CN101349701A - Protein chip for sironi virus detection and preparation method thereof - Google Patents
Protein chip for sironi virus detection and preparation method thereof Download PDFInfo
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- CN101349701A CN101349701A CNA2008101197774A CN200810119777A CN101349701A CN 101349701 A CN101349701 A CN 101349701A CN A2008101197774 A CNA2008101197774 A CN A2008101197774A CN 200810119777 A CN200810119777 A CN 200810119777A CN 101349701 A CN101349701 A CN 101349701A
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Abstract
The invention discloses a protein chip for detecting Silone virus and a process for preparation. The protein chip for detecting Silone virus is fixed with a biological chip of at least a Silone virus proteantigen on the chip base of the biological chip. The invention has the advantages of saving time, high effective and high flux parallel detection, detecting results not only can be qualitatively observed by naked eyes, but also can be qualitatively detected by a scanner, the results can be permanently conserved, the protein chip can not be compared by a traditional detection method, which is the crystallization of the existing biochip technology, the immunological technique and the chemical technology. On one aspect, the protein chip can more accurately do clinical diagnosis for Silone virus, on the other aspect, the protein chip can visually know the function which is played by each protein component of Silone virus in the aspect of immune response, which can provide a theoretical basis for researching subunit vaccine, and can provide technical support for background survey of epidemiology, and can create a precondition for preventing and curing illness.
Description
Technical field
The present invention relates to protein-chip of a kind of Xi Luoni of being used for virus detection and preparation method thereof.
Background technology
West nile virus (West Nile virus, WNV) at first ugandan Xi Niluo area is separated to from fever patient's serum in Africa in nineteen thirty-seven, hence obtains one's name, and this virus can cause heating, is called West Nile fever.
West nile virus belongs to the B Polyzoa matchmaker virus of flaviviridae Flavivirus, single positive chain RNA that genome is made up of 11029 nucleotide.The topmost spinal animals host of west nile virus is a bird, and the bird and the ill bird of band poison are the main infection sources and reservoirs, and mosquito especially culex is topmost communication media.Behind the bird infective virus, do not occur clinical symptoms usually at once, but the viremia virusemia phase of a longer duration, high titre is arranged, can by infect mosquito be propagated by behind the mosquito bite this moment.Virus is propagated by the mode of bird-mosquito-bird, forms endless-chain between bird and mosquito, again infected person, horse and other livestock and poultry once in a while of biting by mosquito.People, horse and other mammals are behind the bite by mosquitos that is infected by west nile virus and fall ill, and influenza-like symptoms (West Nile fever) such as heating, headache appear in the lighter, and weight person can show as central nervous system symptom (west nile encephalitis), even dead.Existing evidence shows that behind the mosquito bite people of infective virus, horse and other animal, these animals become the final host of infection, no longer gives other animal with virus disseminating.
West nile virus is distributed widely in Africa, Europe, the Middle East, the Central Asia, west Asia.The popular country of West Nile fever encephalitis took place in recent years, and Algeria, Romania, Czechoslovakia, Congo, Russia, Israel etc. were arranged, last century end is imported the North America again into, and rapid spread, almost feed through to each state of the U.S., to in June, 2005 the whole America reported the case that 1.7 ten thousand routine human infections should virus, people more than 650 death altogether.
China does not find the report of West Nile fever or west Nile encephalitis case as yet so far, does not arrive west nile virus in isolated in China yet.States such as China circumjacent state such as Israel, repeatedly found the popular of West Nile fever, the popular of West Nile fever also took place repeatedly in some countries (comprising Russia) northern with China and the USSR (Union of Soviet Socialist Republics) that the northwestward borders on, very likely import China by all means into, so there is the huge potential threat that the Xi Niluo infectious disease takes place in China.Along with China's rapid economy development, make people-to-people contacts and international trade, tourism etc. movable flourishing as never before especially, increased the possibility that disease is imported into greatly, importing into popular of disease all may be taken place at any time.China region is wide in addition, environment is various, the animals and plants fauna is abundant, insect vector is numerous, and for the Natural Circulation of west nile virus provides necessary host, media and favourable habitat, virus is in case import into and very easily form circulating ring and remain.In addition, existing result of study shows that there is west nile virus IgG antibody in China normal person, in recent years find also that there is higher west nile virus IgG antibody horizontal in China some areas crowd, prompting China has existed west nile virus to infect, and does not just find case as yet.For successfully managing the outbreak of epidemic that the contingent west nile virus of China infects, must at first set up effective detection and prevent and treat method.
At present, the laboratory diagnosis infected of west nile virus is mainly based on experimental techniques such as virus separation and foranalysis of nucleic acids, serodiagnosises.The separation of west nile virus needs carry out in professional Experimental Establishment with cultivation, as BSL-3 or BSL-4 laboratory, and note bio-safety, and China does not also have the laboratory that meets this bio-safety grade of sufficient amount at present.Foranalysis of nucleic acids generally adopts the RT-PCR method, detects viral gene from patient's serum, spinal fluid, but because the final host of animals such as people, horse for infecting, viral level is low, even if patients during acute stage is also only had an appointment and 40% can be detected viral gene.Serodiagnosis is the laboratory clinical examination method of at present the most frequently used west nile virus, mainly utilizes the specific binding reaction of the antigen protein of west nile virus and corresponding antibodies to reach the purpose of laboratory diagnosis.
West nile virus albumen comprises 3 kinds of structural proteins (C, prM/M, E albumen) and 7 kinds of non-structural proteins.Wherein nucleocapsid protein (C) is a basic protein and is attached to viral genome; Glycoprotein E is the most crucial immunological structural proteins, is the plain mediation of viral erythrocyte agglutination virus-host's combination.E albumen participates in that virus is affine with host cell, absorption and cell fusion process, is the major decision albumen of viral close preferendum and virulence.PrM is the precursor forms of M albumen in the ripe virion, before virus discharges, contains prM in the intracytoplasmic virion.PrM helps the location of E albumen in endoplasmic reticulum and the formation of space conformation, and prevents that E albumen from being cut by proteinase in cytoplasm.These three kinds of albumen all can produce specific antibody in vivo.
Specific antibody enzyme connection absorption immunization (ELISA) is mainly adopted in serodiagnosis at present.Advantages such as it is higher that ELISA has specificity, easy and simple to handle have been widely used in clinical diagnosis and epidemiology survey that west nile virus infects in the popular area of numerous disease.
Protein chip technology (Protein-chip) is the biological study technology that development in recent years is got up, it is the method for a kind of high specificity, susceptibility height, good reproducibility, its outstanding characteristics are the high flux that it has, can be simultaneously at the antibody of synantigen or antigen subunit composition not, thereby be usually used in the examination of same type disease or seek at the detection of antibodies of synantigen subunit composition not.
Summary of the invention
The purpose of this invention is to provide protein-chip of a kind of Xi Luoni of being used for virus detection and preparation method thereof.
The protein-chip of detection Xi Luoni virus provided by the invention is the biochip that is fixed with at least a west nile virus proteantigen on biological chip base.
Also can be fixed with the Quality Control contrast on the described base; Described Quality Control contrast is anti-IgG antibody or anti-IgM antibody.
Described base can be any biochip sheet base commonly used, as glass chip, diaphragm base, macromolecule silicon wafer-based or plastic base, and preferred glass sheet base.For fixing Xi Luoni viral antigen, described base can pass through chemical treatment, as glass chip through the aldehyde radical processing.
Described west nile virus proteantigen comprises: west nile virus C albumen, west nile virus M albumen, west nile virus prM albumen and west nile virus E albumen.
The described protein-chip that is used for the detection of Xi Luoni virus specifically can be the biochip that is fixed with west nile virus M albumen, west nile virus prM albumen, west nile virus E albumen and described Quality Control contrast on the glass chip of aldehyde radicalization.
The present invention also provides a kind of Xi Luoni of preparation virus to detect the method for protein-chip, comprises the steps:
1) with the carbonate buffer solution that contains glycerine with antigen diluent to 0.1-1.0mg/ml; The content of glycerine is 30-70% in the described carbonate buffer solution, preferred 50%; Described carbonate buffer solution, concentration are 0.01-0.2mol/L, preferred 0.05mol/L, and pH is 9.0-10.0, preferred 9.6;
2) antigen after will diluting is fixed on the sheet base;
3) with confining liquid with step 2) the sheet base seal, obtain described protein-chip; Described confining liquid is the PBS solution that contains 0.01-2%BSA and 0.01-2%Tween 20, preferably contains the PBS solution of 0.5%BSA and 0.05%Tween20; Described PBS solution, concentration are 0.001-0.2mol/L, preferred 0.01mol/L, and pH is 6.5-8.5, preferred 7.4.
Described step 2) also can comprise in Quality Control is contrasted the step of solidifying on the sheet base; Described Quality Control contrast is anti-IgG antibody or anti-IgM antibody.
Described base can be any biochip sheet base commonly used, as glass chip, diaphragm base, macromolecule silicon wafer-based or plastic base, and preferred glass sheet base.For fixing Xi Luoni viral antigen, described base can pass through chemical treatment, can pass through the aldehyde radical processing as glass chip.
Described west nile virus proteantigen comprises: west nile virus C albumen, west nile virus M albumen, west nile virus prM albumen and west nile virus E albumen.
When being fixed in on-chip west nile virus proteantigen and being west nile virus M albumen, west nile virus prM albumen and west nile virus E albumen, the concentration after the described west nile virus E albumen dilution is 0.005-5.0mg/ml, preferred 0.05mg/ml when described; Concentration after the described west nile virus prM albumen dilution is 0.005-5.0mg/ml, preferred 0.04mg/ml; Concentration after the described west nile virus M albumen dilution is 0.005-5.0mg/ml, preferred 0.2mg/ml.
The present invention also provides a kind of west nile virus detection kit, comprises described protein chip.
Described kit also can comprise dilution and cleansing solution; Described dilution is for containing 0.01-2%BSA and 0.05-1.0%NaN
3Phosphate buffer, preferably contain 0.5%BSA and 0.1%NaN
3Phosphate buffer; Described cleansing solution is for containing 0.001-2% Tween-20 and 0.05-1.0%NaN
3Phosphate buffer, preferably contain 0.05% Tween-20 and 0.1%NaN
3Phosphate buffer.
Also comprise the reagent that is used for chromogenic reaction in the described kit; Described chromogenic reaction is nm of gold chromogenic reaction, magnetic bead chromogenic reaction, fluorescence developing reaction or enzyme mark chromogenic reaction.
When described chromogenic reaction was enzyme mark chromogenic reaction, the described reagent that is used for chromogenic reaction comprised tracer and developer; Described developer is a tetramethyl benzidine colour developing liquid; Described tracer is that two of horseradish peroxidase mark resists; Described two anti-are anti-human IgG antibody or anti-human IgM antibody.
Described tracer specifically can be the anti-human IgG antibody of the horseradish peroxidase mark that contains 5%BSA (mass percent) and 0.02% thimerosal (mass percent); Described developer specifically can be the acetate buffer solution (pH3.3) that contains 0.005% tetramethyl benzidine (mass percent).
Described Quality Control contrast has two effects, and the one, as the reaction anchor point, the 2nd, as positive control, described Quality Control contrast is anti-IgG antibody or anti-IgM antibody, sample source that inspection is surveyed and target and decide.When described chromogenic reaction being enzyme mark chromogenic reaction, described Quality Control contrast can be the anti-horse IgG antibody of horseradish peroxidase mark or the anti-horse IgM antibody of horseradish peroxidase mark, and described Quality Control contrast is preferably the anti-human IgG antibody of horseradish peroxidase mark, the anti-human IgM antibody of horseradish peroxidase mark.
Use protein chip provided by the invention and can detect west nile virus by different chromogenic reactions.Difference according to chromogenic reaction, described kit can comprise different chromogenic reaction reagent, as nm of gold, magnetic bead, fluorescent reagent etc., different developers needs to be complementary with the mark of tracer, preferred tracer agent is that two of horseradish peroxidase mark resists, and preferred developer is the tetramethyl benzidine liquid with the preparation of pH3.3 acetate buffer solution;
The concrete grammar of using kit detection Xi Luoni virus provided by the invention is as follows:
1, sample dilution: serum specimen is diluted (being that the 15ul serum specimen is added in the 300ul dilution), mixing at 1: 21 with dilution.
2, application of sample: described protein chip is sealed off and lain against on the experiment table, in every grid, add the sample that 300ul has diluted, note making sample be covered with grid.
3, hatch: chip is placed wet box and placed 37 ℃ of incubations 30 minutes, get rid of liquid in the grid, in every lattice, add 4 cleansing solutions, get rid of washing lotion after 1 second of pausing.Continuous washing 3 times washed for 1 second with current from the beginning at last, dries the back and blot water droplet around the grid on thieving paper.
4, add tracer: every lattice drip 2 tracers, do not overflow outside the grid.37 ℃ of incubations are 30 minutes in the wet box, and water droplet is on every side blotted in the same method washing 3 times on thieving paper.
5, colour developing: every lattice add 2 developers, and 37 ℃ of incubators developed the color 10-12 minute.Get rid of liquid in the grid, wash 1 second kind with the distillation current.Dry, thieving paper blots drop.
6, interpretation as a result: under white background, observe colour developing situation in each grid, the positive blue dot that shows, negative colourless; Or on reading apparatus, carry out, analyze automatically by software, generate the gray-scale value of blue spot automatically, T/cutoff 〉=1.0 are negative, and T/cutoff＜1.0 are positive.
During interpretation as a result, need all Quality Control points all to develop the color, otherwise invalidate the test.
The present invention is fixed in highly purified gene engineering antigen on the biological chip base, after adding the patients serum, antibody in the serum combines with antigen on the sheet base, add tracer again, form the figure of pressing the point sample design, once experiment can detect the serum situation of west nile virus M albumen, prM albumen and E protein antibodies.The present invention have save time, efficiently, the advantage of the parallel detection of high flux, but testing result is the naked eyes qualitative observation both, also available scanner detection by quantitative, and the result can forever preserve, being that traditional detection method is unrivaled, is the crystallization of modern biochip technology, immunological technique and chemical technology.Use protein chip of the present invention and can carry out clinical diagnosis to west nile virus more accurately on the one hand, can get information about each protein ingredient of west nile virus role aspect the stimulation immune response on the other hand, development provides theoretical foundation to subunit vaccine, also for the epidemiology background survey provides technical support, for precondition is created in the control of disease.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 is the dot matrix arranging situation of protein chip.
Fig. 2 is the testing result of embodiment 1.
Fig. 3 is the testing result of embodiment 2.
Fig. 4 is the testing result of embodiment 3.
Fig. 5 is the testing result of embodiment 4.
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
One, the preparation of west nile virus antibody multi-disc section protein chip
1, the preparation of material
CBS-G solution: contain carbonate buffer solution in 50% glycerine (mass percent); The concentration of described carbonate buffer solution is 0.05mol/L, and pH is 9.6;
Quality Control contrast (the anti-human IgG antibody of horseradish peroxidase mark): available from the green skies, Shanghai Bioisystech Co., Ltd (A0201);
West nile virus E albumen: available from U.S. abcam company (ab48960);
West nile virus prM albumen: available from U.S. abcam company (ab39905);
West nile virus M albumen: available from U.S. abcam company (ab39904);
Confining liquid: the PBS solution that contains 0.5%BSA (mass percent) and 0.05%Tween 20 (mass percent); The concentration of described PBS solution is 0.01mol/L, and pH is 7.4.
Glass substrate: Shenzhen City Saier Bioisystech Co., Ltd (B 002);
2, the preparation of protein chip
1) with CBS-G solution E albumen is diluted to 0.05mg/ml; With CBS-G solution prM albumen is diluted to 0.04mg/ml; With CBS-G solution M albumen is diluted to 0.2mg/ml; The Quality Control contrast is diluted to 0.5mg/ml with CBS-G;
2) be fixed on the glass substrate of aldehyde radicalization with biochip point sample instrument 3 kinds of antigen and Quality Control contrast that step 1 dilution is good, the point sample amount is 2ul/dot, and lucifuge is put room temperature and dried in the air 2-3 hour; The dot matrix of 3 kinds of antigen and contrast is arranged and is seen Fig. 1; Among Fig. 1, A1, B1, C1 are the Quality Control contrast, and A2 and A3 are that E proteantigen, B2 and B3 are the M proteantigen, and C1 and C2 are the prM proteantigen;
3) each dot matrix adds the confining liquid of 250ul, and 37 ℃ of wet boxes sealed 90 minutes, cleans with distilled water and dries, and obtains protein chip.
Prepare 50 protein chips altogether.
Two, the assembling of kit
Kit composed as follows:
50 protein chips of step 1 preparation;
Dilution: contain 0.5%BSA (mass percent) and 0.1%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Cleansing solution: contain 0.05% Tween-20 (mass percent) and 0.1%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Tracer: the anti-human IgG antibody who contains the horseradish peroxidase mark of 5%BSA (mass percent) and 0.02% thimerosal (mass percent) is (available from the green skies, Shanghai Bioisystech Co., Ltd, A0201);
Developer: the acetate buffer solution (pH3.3) that contains 0.005% tetramethyl benzidine (mass percent);
Three, detect
1, serum sample
50 parts of human serum samples all come from the personnel that have a medical check-up of port, Shenzhen hospital.
2, detect
Kit with step 2 preparation detects above sample, and each sample is with a protein chip, and it is as follows specifically to detect step:
1, with dilution serum specimen is diluted (being that the 15ul serum specimen is added in the 300ul dilution), mixing at 1: 21.
2, take out protein chip and seal off and lie against on the experiment table, in every grid, add the sample that 300ul has diluted, make sample be covered with grid.
3, chip is placed wet box, 37 ℃ of incubations 30 minutes get rid of liquid in the grid, add 4 cleansing solutions in every lattice, get rid of washing lotion after 1 second of pausing.Continuous washing 3 times washed for 1 second with current from the beginning at last, dries the back and blot water droplet around the grid on thieving paper.
4, every lattice drip 2 tracers, do not overflow outside the grid.37 ℃ of incubations are 30 minutes in the wet box, and water droplet is on every side blotted in the same method washing 3 times on thieving paper.
5, every lattice add 2 developers, and 37 ℃ of incubators developed the color 10-12 minute.Get rid of liquid in the grid, wash 1 second kind with the distillation current.Dry, thieving paper blots drop.
Part test the results are shown in Figure 2.
As seen from the figure, the 20# sample is the E protein positive, and the 32# sample is the prM protein positive, and the 47# sample is prM protein positive and E protein positive, and the 29# sample is negative.Through the ELISA checking, testing result is correct.
Four, result verification
Use 50 parts of serum samples in the U.S. FOCUS Diagnostics EL0300G of the company kit detection step 3, by specification is operated.
The result shows have two samples positive in 50 samples, is respectively 32# sample and 47# sample.Because the ELISA kit generally only is solidified with a kind of antigen, the testing result of kit of the present invention shows that 32# sample and 47# sample are the prM protein positive, can the preliminary judgement FOCUS Diagnostics EL0300G of company kit be only to detect at prM therefore.Confirmatory experiment is the result show: on the one hand the testing result of kit of the present invention is reliably, is embodied in the detection of prM albumen at least and the testing result of the FOCUS Diagnostics EL0300G of company kit is on all four; The detection that shows kit of the present invention on the other hand is better than existing ELISA reagent detection, can detect three albumen simultaneously, and ELISA can only detect at a kind of albumen.
One, the preparation of west nile virus antibody multi-disc section protein chip
1, the preparation of material
CBS-G solution: contain carbonate buffer solution in 50% glycerine (mass percent); The concentration of described carbonate buffer solution is 0.05mol/L, and pH is 9.6;
Quality Control contrast (the anti-human IgM antibody of horseradish peroxidase mark): open up bio tech ltd (HT21007) available from Guangzhou China;
West nile virus E albumen: available from U.S. abcam company (ab48960);
West nile virus prM albumen: available from U.S. abcam company (ab39905);
West nile virus M albumen: available from U.S. abcam company (ab39904);
Confining liquid: the PBS solution that contains 0.5%BSA (mass percent) and 0.05%Tween 20 (mass percent); The concentration of described PBS solution is 0.01mol/L, and pH is 7.4.
Glass substrate: Shenzhen City Saier Bioisystech Co., Ltd (B-002);
2, the preparation of protein chip
1) with CBS-G solution E albumen is diluted to 0.05mg/ml; With CBS-G solution prM albumen is diluted to 0.04mg/ml; With CBS-G solution M albumen is diluted to 0.2mg/ml; The Quality Control contrast is diluted to 0.5mg/ml with CBS G;
2) be fixed on the glass substrate of aldehyde radicalization with biochip point sample instrument 3 kinds of antigen and Quality Control contrast that step 1 dilution is good, the point sample amount is 2ul/dot, and lucifuge is put room temperature and dried in the air 2-3 hour; The dot matrix of 3 kinds of antigen and contrast is arranged and is seen Fig. 1; Among Fig. 1, A1, B1, C1 are the Quality Control contrast, and A2 and A3 are that E proteantigen, B2 and B3 are the M proteantigen, and C1 and C2 are the prM proteantigen;
3) each dot matrix adds the confining liquid of 250ul, and 37 ℃ of wet boxes sealed 90 minutes, cleans with distilled water and dries, and obtains protein chip.
Prepare 2 protein chips altogether.
Two, the assembling of kit
Kit composed as follows:
2 protein chips of step 1 preparation;
Dilution: contain 0.5%BSA (mass percent) and 0.1%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Cleansing solution: contain 0.05% Tween-20 (mass percent) and 0.1%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Tracer: (China opens up bio tech ltd available from Guangzhou, HT21007) to contain the anti-human IgM antibody of horseradish peroxidase mark of 5%BSA (mass percent) and 0.02% thimerosal (mass percent);
Developer: the acetate buffer solution (pH3.3) that contains 0.005% tetramethyl benzidine (mass percent);
Three, detect
Get patient's (patient is port, Shenzhen entry personnel) serum of 2 routine heating paresthesias respectively, as sample 1 and sample 2, with the kit check of step 2 preparation.
Each sample is with a protein chip, and it is as follows specifically to detect step:
1, with dilution serum specimen is diluted (being that the 15ul serum specimen is added in the 300ul dilution), mixing at 1: 21.
2, take out protein chip and seal off and lie against on the experiment table, in every grid, add the sample that 300ul has diluted, make sample be covered with grid.
3, chip is placed wet box, 37 ℃ of incubations 30 minutes get rid of liquid in the grid, add 4 cleansing solutions in every lattice, get rid of washing lotion after 1 second of pausing.Continuous washing 3 times washed for 1 second with current from the beginning at last, dries the back and blot water droplet around the grid on thieving paper.
4, every lattice drip 2 tracers, do not overflow outside the grid.37 ℃ of incubations are 30 minutes in the wet box, and water droplet is on every side blotted in the same method washing 3 times on thieving paper.
5, every lattice add 2 developers, and 37 ℃ of incubators developed the color 10-12 minute.Get rid of liquid in the grid, wash 1 second kind with the distillation current.Dry, thieving paper blots drop.
The results are shown in Figure 3, A representative sample 1; B representative sample 2.Show that the Xi Luoni virus infections does not take place this two routine patient.Through the ELISA checking, testing result is correct.
Four, result verification
Use 2 parts of serum samples in the U.S. FOCUS Diagnostics EL0300M of the company kit detection step 3, by specification is operated.
Testing result shows that two sample standard deviations are negative, and is consistent with kit testing result of the present invention.
One, the preparation of west nile virus antibody multi-disc section protein chip
1, the preparation of material
CBS-G solution: contain carbonate buffer solution in 30% glycerine (mass percent); The concentration of described carbonate buffer solution is 0.01mol/L, and pH is 9.0;
Quality Control contrast (the anti-human IgG antibody of horseradish peroxidase mark): available from the green skies, Shanghai Bioisystech Co., Ltd (A0201);
West nile virus E albumen: available from U.S. abcam company (ab48960);
West nile virus prM albumen: available from U.S. abcam company (ab39905);
West nile virus M albumen: available from U.S. abcam company (ab39904);
Confining liquid: the PBS solution that contains 0.01%BSA (mass percent) and 0.01%Tween 20 (mass percent); The concentration of described PBS solution is 0.001mol/L, and pH is 6.5.
Glass substrate: Shenzhen City Saier Bioisystech Co., Ltd (B-002);
2, the preparation of protein chip
1) with CBS-G solution E albumen is diluted to 0.005mg/ml; With CBS-G solution prM albumen is diluted to 0.005mg/ml; With CBS-G solution M albumen is diluted to 0.005mg/ml; The Quality Control contrast is diluted to 0.005mg/ml with CBS-G;
2) be fixed on the glass substrate of aldehyde radicalization with biochip point sample instrument 3 kinds of antigen and Quality Control contrast that step 1 dilution is good, the point sample amount is 2ul/dot, and lucifuge is put room temperature and dried in the air 2-3 hour; The dot matrix of 3 kinds of antigen and contrast is arranged and is seen Fig. 1; Among Fig. 1, A1, B1, C1 are the Quality Control contrast, and A2 and A3 are that E proteantigen, B2 and B3 are the M proteantigen, and C1 and C2 are the prM proteantigen;
3) each dot matrix adds the confining liquid of 250ul, and 37 ℃ of wet boxes sealed 90 minutes, cleans with distilled water and dries, and obtains protein chip.
Prepare 2 protein chips altogether.
Two, the assembling of kit
Kit composed as follows:
2 protein chips of step 1 preparation;
Dilution: contain 0.01%BSA (mass percent) and 0.05%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Cleansing solution: contain 0.001% Tween-20 (mass percent) and 0.05%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Tracer: the anti-human IgG antibody who contains the horseradish peroxidase mark of 5%BSA (mass percent) and 0.02% thimerosal (mass percent) is (available from the green skies, Shanghai Bioisystech Co., Ltd, A0201);
Developer: the acetate buffer solution (pH3.3) that contains 0.005% tetramethyl benzidine (mass percent);
Three, detect
1, serum sample
2 parts of human serum samples all come from the personnel that have a medical check-up of port, Shenzhen hospital.Portion is Xi Luoni virus-positive sample (U.S. EL0300G of a FOCUS Diagnostics company kit detection validation), and another part is Xi Luoni virus negative sample (U.S. EL0300G of FOCUS Diagnostics company kit detection validation).
2, detect
With 2 of the step 3 of embodiment 1.
Testing result is seen Fig. 4.Sample 1 is a prM protein positive sample (A), sample 2 negative samples (B).
Embodiment 4, to the detection of human IgG
One, the preparation of west nile virus antibody multi-disc section protein chip
1, the preparation of material
CBS-G solution: contain carbonate buffer solution in 70% glycerine (mass percent); The concentration of described carbonate buffer solution is 0.2mol/L, and pH is 10.0;
Quality Control contrast (the anti-human IgG antibody of horseradish peroxidase mark): available from the green skies, Shanghai Bioisystech Co., Ltd (A0201);
West nile virus E albumen: available from U.S. abcam company (ab48960);
West nile virus prM albumen: available from U.S. abcam company (ab39905);
West nile virus M albumen: available from U.S. abcam company (ab39904);
Confining liquid: the PBS solution that contains 2%BSA (mass percent) and 2%Tween 20 (mass percent); The concentration of described PBS solution is 0.2mol/L, and pH is 8.5.
Glass substrate: Shenzhen City Saier Bioisystech Co., Ltd (B-002);
2, the preparation of protein chip
1) with CBS-G solution E albumen is diluted to 5.0mg/ml; With CBS-G solution prM albumen is diluted to 5.0mg/ml; With CBS G solution M albumen is diluted to 5.0mg/ml; The Quality Control contrast is diluted to 5.0mg/ml with CBS-G;
2) be fixed on the glass substrate of aldehyde radicalization with biochip point sample instrument 3 kinds of antigen and Quality Control contrast that step 1 dilution is good, the point sample amount is 2ul/dot, and lucifuge is put room temperature and dried in the air 2-3 hour; The dot matrix of 3 kinds of antigen and contrast is arranged and is seen Fig. 1; Among Fig. 1, A1, B1, C1 are the Quality Control contrast, and A2 and A3 are that E proteantigen, B2 and B3 are the M proteantigen, and C1 and C2 are the prM proteantigen;
3) each dot matrix adds the confining liquid of 250ul, and 37 ℃ of wet boxes sealed 90 minutes, cleans with distilled water and dries, and obtains protein chip.
Prepare 2 protein chips altogether.
Two, the assembling of kit
Kit composed as follows:
2 protein chips of step 1 preparation;
Dilution: contain 2%BSA (mass percent) and 1%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Cleansing solution: contain 2% Tween-20 (mass percent) and 1%NaN
3The phosphate buffer of (mass percent) (pH7.4);
Tracer: the anti-human IgG antibody who contains the horseradish peroxidase mark of 5%BSA (mass percent) and 0.02% thimerosal (mass percent) is (available from the green skies, Shanghai Bioisystech Co., Ltd, A0201);
Developer: the acetate buffer solution (pH3.3) that contains 0.005% tetramethyl benzidine (mass percent);
Three, detect
1, serum sample
2 parts of human serum samples all come from the personnel that have a medical check-up of port, Shenzhen hospital.Portion is Xi Luoni virus-positive sample (U.S. EL0300G of a FOCUS Diagnostics company kit detection validation), and another part is Xi Luoni virus negative sample (U.S. EL0300G of FOCUS Diagnostics company kit detection validation).
2, detect
With 2 of the step 3 of embodiment 1.
Testing result is seen Fig. 5.Sample 1 is a prM protein positive sample (A), sample 2 negative samples (B).
Embodiment 5, specific detection
Have research data to show, other kind of west nile virus and yellow fever virus especially encephalitis B and dengue fever exists comparatively serious crossover phenomenon in the serological reaction that detects antibody.In order to verify the confidence level of three protein I gG of above-mentioned west nile virus antibody positive detection result of specimen, it encephalitis B and dengue fever IgG antibody test experiment have been carried out.
This enforcement adopts the ELISA indirect method to three west nile virus antibody (IgG) positive serum sample (20# in 50 serum samples, 32#, 47#) detect, envelope antigen adopts dengue fever virus NS1 albumen (available from U.S. abcam company, ab64456) and japanese encephalitis virus E albumen (available from U.S. abcam company, ab43031), the prescription of other reaction solution is with embodiment 1.
1, (available from U.S. abcam company, ab64456) be diluted to coated elisa plate behind the 5ug/ml with CBS-G solution (Shenzhen City Saier Bioisystech Co., Ltd), 4 ℃ are spent the night with dengue fever virus NS1 albumen; (available from U.S. abcam company, ab43031) be diluted to coated elisa plate behind the 5ug/ml with CBS-G solution (Shenzhen City Saier Bioisystech Co., Ltd), 4 ℃ are spent the night with japanese encephalitis virus E albumen.
2, add confining liquid (Shenzhen City Saier Bioisystech Co., Ltd), place 4 ℃ to spend the night.
When 3, detecting encephalitis B IgG antibody with sample to be measured by 1: 41 the usefulness dilution (Shenzhen City Saier Bioisystech Co., Ltd) dilute; When detecting dengue fever IgG antibody sample to be measured by 1: 41 the usefulness dilution (Shenzhen City Saier Bioisystech Co., Ltd) dilute; Careful absorption has been diluted sample supernatant 100ul to be checked in reacting hole, and it is as follows to establish contrast simultaneously: negative control: normal human serum 100ul (Shenzhen City Saier Bioisystech Co., Ltd); Positive control 1: each 100ul of dengue fever positive serum (Shenzhen City Saier Bioisystech Co., Ltd); Positive control 2: encephalitis B positive serum 100ul (Shenzhen City Saier Bioisystech Co., Ltd); Blank: sample diluent 100ul.Patting mixing hatched 30 minutes for rearmounted 37 ℃.
4, wash orifice plate 3 times with cleansing solution (Shenzhen City Saier Bioisystech Co., Ltd), pat dry.The anti-human IgG that adds the HRP mark two anti-(available from the green skies, Shanghai Bioisystech Co., Ltd, A0201), the 100ul/ hole.Mixing was hatched 30 minutes for rearmounted 37 ℃.
5, wash plate with step 4.Every hole adds the colour developing of 100ulTMB (Shenzhen City Saier Bioisystech Co., Ltd) substrate, and 37 ℃ were reacted 10 minutes.
6, every hole adds the H of 50ul 2mol/L
2SO
4Cessation reaction is measured the OD450 value on microplate reader.
7, data processing: get 2.1 * NC value as cutoff value (annotating: with 0.1 note, remembered with actual value during NC value less than 0.1) greater than 0.1 o'clock.
The result shows that the encephalitis B of 3 routine west nile virus antibody (IgG) positive samples and the IgG antibody test result of dengue fever are negative entirely.The result shows do not have cross reaction between this 3 routine west nile virus prM albumen or E protein I gG antibody positive sample and encephalitis B and the dengue fever virus.
Claims (10)
1, a kind of protein-chip that detects Xi Luoni virus is the biochip that is fixed with at least a west nile virus proteantigen on biological chip base.
2, chip as claimed in claim 1 is characterized in that: also be fixed with the Quality Control contrast on the described base; Described Quality Control contrast is anti-IgG antibody or anti-IgM antibody.
3, chip as claimed in claim 1 or 2 is characterized in that: described base is glass chip, diaphragm base, macromolecule silicon wafer-based or plastic base, preferred glass sheet base; Described west nile virus proteantigen comprises: west nile virus C albumen, west nile virus M albumen, west nile virus prM albumen and west nile virus E albumen.
4, a kind of method for preparing Xi Luoni virus detection protein-chip comprises the steps:
1) with the carbonate buffer solution that contains glycerine with antigen diluent to 0.1-1.0mg/ml; The content of glycerine is 30-70% in the described carbonate buffer solution, preferred 50%; The concentration of described carbonate buffer solution is 0.01-0.2mol/L, preferred 0.05mol/L, and pH is 9.0-10.0, preferred 9.6;
2) antigen after will diluting is fixed on the sheet base;
3) with confining liquid with completing steps 2) the sheet base seal, obtain described protein-chip; Described confining liquid is the PBS solution that contains 0.01-2%BSA and 0.01-2%Tween 20, preferably contains the PBS solution of 0.5%BSA and 0.05%Tween20; The concentration of described PBS solution is 0.001-0.2mol/L, preferred 0.01mol/L, and pH is 6.5-8.5, preferred 7.4.
5, method as claimed in claim 4 is characterized in that: also comprise described step 2) Quality Control is contrasted the step of solidifying on the sheet base; Described Quality Control contrast is anti-IgG antibody or anti-IgM antibody; Described base is glass chip, diaphragm base, macromolecule silicon wafer-based or plastic base, preferred glass sheet base; Described west nile virus proteantigen comprises: west nile virus C albumen, west nile virus M albumen, west nile virus prM albumen and west nile virus E albumen.
6, method as claimed in claim 5 is characterized in that: the concentration after the described west nile virus E albumen dilution is 0.005-5.0mg/ml, preferred 0.05mg/ml; Concentration after the described west nile virus prM albumen dilution is 0.005-5.0mg/ml, preferred 0.04mg/ml; Concentration after the described west nile virus M albumen dilution is 0.005-5.0mg/ml, preferred 0.2mg/ml.
7, a kind of west nile virus detection kit comprises arbitrary described protein-chip in the claim 1 to 3.
8, kit as claimed in claim 7 is characterized in that: described kit also comprises dilution and cleansing solution; Described dilution is for containing 0.01-2%BSA and 0.05-1.0%NaN
3Phosphate buffer, preferably contain 0.5%BSA and 0.1%NaN
3Phosphate buffer; Described cleansing solution is for containing 0.001-2% Tween-20 and 0.05-1.0%NaN
3Phosphate buffer, preferably contain 0.05% Tween-20 and 0.1%NaN
3Phosphate buffer.
9, as claim 7 or 8 described kits, it is characterized in that: also comprise the reagent that is used for chromogenic reaction in the described kit; Described chromogenic reaction is nm of gold chromogenic reaction, magnetic bead chromogenic reaction, fluorescence developing reaction or enzyme mark chromogenic reaction.
10, kit as claimed in claim 9 is characterized in that: described chromogenic reaction is an enzyme mark chromogenic reaction, and the described reagent that is used for chromogenic reaction is tracer and developer; Described developer is a tetramethyl benzidine colour developing liquid; Described tracer is that two of horseradish peroxidase mark resists; Described two anti-are anti-human IgG antibody or anti-human IgM antibody;
Described tracer is preferably the anti-human IgG antibody of the horseradish peroxidase mark that contains 5%BSA and 0.02% thimerosal; Described developer is preferably the acetate buffer solution that contains 0.005% tetramethyl benzidine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175874A (en) * | 2011-01-14 | 2011-09-07 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation and application of vaccine immunity effect evaluation protein chip |
| CN102808042A (en) * | 2012-03-14 | 2012-12-05 | 中华人民共和国天津出入境检验检疫局 | Method for detecting flavivirus by gene chip method |
| CN103983640A (en) * | 2014-05-29 | 2014-08-13 | 上海理工大学 | Protein chip sample point quality control and tracing method |
| CN109596589A (en) * | 2018-12-27 | 2019-04-09 | 南京祥中生物科技有限公司 | A kind of fluorescence solid phase biological chip detecting method based on LED light source |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1239907C (en) * | 2003-06-23 | 2006-02-01 | 林连成 | Process for preparing protein chips |
| CN101000340B (en) * | 2006-01-12 | 2011-01-19 | 深圳市赛尔生物技术有限公司 | Method of biochip for simultaneous testing avian influenza infection of human and fowls |
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2008
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175874A (en) * | 2011-01-14 | 2011-09-07 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation and application of vaccine immunity effect evaluation protein chip |
| CN102808042A (en) * | 2012-03-14 | 2012-12-05 | 中华人民共和国天津出入境检验检疫局 | Method for detecting flavivirus by gene chip method |
| CN103983640A (en) * | 2014-05-29 | 2014-08-13 | 上海理工大学 | Protein chip sample point quality control and tracing method |
| CN109596589A (en) * | 2018-12-27 | 2019-04-09 | 南京祥中生物科技有限公司 | A kind of fluorescence solid phase biological chip detecting method based on LED light source |
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