspending chip detector detected, and per step, all with microballoon cleaning fluid washing microballoon, making did not have the material of combination to remove system in conjunction with afterwards.
The ultimate principle of suspending chip is to utilize the microballoon of polystyrene (polystyrene) made, coat the ruddiness and the infrared light colour former of different proportion, and produce 100 kinds of different proportion colors, color numbers as 100 kinds of uniquenesses, every about 5.6 μ m of microballoon size, different research purposes such as immunoassay, nucleic acids research, enzyme analysis, acceptor and part discriminance analysis etc. be can comply with, and specific antibodies, nucleic acid probe and various acceptor probe demarcated according to different research purposes.The microballoon of label probe and determinand react in 96 orifice plates.After the reaction, utilize machine automatically reactant liquor to be picked up and by a microcapillary sense channel, only allow a microballoon to pass through sense channel at every turn.Being provided with twice laser in the sense channel, is red together, excites the color in the microballoon matrix, and the sorting code number of identification microballoon is to determine test item; Be green together, excite the color of reporter molecules, the tracer signal power is to detect the content of determinand.When the probe of sample to be tested and specific microballoon was attached together, the light that twice laser is excited all can be detected.And, then only have the exciting light in the microballoon to be detected if do not contain this subject matter in the sample.Microballoon kind and the quantity that is excited by machine and computing machine automatic statistical analysis twice laser again, thereby several test target things are arranged therein in the judgement sample to be tested, learn to have or not cause of disease to be measured to exist in the test sample book, or have several simultaneously to tens of kinds of cause of diseases.The suspending chip technology is owing to utilize microballoon to react in solution, overcome sheet film chip and when big Molecular Detection, be subjected to the influence to reaction kinetics such as surface tension, steric effect, utilize laser measuring technology simultaneously, improve the accuracy and the repeatability of sample detection greatly, had characteristics such as easy and simple to handle, the good reproducibility that is better than sheet film chip.
The protein suspension chip system of a kind of important heating pathogen antibody of the utility model rapid screening be described further by following embodiment, but the utility model is subjected to the qualification of this embodiment never in any form.
One, material
1. the relevant damping fluid of protein suspension chip:
(1) PBS damping fluid (pH7.4): NaCl 137mmol/L; KCl 2.7mmol/L; Na
2HPO
410mmol/L; KH
2PO
42mmol/L.With 800mL dissolved in distilled water 8gNaCl, 0.2gKCl, 1.44g Na
2HPO
4With 0.24g KH
2PO
4PH value to 7.4 with the HCl regulator solution adds water to 1L.After the packing at 15psi (1.05kg/cm
2) high pressure steam 20 minutes, or filtration sterilization, be stored in room temperature.
(2) microballoon cleaning fluid: PBS (pH7.4), 0.05%TWEEN-20.
(3) microballoon activation damping fluid 100mM NaH
2PO
4: 3g NaH
2PO
4, 5N NaOH 1.5mL, constant volume are in 250mL, and pH 6.2.
(4) the microballoon bag is cushioned liquid 0.05M MES, pH 5.0:2.44g MES, and 5N NaOH0.15mL, constant volume is in 250mL.
(5) microballoon is preserved liquid PBS-TBN:PBS, 0.1%BSA, and 0.02%TWEEN, 0.05% azide, pH 7.4.
(6) microballoon confining liquid PBS-BN:PBS, 1%BSA, 0.05% azide, pH7.4.
(7) detect damping fluid: PBS, 1%BSA, pH7.4.
(8) antibody diluent PBS, pH7.4.。
(9) microballoon dilution: PBS, 1%BSA, pH7.4.
(10) sample diluting liquid PVX:PBS, 0.5%PVA, 0.8%PVP;
(11) SA-PE dilution: PBS (pH7.4), 1%BSA.
2. antigen and antibody
The employed antigen of the utility model is the 38KD albumen of Much's bacillus and 16KD albumen, LAM antigen, influenza NP albumen, F 1 antibody of plague bacterium, SARS-CoV N albumen can be available from institute of microbiology of thing medical scientific institute, and bird flu H5 albumen can be available from the Chinese Academy of Agricultural Sciences's Harbin veterinary institute.
The utility model uses detection antibody to draw IgG as the anti-soil of rabbit, can be available from China Inst. of Quarantine Inspection Sciences, rabbit anti-dengue 2 type antibody can be available from institute of microbiology of military medicine research institute, it is biotinylation goat anti-rabbit igg and biotinylation goat anti-human igg that detection two resists, and described goat anti-rabbit igg can be available from Beijing ancient cooking vessel state biotech company.
Two, the preparation of testing sample
1, the preparation of target analysis matter sample
Target analytes is that rabbit approach antibody, mouse-anti influenza NP IgG, mouse-anti H 5 N 1 avian influenza serum, the anti-plague IgG of rabbit, the anti-SARS IgG of rabbit are antibody to be measured, is capture antibody with biotinylated goat anti-rabbit igg/sheep anti-mouse igg.
Is variable concentrations sample with sample diluting liquid with 4 times of multiple proportions serial dilutions with antibody to be analyzed, to draw the typical curve of sample detection dose-response, wherein several sample concentrations are lower than the susceptibility of detection, and enriched sample should make the binding site of coding microball be in state of saturation.
2, the processing of human serum sample
The human serum sample with diluted sample dilution in 1: 10, after for example human serum sample 5 μ L add sample dilution 50 μ L mixings, is carried out the detection of suspension chip method as sample to be checked.
Embodiment 1: a kind of preparation of protein suspension chip of important heating pathogen antibody rapid screening
1. get 27,31,32,33 respectively, 43, No. 44 coding microballs, with vortex oscillation device vibration microballoon suspension, time 30s ultrasonic 30 seconds, mixes microballoon;
2. get 1.25 * 106 of above-mentioned each coding microballs respectively in the 1.5mL centrifuge tube, the centrifugal 4min of 10000g～14000g, careful sucking-off supernatant also discards;
3. add 100 μ L pearl ball lavation buffer solution (0.05%TWEEN-20) suspension microballoon shook 30 seconds for PBS, pH7.4, ultrasonic 30 seconds, the centrifugal 4min of 10000g～14000g, careful sucking-off supernatant also discards;
4. pearl ball activation damping fluid (the 3g NaH that adds 80 μ L
2PO
4, 5N NaOH40drops/250mL H
2O), with vortex oscillation device vibration microballoon suspension;
5. and then the EDC (50mg/mL) that adds the fresh configuration of 10 μ L adds the Sulfo-NHS (50mg/mL) of the fresh configuration of 10 μ L, shakes at a high speed after 30 seconds with the aluminium foil parcel, jolts 20min in room temperature;
6. the PBS (pH7.4) that adds 150 μ L shook after 10 seconds, the centrifugal 4min of 10000g～14000g, and careful sucking-off supernatant also discards;
7. repeat step once;
8. PBS (pH7.4) the suspended coding microballoon that adds 100 μ L, middling speed shook after 30 seconds, ultrasonic 15 seconds;
9. get antigen respectively: the 38KD albumen of Much's bacillus and 16KD albumen, influenza NP albumen, LAM antigen, bird flu H5 antigen, F 1 antibody of plague bacterium, SARS-CoV N albumen 1-10 μ g join in the coding microball after the activation, and be fixed molten to 500 μ L with PBS (pH7.4);
10. Aluminium Foil Package is rolled in room temperature and jolts 2h, the centrifugal 4min of 10000g～14000g, and careful sucking-off supernatant discards;
11. the PBS (pH7.4) with 500 μ L washes once, the centrifugal 4min of 10000g～14000g, and careful sucking-off supernatant discards;
12. add 250 μ L the sealing damping fluid (PBS (pH7.4), 1%BSA, 0.05%Azide) suspended coding microballoon, middling speed concussion 15 seconds with the aluminium foil parcel, jolts 30min in room temperature, the centrifugal 4min of 10000g～14000g, careful sucking-off supernatant discards;
13. add deposit damping fluid (PBS (pH7.4), 0.1%BSA, 0.02%TWEEN, 0.05% azide) the washing coding microball of 500 μ L, the centrifugal 6min of 10000g～16000g, careful sucking-off supernatant discards;
14. use the deposit damping fluid suspended coding microballoon of 150 μ L at last, promptly get 38KD albumen and 16KD albumen and No. 27 carboxyl microballoons formation couplets of Much's bacillus, influenza NP albumen and No. 31 carboxyl microballoons form couplet, LAM antigen and No. 33 carboxyl microballoons form couplet, bird flu H5 antigen and No. 32 carboxyl microballoons form couplet, F 1 antibody of plague bacterium and No. 43 carboxyl microballoons form couplet, and SARS-CoV N albumen and No. 44 carboxyl microballoons form couplet;
15. draw an amount of microballoon, after the dilution, under fluorescent microscope, count with blood counting chamber, converse the concentration of every kind of microballoon;
Be placed on 4 ℃ and keep in Dark Place 16. even chain store is got microballoon, the microballoon of general every kind of antigen couplet is preserved separately, during use, selects to mix according to test item.
Embodiment 2: the protein suspension chip of sample detects:
Adopt the immunology detection pattern of indirect method, total overall reaction is all carried out on 96 hole filter plates in the testing process.
1. every hole adds 150 μ L and detects the pre-wet hole of damping fluid, uses the vacuum pump suction filtration;
2. every hole adds the working solution that 50 μ L contain the corresponding encoded microballoon, washing lotion washing and with twice of vacuum pump suction filtration;
3. add the test sample of 50 μ L dilution, the room temperature lucifuge jolts 30min behind the mixing, washing lotion washing and suction filtration three times;
4. add the anti-with the biotinylation after the antibody diluent dilution two of 50 μ L debita spissitudos, the room temperature lucifuge jolts 30min behind the mixing, washing lotion 100 μ L washing 3 times, and vacuum pump suction filtration;
5. the SA-PE that adds 50 μ L, the room temperature lucifuge jolts 10min behind the mixing.Washing lotion 100 μ L washing 3 times, and vacuum pump suction filtration;
6. the detection damping fluid that adds 125 μ L makes bead resuspended evenly through vibration;
7. read FMI numerical value and analyze data with the Bio-Plex suspension chip system.
Embodiment 3: the protein suspension chip system is to the detection of the anti-TB IgG of variable concentrations rabbit
1. each antigen and the coupling of different coding microballoon, concrete operation method such as embodiment 1 obtain the not couplet of synantigen and coding microball;
2. get the 38KD albumen that is coated with the antigen Much's bacillus and No. 27 carboxyl microballoon couplets of 16KD albumen, No. 32 carboxyl microballoon couplets of bird flu H5 antigen, No. 43 carboxyl microballoon couplets of F 1 antibody of plague bacterium, No. 44 carboxyl microballoon couplets of SARS-CoV N albumen, each 3500, add in the same centrifuge tube, add the microballoon dilution to the testing solution of cumulative volume 50 μ L as multiple microsphere system;
3. the anti-TB IgG of rabbit is done 4 times of gradient dilutions as the sample that detects with sample diluting liquid, the concentration of the anti-TB IgG of rabbit is respectively: 0.147ng/ml, 0.588ng/ml, 2.354ng/ml, 9.418ng/ml, 37.672ng/ml, 150.687ng/ml, 602.75ng/ml, 2.411 μ g/ml;
4. concrete detecting operation step such as embodiment 2;
5. the MFI value that obtains of the anti-TB of each microballoon detection rabbit sees the following form 1, and the protein suspension chip system detects the typical curve of the anti-TB of rabbit:
FI＝2.32671+(5582.99-2.32671)/[1+(Conc/331.934)
-1.42723]
0.78545
Detection sensitivity is 1.5815ng/ml.
Table 1 protein suspension chip system detects the MFI value of the anti-TB of rabbit
Embodiment 4: the protein suspension chip system is to variable concentrations mouse-anti influenza NP detection of antibodies
1. each antigen and the coupling of different coding microballoon, concrete operation method such as embodiment 1; Obtain the not couplet of synantigen and coding microball;
2. get the 38KD albumen that is coated with the antigen Much's bacillus and No. 27 carboxyl microballoon couplets of 16KD albumen, No. 31 carboxyl microballoon couplets of influenza NP albumen, No. 43 carboxyl microballoon couplets of F 1 antibody of plague bacterium, No. 44 carboxyl microballoon couplets of SARS-CoV N albumen, each 3500, add in the same centrifuge tube, add the microballoon dilution to the testing solution of cumulative volume 50 μ L as multiple microsphere system;
3. mouse-anti influenza NP antibody is done 4 times of gradient dilutions as the sample that detects with sample diluting liquid, the concentration of mouse-anti influenza NP antibody is respectively: 3.052ng/ml, 12.207ng/ml, 48.828ng/ml, 195.312ng/ml, 781.25ng/ml, 3125ng/ml, 12500ng/ml, 50000ng/ml;
4. concrete detecting operation step such as embodiment 2;
5. each microballoon detects the MFI value that mouse-anti influenza NP antibody obtains and sees the following form 2, the typical curve of protein suspension chip system detection mouse-anti bird flu NP:
FI＝20.3844+(583.981-20.3844)/[1+(Conc/6113.06)
-2.38124]
0.349701
Sensitivity is 968.8ng/ml.
Table 2 protein suspension chip system detects the MFI value of mouse-anti bird flu NP
Embodiment 5: the protein suspension chip system is to variable concentrations mouse-anti H 5 N 1 avian influenza detection of antibodies
1. each antigen and the coupling of different coding microballoon, concrete operation method such as embodiment 1; Obtain the not couplet of synantigen and coding microball;
2. get the 38KD albumen that is coated with the antigen Much's bacillus and No. 27 carboxyl microballoon couplets of 16KD albumen, No. 32 carboxyl microballoon couplets of bird flu H5 antigen, No. 43 carboxyl microballoon couplets of F 1 antibody of plague bacterium, No. 44 carboxyl microballoon couplets of SARS-CoV N albumen, each 3500, add in the same centrifuge tube, add the microballoon dilution to the testing solution of cumulative volume 50 μ L as multiple microsphere system;
3. mouse-anti H 5 N 1 avian influenza antibody is done 4 times of gradient dilutions as the sample that detects with sample diluting liquid, the concentration of mouse-anti H 5 N 1 avian influenza antibody is respectively: 0.305ng/ml, 1.221ng/ml, 4.883ng/ml, 19.531ng/ml, 78.125ng/ml, 312.5ng/ml, 1250.0ng/ml, 5000.0ng/ml, 20000ng/ml;
4. concrete detecting operation step such as embodiment 2;
5. the MFI value of each microballoon detection mouse-anti H 5 N 1 avian influenza antibody sees the following form 3, and multiple system microballoon detects the typical curve of mouse-anti H 5 N 1 avian influenza:
FI＝11.2281+(1516.12-11.2281)/[1+(Conc/628.306)
-1.0296]
1.06526
Sensitivity is 69.4ng/ml.
Table 3 protein suspension chip system detects the MFI value of mouse-anti H 5 N 1 avian influenza
Embodiment 6: the protein suspension chip system is to the anti-pestis F 1 detection of antibodies of variable concentrations rabbit
1. each antigen and the coupling of different coding microballoon, concrete operation method such as embodiment 1; Obtain the not couplet of synantigen and coding microball;
2. get the 38KD albumen that is coated with the antigen Much's bacillus and No. 27 carboxyl microballoon couplets of 16KD albumen, No. 32 carboxyl microballoon couplets of bird flu H5 antigen, No. 43 carboxyl microballoon couplets of F 1 antibody of plague bacterium, No. 44 carboxyl microballoon couplets of SARS-CoV N albumen, each 3500, add in the same centrifuge tube, add the microballoon dilution to the testing solution of cumulative volume 50 μ L as multiple microsphere system;
With the anti-pestis F 1 antibody of rabbit do 4 times of gradient dilutions as the sample that detects with sample diluting liquid, the concentration of the anti-pestis F 1 antibody of rabbit is respectively: 0.305ng/ml, 1.221ng/ml, 4.883ng/ml, 19.531ng/ml, 78.125ng/ml, 312.5ng/ml, 1250.0ng/ml, 5000.0ng/ml, 20000.0ng/ml, 80000.0ng/ml;
4. concrete detecting operation step such as embodiment 2;
5. the MFI value of the anti-pestis F 1 antibody of each microballoon detection rabbit sees the following form 4, and the protein suspension chip system detects the typical curve of the anti-pestis F 1 antibody concentration of rabbit:
FI＝4.423+(4130.55-4.423)/(1+(Conc/1853.93)^-0.986615)
Sensitivity is 6.29475ng/ml.
Table 4 protein suspension chip system detects the MFI value of the anti-pestis F 1 antibody of rabbit
Embodiment 7: the protein suspension chip system is examined the anti-SarsN13 detection of antibodies of variable concentrations rabbit
1. each antigen and the coupling of different coding microballoon, concrete operation method such as embodiment 1; Obtain the not couplet of synantigen and coding microball;
2. get the 38KD albumen that is coated with the antigen Much's bacillus and No. 27 carboxyl microballoon couplets of 16KD albumen, No. 32 carboxyl microballoon couplets of bird flu H5 antigen, No. 43 carboxyl microballoon couplets of F 1 antibody of plague bacterium, No. 44 carboxyl microballoon couplets of SARS-CoV N albumen, each 3500, add in the same centrifuge tube, add the microballoon dilution to the testing solution of cumulative volume 50 μ L as multiple microsphere system;
With the anti-SarsN13 antibody of rabbit do 4 times of gradient dilutions as the sample that detects with sample diluting liquid, the concentration of the anti-SarsN13 antibody of rabbit is respectively: 0.034ng/ml, 0.137ng/ml, 0.549ng/ml, 2.197ng/ml, 8.789ng/ml, 35.156ng/ml, 140.625ng/ml, 562.5ng/ml, 2250.0ng/ml;
4. concrete detecting operation step such as embodiment 2;
5. the MFI value of the anti-SarsN13 antibody of each microballoon detection rabbit sees the following form 5, and the protein suspension chip system detects the typical curve of the anti-SarsN13 antibody concentration of rabbit:
FI＝8.9686+(2204.42-8.9686)/((1+(Conc/287.286)^-1.2352))^1.10117
Sensitivity is 15.5265ng/ml.
Table 5 protein suspension chip system detects the MFI value of the anti-SarsN13 of rabbit
Embodiment 8: the application of the protein suspension chip system of important heating pathogen antibody rapid screening of the present invention tuberculosis antibody in detecting human serum.
1. get tuberculosis LAM antigen and No. 33 coding microball couplings, concrete operation method such as embodiment 1; Obtain the couplet of LAM antigen and No. 33 coding microballs;
2. 35 parts of serum and 53 parts of normal health human serums that will be diagnosed as tuberculosis patient are done 1: 10 times of ladder dilution with sample diluting liquid respectively;
3. get 96 hole filter plates, add 150 μ L in every hole and detect the pre-wet hole of damping fluid, use the vacuum pump suction filtration;
4. every hole adds 50 μ L and contains 3500 working solutions that are coated with No. 33 coding microballs of LAM antigen, washing lotion washing and with twice of vacuum pump suction filtration;
5. add the test sample of 50 μ L dilution, the room temperature lucifuge jolts 30min behind the mixing, washing lotion washing and suction filtration three times;
6. add 50 μ L debita spissitudos with the biotinylation goat anti-human igg after the antibody diluent dilution, the room temperature lucifuge jolts 30min behind the mixing, washing lotion 100 μ L washing 3 times, and vacuum pump suction filtration;
7. the SA-PE that adds 50 μ L, the room temperature lucifuge jolts 10min behind the mixing.Washing lotion 100 μ L washing 3 times, and vacuum pump suction filtration;
8. the detection damping fluid that adds 125 μ L makes bead resuspended evenly through vibration;
9. detect with the Bio-Plex suspension chip system;
10. same serum sample is used for commercially available ELISA kit and detects.
Testing result sees Table 6,7, this shows that detection sensitivity by the tuberculosis antibody of lam Detection of antigen is 68.57%, specificity is 83.02%, accuracy is 77.27%; The detection sensitivity that the ELISA kit detects tuberculosis antibody in the serum is 62.86%, specificity is 94.34%, accuracy is 81.82%, the antigen coated microballoon of lam detects the commercially available ELISA method of remolding sensitivity highly sensitive of tuberculosis antibody with suspending chip, verification and measurement ratio is also higher.
The testing result of tuberculosis antibody in the table 6 lam Detection of antigen human serum sample
Table 7 EILSA kit detects the testing result of tuberculosis antibody in the human serum
Embodiment 9: the application of the protein suspension chip system of important heating pathogen antibody rapid screening of the present invention tuberculosis antibody, avian influenza antibody, plague antibodies, SARS antibody in detecting human serum.
1. each antigen and the coupling of different coding microballoon, concrete operation method such as embodiment 1; Obtain the not couplet of synantigen and coding microball;
2. get the 38KD albumen that is coated with the antigen Much's bacillus and No. 27 carboxyl microballoon couplets of 16KD albumen, No. 32 carboxyl microballoon couplets of bird flu H5 antigen, No. 43 carboxyl microballoon couplets of F 1 antibody of plague bacterium, No. 44 carboxyl microballoon couplets of SARS-CoV N albumen, each 3500, add in the same centrifuge tube, add the microballoon dilution to the testing solution of cumulative volume 50 μ L as multiple microsphere system;
3. 94 parts of human serums are done 1: 10 times of ladder dilution with sample diluting liquid respectively;
4. get 96 hole filter plates, add 150 μ L in every hole and detect the pre-wet hole of damping fluid, use the vacuum pump suction filtration;
5. every hole adds the working solution in the 50 μ L above-mentioned steps 2, washing lotion washing and with twice of vacuum pump suction filtration;
6. add the test sample of 50 μ L dilution, the room temperature lucifuge jolts 30min behind the mixing, washing lotion washing and suction filtration three times;
7. add 50 μ L debita spissitudos with the biotinylation goat anti-human igg after the antibody diluent dilution, the room temperature lucifuge jolts 30min behind the mixing, washing lotion 100 μ L washing 3 times, and vacuum pump suction filtration;
8. the SA-PE that adds 50 μ L, the room temperature lucifuge jolts 10min behind the mixing.Washing lotion 100 μ L washing 3 times, and vacuum pump suction filtration;
9. the detection damping fluid that adds 125 μ L makes bead resuspended evenly through vibration;
10. detect with the Bio-Plex suspension chip system.
The result judges: in the single test item standard deviation of the mean value of 94 people detection fluorescent value MFI and 94 people detection fluorescent value MFI of 3 times and as the cutoff value of this test item, if the MFI value＞cutoff value of detection is positive, on the contrary negative.Testing result sees the following form 8.
The multiple microsphere system of table 8 detects the human serum result
| Test item | Serum example number | Cutoff value (MFI) | Positive routine number | Positive rate |
| Tuberculosis antibody | 94 | 1322.94 | 18 | 20.0％ |
| Avian influenza antibody | 94 | 2286 | 7 | 8.0％ |
| Plague antibodies | 94 | 632.35 | 5 | 6.0％ |
| SARS antibody | 94 | 3123.355 | 8 | 9.0％ |
Claims (8)
1. the protein suspension chip system of an important heating pathogen antibody rapid screening, it is characterized in that, this chip system comprises: chip basal body, add liquid systems, suspending chip detector and computing machine, wherein, relevant buffer solution and antibody to be measured are housed in the chip basal body, be provided with No. 027 in the relevant damping fluid, No. 031, No. 032, No. 033, No. 043, No. 044 coding microball, be coated with corresponding capture antigen on the various coding microballs respectively, adding liquid systems adds biotin labeled two anti-successively in chip basal body, fluorescence signal detects thing, the suspending chip detector comprises microcapillary sense channel and detection laser, reacted solution is drawn in the microcapillary sense channel, so that each coding microball is successively by the microcapillary sense channel, detection laser excites and the color of recognition coding microballoon and the fluorescence signal of last determinand thereof, computing machine links to each other with the suspending chip detector, and record, analyze the measurement result of suspending chip detector.
2. protein suspension chip as claimed in claim 1 system, it is characterized in that described relevant buffer solution comprises that used microballoon dilution, the dilution described biotin labeled two of sample diluting liquid, the described coding microball of dilution that is used for described antibody dilution to be measured resists used antibody diluents, each reaction to wash the used microballoon cleaning fluid of coding microball, SA-PE dilution afterwards, detect damping fluid.
3. protein suspension chip as claimed in claim 2 system, it is characterized in that, the 38KD albumen of Much's bacillus and 16KD proteantigen wrap by described No. 027 coding microball as capture antigen, influenza NP proteantigen wraps by described No. 031 coding microball as capture antigen, LAM antigen wraps by described No. 033 coding microball as capture antigen, bird flu H5 antigen wraps by described No. 032 coding microball as capture antigen, F 1 antibody of plague bacterium wraps by described No. 043 coding microball as capture antigen, and SARS-CoV N proteantigen wraps by described No. 044 coding microball as capture antigen.
4. protein suspension chip as claimed in claim 2 system is characterized in that described antibody to be measured is blood serum sample.
5. protein suspension chip as claimed in claim 2 system is characterized in that, described biotin labeled two anti-ly are preferably Biotin-goat anti-rabbit igg and/or Biotin-goat anti-human igg.
6. protein suspension chip as claimed in claim 2 system is characterized in that, it is streptavidin-phycoerythrin that described fluorescence signal detects thing.
7. protein suspension chip as claimed in claim 2 system is characterized in that described chip basal body is 96 hole filter plate or microcentrifugal tubes.
8. protein suspension chip as claimed in claim 2 system, it is characterized in that, described detection laser comprises with exciting color in the described coding microball matrix, the sorting code number of recognition coding microballoon to determine the red laser of test item, and the color signal, tracer signal power that is used to excite and discern testing molecule is with the green laser of the content that detects described antibody to be measured.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107677803A (en) * | 2017-08-25 | 2018-02-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of coding/decoding system and method for liquid-phase chip analyzer |
| CN111308075A (en) * | 2020-03-18 | 2020-06-19 | 杭州广科安德生物科技有限公司 | Multi-tumor combined detection kit and use method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107677803A (en) * | 2017-08-25 | 2018-02-09 | 中国科学院苏州生物医学工程技术研究所 | A kind of coding/decoding system and method for liquid-phase chip analyzer |
| CN107677803B (en) * | 2017-08-25 | 2019-12-17 | 中国科学院苏州生物医学工程技术研究所 | coding and decoding system and method for liquid phase chip analyzer |
| CN111308075A (en) * | 2020-03-18 | 2020-06-19 | 杭州广科安德生物科技有限公司 | Multi-tumor combined detection kit and use method thereof |
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