CN105738629B - A kind of indirect immunofluorescene assay method of yellow fever virus IgG antibody - Google Patents
A kind of indirect immunofluorescene assay method of yellow fever virus IgG antibody Download PDFInfo
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- CN105738629B CN105738629B CN201511002933.5A CN201511002933A CN105738629B CN 105738629 B CN105738629 B CN 105738629B CN 201511002933 A CN201511002933 A CN 201511002933A CN 105738629 B CN105738629 B CN 105738629B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The present invention relates to a kind of indirect immunofluorescene assay methods of quick, easy, sensitive yellow fever virus ((YellowFever Virus)) IgG antibody.The detection method principle of the present invention is to detect people's yellow fever poison antiviral antibody with indirect immunofluorescence.It is the 293F cells that coating can express yellow fever virus NS1 albumen on glass slide, positive control or positive moderate resistance NS1 protein antibodies are after cell surface is attached to, in fluorescence microscopy Microscopic observation after being captured with fluorochrome label secondary antibody, the fluorecyte of visible green is determined as the yellow fever virus IgG antibody positive.If fluorescence is not shown without yellow fever virus antibody, cell in sample.Detection method includes the following steps：The preparation of 293F cell wave carrier pieces adds in sample, the combination of fluorescent marker secondary antibody, washing, fluoroscopic examination, result judgement on wave carrier piece.The present invention can carry out yellow fever virus the detection of rapid sensitive, easy to operate, the detection efficiency of one X -ray inspection X quarantine functionary of entry and exit port can be increased substantially by the present invention, the input of external deadly infectious disease is of great significance to prevention and control.
Description
Technical field
The present invention relates to external diagnosis reagent technical fields, and in particular to a kind of yellow fever virus IgG antibody is immunized indirectly
Fluorescence detection method.
Background introduction
With the rapid development of China's economic trade, the ecology of Disease outbreaks has been broken in increasingly frequent international trade
Territory restriction has become and disseminates infection and its important channel of Vector factors, exacerbates the wind of the transnational propagation of infectious disease
Danger.According to the statistics of State General Administration for Quality Supervision, every year by the entry and exit number of frontier port more than 200,000,000 person-times.Infectious disease is in state
Wide-scale distribution between border is with popular, Xin Fa and the emergence for sending out infectious disease again, has increasingly becomed great public in the whole world and has defended
Raw problem, thus the Introduced cases epidemic of frontier port reply at this stage is faced with huge challenges and tests.
Flavivirus is RNA virus, has thermophilic visceral and Neural invasion, after infecting mosquito bite people, will contain yellow fever
The saliva injection human body subcutaneous capillary of virus, rapidly diffuses into regional nodes, constantly breeds, blood flow is entered after a few days,
Form viremia virusemia.Then Virus localization is in histoorgans such as liver,kidney,spleen, the heart, marrow and lymph nodes, even if virus is in blood
Through disappearing, and virus can still remain in histoorgan.Due to the direct detrimental effect of virus, cause extensive lesion tissue,
The specificity that middle hepatic pathology variation most diagnoses.The major source of infection of urban yellow fever is patient, infectiousness in onset 3 days
It is most strong；The major source of infection of jungle type yellow fever is monkey and other primates in matto.Communication media city
The primary vehicle of city's type yellow fever is Aedes aegypti, and the communication media of jungle type yellow fever mainly has Haemagogus, evil spirit mosquito
Belong to, mosquito is sucked blood after infection, and 37 DEG C can propagate through 4 days.Infected mosquito can band poison, and being transmitted through ovum throughout one's life.Susceptible population
Crowd without immunity is generally susceptible to yellow fever, can obtain long-lasting immunity after subclinical infection or morbidity, generate in vivo
Neutralizing antibody can keep lifelong, do not find the infected again.Be grown up in Endemic Area has immunity mostly, therefore is accounted for children's morbidity more
Number.Yellow fever is acute infectious disease caused by yellow fever virus.Clinic is to generate heat, yellow subcutaneous ulcer, albuminuria, relative infrequent pulse and bleeding etc. are
Feature.Yellow fever quilt in 1907《International Sanitary Convention》It is classified as international quarantine infectious disease.Nearly ten years, in Africa and Latin America
More than ten of country successively yellow fever for several times have occurred distribute prevalence, and there is no morbidity in China.
Yellow fever virus is not yet popular in China, but with China's commerce and trade, the fast development of tourist industry, local and overseas personnel's contacts
The influences such as increasingly close and animal import, the danger of potent virus input increasingly increase, and therefore, establish as early as possible quick
The detection method of easy, special sensitivity takes precautions against yellow fever virus and inputs and its outbreak of epidemic is controlled to be of great significance.To yellow fever
The detection method of poison is less using immunization method at present largely using gene tester, between IgG antibody provided by the invention
It connects immunofluorescence technique and can detect people's yellow fever poison NS1 protein antibodies, the detection for yellow fever virus provides quickly, easy, spirit
Quick immunofluorescent detection method, the present invention can increase substantially the detection efficiency of one X -ray inspection X quarantine functionary of import-export ports,
The generation of external input sexually transmitted disease is prevented to the maximum extent.
Invention content
The technical problems to be solved by the invention are to provide a kind of exempting from indirectly for quick, sensitive yellow fever virus IgG antibody
Epidemic disease fluorescence detection method.The indirect immunofluorescene assay method of the yellow fever virus IgG antibody of the present invention, includes the following steps：
1. express the preparation of the 293F cell wave carrier pieces of yellow fever virus NS1 albumen:
By Expi293F cells through adhere-wall culture to cell state it is good when, the cell count after pancreatin digests, by cell
Density is adjusted to 4X105After a/milliliter, cell suspension is added in 250ul/ holes on Chamber Slide plates, is softly rocked,
It is placed in cell incubator and cultivates 24 hours.Cell is paved with bottom hole, then the full-length gene with yellow fever virus NS1 albumen substantially at this time
Expression plasmid carries out cell transfecting.With 2000 transfected plasmids of liposome, with the ratio of 1ug plasmids/2000 transfection reagent of 2ul liposomes
Example carries out.After cell transfecting 48 hours, the yellow fever virus NS1 albumen expressed carries out cell with paraformaldehyde and fixes, fixed
Completion carries out 5% lowlenthal serum closing again, stored refrigerated with 10% 2-8 DEG C of glycerine mounting after closing.
2. the configuration of sample diluting liquid and washing lotion；
3. express the balance of the 293F cell wave carrier pieces of yellow fever virus NS1 albumen：Expression there is into yellow fever virus NS1 albumen
293F cell wave carrier piece Chamber Slide are taken from 2-8 DEG C into room temperature, are placed 10 minutes.
4. add in sample to be tested, positive control, negative control：200ul is added in per hole, covers Chamber Slide lids,
It is placed at room temperature for 60 minutes.
5. sopping up supernatant, 300ul washing lotions are added in per hole, sop up washing lotion within 10 seconds or so, repeated primary.
6. add in fluorescent marker secondary antibody：200ul, room temperature avoid light place 1 hour are added in per hole, this step is both needed to keep away later
Light processing prevents fluorescence from failing.
7. sopping up sample solution, 300ul washing lotions are added in per hole, washing lotion is sopped up within 10 seconds or so, repeats.
8. result judgement：Chamber Slide are placed in fluorescence microscopy Microscopic observation fluorescence, judgement is as a result, have green glimmering
Light is positive reaction, and redgreen fluorescence does not have immune response then, represents not containing yellow fever virus antibody in sample.
Yellow fever virus described in step 1 is Strain AEQ35299；
Sample diluting liquid described in step 2 is dissolved in for 5% lowlenthal serum in 0.01M PBS buffer solution；Washing lotion is 0.05%
Polysorbas20 be dissolved in 0.01M PBS buffer solution.
The rabbit positive serum that positive control described in step 4 is yellow fever poison NS1 Protein Gs P prepares and includes following step
Suddenly：The method of full-length gene (AEQ35299 strains) through molecular cloning for expressing yellow fever virus NS1 Protein Gs P is cloned into
GIneratorTMAfter carrier, new zealand white rabbit is immunized with the method for genetic immunization, after first immunisation, secondary immunity, ear is quiet
A small amount of antiserum is collected in arteries and veins blood sampling, the sero-fast potency of method detection through indirect ELISA, after bioactivity qualification (>1:
64000) booster immunization (behind secondary immunity interval 3 weeks) is carried out, antiserum is collected after 10 days；Not immune new zealand white rabbit is made
For negative control, be carried out at the same time with positive control prepare operation and detection.
The dilution 1 of positive control described in step 4：100 dilutions, negative control dilution 1：100 dilutions.
Fluorescent marker secondary antibody described in step 6 is goat-anti rabbit and goat-anti people's secondary antibody mixed liquor；Fluorescence secondary antibody mixed liquor adds in
It is preceding to use sample diluting liquid 1:2000 times of dilutions.
Yellow fever virus is not yet popular in China, and major part uses gene tester at present, less to use immunization method, this
Invention detects people's yellow fever poison NS1 protein antibodies with indirect immunofluorescence.It is that coating can express yellow fever virus on glass slide
The 293F cells of NS1 albumen, positive control or positive moderate resistance NS1 protein antibodies use fluorescence after cell surface is attached to
In fluorescence microscopy Microscopic observation after the secondary antibody capture of dye marker, it is seen that green fluorecyte is determined as that yellow fever virus IgG resists
Body is positive.This detection method is easy to operate, save the time, high sensitivity, specificity are good, and the detection for yellow fever virus provides soon
Speed, easy, sensitive immunofluorescent detection method.Import-export ports one X -ray inspection X quarantine people can be increased substantially by the present invention
The detection efficiency of member can not only reduce workload but also can solve traditional detection method positive missing inspection that may be present to the maximum extent
Problem, so as to prevent the generation of external input sexually transmitted disease to the maximum extent.
Description of the drawings
The protein electrophoresis figure purified after the outer yellow fever virus NS1 protein expressions of wave carrier piece in Fig. 1 embodiments 1.
Yellow fever virus detects positive control fluoroscopic examination figure in Fig. 2 embodiments 1.
In figure, the left side is the visible ray figure of positive control, and the right is the fluorogram of positive control.
Yellow fever virus detects negative control fluoroscopic examination figure in Fig. 3 embodiments 1.
In figure, the left side is the visible ray figure of negative control, and the right is the fluorogram of negative control.
The positive analog sample fluoroscopic examination result of yellow fever virus detection in Fig. 4 embodiments 1.
In figure, the left side is the visible ray figure of positive analog sample, and the right is the fluorogram of positive analog sample.
Yellow fever virus detects negative sample fluoroscopic examination result figure in Fig. 5 embodiments 1.
In figure, the left side is the visible ray figure of Healthy Human Serum sample, and the right is that corresponding Healthy Human Serum sample is glimmering
Light figure.
Specific embodiment
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The indirect immunofluorescene assay method of 1 yellow fever virus IgG antibody of embodiment
First, experimental procedure
1. sample prepares
In the present embodiment, since positive sample source is limited, therefore used with the rabbit positive serum of yellow fever poison NS1 Protein Gs P
For different diluted concentrations as simulation positive sample and positive control, negative control sample is the serum of Healthy People.
2. material, reagent, instrument
Lab-Tek II Chamber Slide chamber slides are purchased from Nunc companies, GIneratorTMExpression vector is purchased from
Immune Technology companies, Expi293FTMAll reagents of expression system are public purchased from Thermo Fisher Scientific
Department, photofulorography microscope AXIO SCOPE A1 are purchased from Zeiss company, and the mixing fluorescence secondary antibody of goat-anti rabbit and people are purchased from
Jackson ImmunoResearch companies.
3. method
The preparation and purification of 3.1 yellow fever virus NS1 albumen
It is prepared by the rabbit positive serum of 3.1 yellow fever poison NS1 albumen
The method of full-length gene (AEQ35299) through molecular cloning for expressing yellow fever virus NS1 albumen is cloned into
GIneratorTMAfter carrier, new zealand white rabbit is immunized with the method for genetic immunization, after first immunisation, secondary immunity, ear is quiet
A small amount of antiserum is collected in arteries and veins blood sampling, the sero-fast potency of method detection through indirect ELISA, after bioactivity qualification (>1:
64000) booster immunization (behind secondary immunity interval 3 weeks) is carried out, antiserum is collected after 10 days.Not immune new zealand white rabbit is made
For negative control, it is carried out at the same time duplicate operation and detection.
The foundation of 3.2 yellow fever virus antibody mediated immunity fluorescence detection methods
(1) preparation of the 293F cell wave carrier pieces of expression yellow fever virus NS1 albumen:
By 293F cells through adhere-wall culture to cell state it is good when, the cell count after pancreatin digests, by cell density
It adjusts to 4X105After a/milliliter, cell suspension is added in 250ul/ holes on Chamber Slide plates, softly rocks, is placed in
It is cultivated 24 hours in cell incubator.Cell is paved with bottom hole substantially at this time, then is expressed with the full-length gene of yellow fever virus NS1 albumen
Plasmid carries out cell transfecting.With 2000 transfected plasmids of liposome, with the ratio of 1ug plasmids/2000 transfection reagent of 2ul liposomes into
Row.After cell transfecting 48 hours, the yellow fever virus NS1 albumen expressed carries out cell with paraformaldehyde and fixes, fixed to complete
5% lowlenthal serum closing is carried out again, it is stored refrigerated with 10% 2-8 DEG C of glycerine mounting after closing.
(2) configuration of sample diluting liquid and washing lotion：5% lowlenthal serum is dissolved in 0.01M PBS buffer solution.Washing lotion is
0.05% polysorbas20 is dissolved in 0.01M PBS buffer solution.
(3) balance of the 293F cell wave carrier pieces of expression yellow fever virus NS1 albumen：Expression there is into yellow fever virus NS1 albumen
293F cell wave carrier piece Chamber Slide taken from 2-8 DEG C into room temperature, place 10 minutes.
(4) simulation positive sample, negative sample, positive control, negative control are separately added into：Simulation positive sample is rabbit-anti
Positive serum makees 1 with sample diluting liquid：10 dilutions, negative control sample are the serum of Healthy People, and positive control is positive for rabbit-anti
Serum makees 1 with sample diluting liquid：100 dilutions, negative control are to make 1 with sample diluting liquid：100 diluted rabbit control serums.Often
Hole adds in 200ul.Chamber Slide lids are covered, are placed at room temperature for 60 minutes.
(5) supernatant is sopped up, 300ul washing lotions are added in per hole, sop up washing lotion within 10 seconds or so, is repeated primary.
(6) with sample diluting liquid 1:2000 times of dilution fluorescent marker secondary antibodies, 200ul are added in per hole, room temperature avoid light place 1 is small
When.It is both needed to be protected from light processing after this step, prevents fluorescence from failing.Fluorescent marker secondary antibody is mixed for goat-anti rabbit with goat-anti people's secondary antibody
Liquid, the mixed liquor of this secondary antibody can ensure people's yellow fever poison NS1 eggs in rabbit-anti positive serum controls and real positive sample
White IgG, which can combine to be labeled, shows Positive fluorescence signal.
(7) sample solution is sopped up, 300ul washing lotions are added in per hole, washing lotion is sopped up within 10 seconds or so, repeats.
(8) Chamber Slide are placed in fluorescence microscopy Microscopic observation fluorescence, judged as a result, it is sun to have green fluorescence
Property reaction, redgreen fluorescence do not have immune response then, represents not containing yellow fever virus antibody in sample.
The verification of yellow fever virus NS1 protein expressions on 3.3 293F cell wave carrier pieces：
In order to verify the expression feelings of NS1 albumen on the 293F cell wave carrier pieces of prepared expression yellow fever virus NS1 albumen
Condition has carried out the synchronous experiment of yellow fever virus NS1 albumen preparation and purifications outside wave carrier piece, it is contemplated that real the problem of expression quantity
Experiment condition during testing slightly changes, but does not influence last expression of results, and specific experiment is as follows：
By Expi293FTMCell is in Expi293FTMSuspension culture is carried out in expression culture medium, treats that cell concentration rises to
Cell transfection assays are carried out during suitable concentration.Cell uses ExpiFectamineTM293 transfection reagents carry out cell transfecting.With
When the cultivating system of 30ml is transfected, take added in centrifuge tube A/B, the A pipe of two 1.5ml RPMI 1640 cell culture fluids,
Strain AEQ35299DNA30ug, total volume 1.5ml, soft mixing.Added in B pipes RPMI 1640 cell culture fluids,
ExpiFectamineTMTransfection reagent 80ul, total volume 1.5ml, soft mixing, incubation at room temperature after five minutes, A are added in B, gently
Soft mixing is incubated at room temperature 20 minutes and adds in cell.96 hours or so rear collection albumen after transfection.In albumen preparation process
Culture medium or addition culture medium need not be changed.The cell conditioned medium of collection is through Ni column purifications, the yellow fever virus NS1 eggs expressed
In vain.
2nd, experimental result
The verification result of yellow fever virus NS1 protein expressions on 1.293F cell wave carrier pieces：
In the synchronous experiment of the yellow fever virus NS1 albumen preparation and purifications carried out outside wave carrier piece, in the Expi293F of 30mlTM
Yellow fever virus NS1 albumen is prepared in expression system, the cell conditioned medium of collection carries out the egg that protein electrophoresis obtains after Ni column purifications
White electrophoretogram, the molecular weight of yellow fever strain AEQ35299 albumen should be 43KD, and protein electrophoresis figure is shown in apparent item between 40-50KD
Band is specifically shown in Fig. 1.
2. the foundation of yellow fever virus antibody mediated immunity fluorescence detection method
(1) positive control testing result
Utilize positive rabbit anti-serum 1:100 dilutions are as the positive control in this detection method.Fig. 2 left sides are visible ray
Figure, the right is fluorogram.Figure it is seen that Positive control wells fluorescence signal is very strong.
(2) negative control testing result
Rabbit anteserum 1 not to be immunized:100 dilutions are visible ray figure as negative control, Fig. 3 left sides, and the right is fluorogram.From
Fig. 3 can be seen that negative control hole and not detect any fluorescence signal.
(2) positive analog sample testing result
Positive analog sample is carried out 1:500 dilutions, obtain fluorescence signal figure, are specifically shown in Fig. 4 after testing.Fig. 4 left sides are
Visible ray figure, the right are fluorogram.From fig. 4, it can be seen that simulation Positive control wells can detect stronger fluorescence signal.
(3) yellow hot negative sample testing result
Healthy Human Serum sample is detected for negative sample, obtains fluorescence signal figure.All negative samples do not detect
To fluorescence, illustrate detection specificity preferably, no cross reaction is specifically shown in Fig. 5.Fig. 5 left sides are visible ray figure, and the right is glimmering
Light figure.
Present invention uses the 293F cells of expression yellow fever virus NS1 albumen to provide detection target position, eukaryotic cell expression
NS1 albumen there is native conformation, antiserum moderate resistance yellow fever virus NS1 protein antibodies can be effectively detected.This
Method has specificity strong (the human serum negative control 10 of detection different background, no cross reaction problem), and sensitivity is higher
(antiserum is 1:500 dilution be still to have compared with strong signal), using it is simple the characteristics of, for detect yellow fever virus infection provide one
It is a simple, quickly, accurate method.This shows that the detection performance of the yellow fever virus IgG antibody immunofluorescence method of the present invention is good
It is good, negative and positive sample can be accurately distinguished, specificity is good, misjudgement phenomenon does not occur.
Claims (8)
- A kind of 1. indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose, it is characterised in that：Including with Lower detecting step：(1) preparation of the 293F cell glass slides of expression yellow fever virus NS1 albumen:By Expi293F cells through adhere-wall culture to cell state it is good when, the cell count after pancreatin digests, by cell density It adjusts to 4X105After a/milliliter, cell suspension is added in 250ul/ holes on chamber slides Chamber Slide plates, is softly shaken It shakes, is placed in cell incubator and cultivates 24 hours；Cell is paved with bottom hole, then the overall length with yellow fever virus NS1 albumen substantially at this time Gene expression plasmid carries out cell transfecting；With 2000 transfected plasmids of liposome, with 1ug plasmids/2000 transfection reagent of 2ul liposomes Ratio carry out；After cell transfecting 48 hours, the yellow fever virus NS1 albumen expressed carries out cell with paraformaldehyde and fixes, It is fixed to complete to carry out 5% lowlenthal serum closing again, it is stored refrigerated with 10% 2-8 DEG C of glycerine mounting after closing；Yellow fever used Poison is Strain AEQ35299；(2) configuration of sample diluting liquid and washing lotion；(3) balance of the 293F cell glass slides of expression yellow fever virus NS1 albumen：Expression there is into yellow fever virus NS1 albumen 293F cell glass slide Chamber Slide are taken from 2-8 DEG C into room temperature, are placed 10 minutes；(4) sample to be tested, positive control, negative control are added in；Rabbit sun of the positive control for yellow fever poison NS1 albumen Property serum, preparation include the following steps：The full-length gene of yellow fever virus strain AEQ35299 albumen will be expressed through molecular cloning Method be cloned into IneratorTMAfter carrier, new zealand white rabbit is immunized with the method for genetic immunization, through first immunisation, secondary After immune, a small amount of antiserum is collected in ear vein blood sampling, and the method through indirect ELISA detects sero-fast potency, bioactivity>1: Booster immunization is carried out after 64000, antiserum is collected in secondary immunity interval after 3 weeks after 10 days；The negative control is rather Epidemic disease new zealand white rabbit, the same positive control of preparation process；(5) supernatant is sopped up, 300ul washing lotions are added in per hole, sop up washing lotion within 10 seconds or so, is repeated primary；(6) fluorescent marker secondary antibody is added in；(7) sample solution is sopped up, 300ul washing lotions are added in per hole, washing lotion is sopped up within 10 seconds or so, repeats；(8) result judgement：Glass slide Chamber Slide are placed in fluorescence microscopy Microscopic observation fluorescence, are judged as a result, there is green Fluorescence is positive reaction, and redgreen fluorescence does not have immune response then, represents not containing yellow fever virus antibody in sample.
- 2. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：Diluent ingredient described in step (2) is dissolved in for 5% lowlenthal serum in the phosphate buffer PBS of 0.01M, institute The washing lotion ingredient stated is dissolved in for 0.05% polysorbas20 in 0.01M phosphate buffers.
- 3. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：Step (4) positive control dilution 1：100 dilutions, negative control dilution 1：100 dilutions.
- 4. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：Sample to be tested, positive control, negative control described in step (4) add in 200ul per hole, cover Chamber Slide lids are placed at room temperature for 60 minutes.
- 5. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：Fluorescent marker secondary antibody described in step (6) is goat-anti rabbit and goat-anti people's secondary antibody mixed liquor.
- 6. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：Fluorescence secondary antibody mixed liquor described in step (6) uses sample diluting liquid 1 before adding in:2000 times of dilutions.
- 7. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：Fluorescence secondary antibody described in step (6) adds in 200ul, room temperature avoid light place 1 hour per hole, this step is done later It is protected from light processing.
- 8. the indirect immunofluorescene assay method of the yellow fever virus IgG antibody of non-diagnostic purpose according to claim 1, It is characterized in that：With carrying out the yellow fever virus NS1 albumen preparation and purification confirmatory experiment synchronous with step (1), packet outside glass slide Include following process：By Expi293FTMCell is in Expi293FTMSuspension culture is carried out in expression culture medium, it is suitable dense to treat that cell concentration rises to Cell transfection assays are carried out when spending；Cell uses ExpiFectamineTM293 transfection reagents carry out cell transfecting；With the training of 30ml When foster system is transfected, take and RPMI1640 cell culture fluids, Strain are added in centrifuge tube A/B, the A pipe of two 1.5ml AEQ35299 DNA 30ug, total volume 1.5ml, soft mixing；Added in B pipes RPMI1640 cell culture fluids, ExpiFectamineTMTransfection reagent 80ul, total volume 1.5ml, soft mixing, incubation at room temperature after five minutes, A are added in B, gently Soft mixing is incubated at room temperature 20 minutes and adds in cell；96 hours or so rear collection albumen after transfection；In albumen preparation process Culture medium or addition culture medium need not be changed, the cell conditioned medium of collection is through Ni column purifications, the yellow fever virus NS1 eggs expressed In vain.
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| CN113372441B (en) * | 2018-04-04 | 2023-03-10 | 中国科学院微生物研究所 | High-sensitivity yellow fever virus humanized monoclonal antibody and application thereof |
| CN111398594B (en) * | 2020-04-16 | 2023-01-24 | 中国人民解放军南部战区疾病预防控制中心 | Immunodiagnosis kit for specifically detecting yellow fever virus NS1 antigen |
| CN112415188B (en) * | 2020-11-25 | 2022-10-04 | 首都医科大学宣武医院 | Magnetic cell and preparation method and application thereof |
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| CN202024998U (en) * | 2010-12-28 | 2011-11-02 | 中国检验检疫科学研究院 | Protein suspension array system for detecting yellow fever antibodies |
| CN102952900A (en) * | 2012-11-22 | 2013-03-06 | 浙江国际旅行卫生保健中心 | Reagent and method for detecting yellow fever virus |
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| CN202024998U (en) * | 2010-12-28 | 2011-11-02 | 中国检验检疫科学研究院 | Protein suspension array system for detecting yellow fever antibodies |
| CN102952900A (en) * | 2012-11-22 | 2013-03-06 | 浙江国际旅行卫生保健中心 | Reagent and method for detecting yellow fever virus |
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