CN105548539A - Indirect immunofluorescence detection method for Lassa Virus IgG antibody - Google Patents
Indirect immunofluorescence detection method for Lassa Virus IgG antibody Download PDFInfo
- Publication number
- CN105548539A CN105548539A CN201511005030.2A CN201511005030A CN105548539A CN 105548539 A CN105548539 A CN 105548539A CN 201511005030 A CN201511005030 A CN 201511005030A CN 105548539 A CN105548539 A CN 105548539A
- Authority
- CN
- China
- Prior art keywords
- lassa virus
- cell
- igg antibody
- sample
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention relates to a fast, simple and sensitive indirect immunofluorescence detection method for a Lassa Virus IgG antibody. The detection method provided by the invention has the principle of detecting a human-anti-Lassa Virus glycoprotein antibody by indirect immunofluorescence. 293F cells capable of expressing Lassa Virus glycoprotein are coated on a glass slide; after a glycoprotein antibody in a positive control or positive sample is combined with a cell surface, a fluorescent dye is used for marking a second antibody, and then an operator can observe the second antibody after capturing under a fluorescent microscope; if green fluorescent cells are observed, the Lassa Virus IgG antibody can be confirmed as positive; and if no Lassa Virus antibody exists in the sample, the cells are not fluorescent. The detection method comprises the following steps: preparing a 293F cell glass slide; loading the sample onto the glass slide; combining the fluorescently marked second antibody; washing; performing fluorescence detection; and judging the result. The method provided by the invention is used for quickly and sensitively detecting Lassa Virus; the operation is simple and convenient; the detection efficiency of the on-site inspection and quarantine personnel at entry and exit ports can be greatly increased according to the method; and the method has significance in preventing and controlling the spreading of foreign fulminating infectious diseases.
Description
Technical field
The present invention relates to external diagnosis reagent technical field, be specifically related to a kind of indirect immunofluorescene assay method of Lassa virus IgG antibody.
background introduction
Along with global economic integration and liberalization of trade, external Medical Vectors, by the advanced vehicles and international trade, can be transmitted to the whole world infectious disease from a country rapidly, cause international propagation.In recent years, potent virus sexually transmitted disease is frequent outbreak of epidemic in countries in the world, brings serious threat to human health, causes tremendous economic to lose to society.Taking place frequently of these potent virus sexually transmitted diseases has beaten alarm bell to us, and it is vital for strengthening its research and monitoring with prevention and control early.
Lassa fever is the acute viral infection that a kind of by name Lassa virus (LassaVirus) causes, is popular in the country in continent, West Africa, as Guinea, Liberia, Sierra Leone and Nigeria.The host of virus is the field rodent in continent, West Africa.Lassa virus property Hemorrhagic fever is the acute illness in a kind of course of disease 1-4 week occurring in West Africa.Although describe, until within 1969, just determine the virus causing this disease in generation nineteen fifty first.This virus is a kind of singlestranded RNA virus belonging to Arenaviridae.Known Lassa fever is popular in Guinea, Liberia, Sierra Leone and Nigeria some areas, but also may exist at other the west African state.Because the symptom of Lassa fever is so different and non-specific, be often difficult to carry out clinical diagnosis, especially at the course of disease initial stage.Lassa fever is difficult to distinguish with causing many Other diseases of fever, comprises malaria, shigellosis, typhoid fever, yellow fever and other viral hemorrhagic fever.Make a definite diagnosis the deployable detection in laboratory that Lassa fever needs to only have highly-specialised.Laboratory sample may be harmful to, and must process extremely modestly.Lassa fever is by finding that the husky antiviral antibody of Lassa virus antigen, tension or virus isolation techniques are diagnosed.
Lassa virus is not yet popular in China, but along with the fast development of China's commerce and trade, tourist industry, local and overseas personnel's contacts are day by day close, and the impact such as the import of animal, the danger of potent virus input increases day by day, therefore, the detection method setting up fast and convenient, special sensitivity is as early as possible taken precautions against Lassa virus and to be inputted and to control its outbreak of epidemic significant.Still belong to blank to the research of the detection technique of Lassa virus is domestic, IgG antibody indirect immunofluorescence provided by the invention can detect the husky VGP antibody of people's tension, detection for Lassa virus provides fast, easy, sensitive immunofluorescent detection method, the present invention can increase substantially the detection efficiency of import-export ports one X-ray inspection X quarantine functionary, prevents the generation of external Introduced cases infectious disease to greatest extent.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of indirect immunofluorescene assay method of quick, sensitive Lassa virus IgG antibody.The indirect immunofluorescene assay method of Lassa virus IgG antibody of the present invention, comprises the steps:
1. express the preparation of the 293F cell wave carrier piece of Lassa virus glycoprotein:
By Expi293F cell when adhere-wall culture is good to cell state, cell count after trypsinization, is adjusted to 4X10 by cell density
5after individual/milliliter, ChamberSlide plate adds cell suspension with 250ul/ hole, softly rocks, be placed in cell culture incubator and cultivate 24 hours.Now cell is paved with at the bottom of hole substantially, then carries out cell transfecting with the full-length gene expression plasmid of Lassa virus glycoprotein.With liposome 2000 transfected plasmids, carry out with the ratio of 1ug plasmid/2ul liposome 2000 transfection reagent.Cell transfecting is after 48 hours, obtains the Lassa virus Glycoprotein G P expressed, carries out cell fix with paraformaldehyde, fixedly completes to carry out 5% lowlenthal serum again and close, and closes afterwards by 10% glycerine mounting 2-8 DEG C of stored refrigerated.
2. the configuration of sample diluting liquid and washing lotion;
3. express the balance of the 293F cell wave carrier piece of Lassa virus glycoprotein: have the 293F cell wave carrier piece ChamberSlide of Lassa virus glycoprotein to be taken to room temperature from 2-8 DEG C expression, place 10 minutes.
4. add testing sample, positive control, negative control: every hole adds 200ul, and cover ChamberSlide lid, room temperature places 60 minutes.
5. sop up supernatant, every hole adds 300ul washing lotion, within about 10 seconds, sops up washing lotion, then repeats once.
6. add fluorescence labeling two to resist: every hole adds 200ul, and room temperature lucifuge places 1 hour, all needs to do lucifuge process, prevent fluorescence from failing after this step.
7. sop up sample solution, every hole adds 300ul washing lotion, within about 10 seconds, sops up washing lotion, repeats.
8. result judges: ChamberSlide is placed in fluorescence microscopy Microscopic observation fluorescence, result of determination, and what have green fluorescence is positive reaction, and redgreen fluorescence does not then have immune response, represents in sample not containing Lassa virus antibody.
Lassa virus described in step 1 is Strain Nig08-A19;
Sample diluting liquid described in step 2 is that 5% lowlenthal serum is dissolved in 0.01MPBS damping fluid; Washing lotion be 0.05% polysorbas20 be dissolved in 0.01MPBS damping fluid.
Positive control described in step 4 is the rabbit positive serum of the husky VGP GP of tension, and its preparation comprises the steps: the full-length gene (Nig08-A19 strain) of expressing Lassa virus Glycoprotein G P to be cloned into GInerator through the method for molecular cloning
tMafter carrier, with the method immunize New Zealand White Rabbit of genetic immunization, after first immunisation, secondary immunity, a small amount of antiserum is collected in ear vein blood sampling, method through indirect ELISA detects sero-fast tiring, after bioactivity is qualified, (>1:64000) carries out booster immunization (secondary immunity interval is after 3 weeks), collects antiserum after 10 days; Not immune new zealand white rabbit, as negative control, carries out preparing the same operation and detection with positive control simultaneously.
The dilution 1:100 of positive control described in step 4 dilutes, and negative control dilution 1:100 dilutes.
Fluorescence labeling two described in step 6 resists for goat-anti rabbit and the anti-mixed liquor of goat-anti people two; Doubly dilute with sample diluting liquid 1:2000 before the anti-mixed liquor of fluorescence two adds.
Lassa virus is not yet popular in China, source due to positive is limited makes the correlative study of the Fast Detection Technique to Lassa virus less, especially immunologic detection method, but as the quarantine of port one X-ray inspection X, the tachnical storage of Lassa virus detection method need be had.The present invention detects the husky VGP antibody of people's tension with indirect immunofluorescence.Microslide is bag can be expressed the 293F cell of Lassa virus glycoprotein, in positive control or positive, anti-glycoprotein antibody is after being attached to cell surface, with two of fluorochrome label anti-catch after at fluorescence microscopy Microscopic observation, the fluorecyte of visible green, is judged to be that Lassa virus IgG antibody is positive.This detection method is simple to operate, save time, highly sensitive, specificity good, and the detection for Lassa virus provides fast, easy, sensitive immunofluorescent detection method.The detection efficiency of import-export ports one X-ray inspection X quarantine functionary can be increased substantially by the present invention, not only can reduce workload, but also the undetected problem of the positive that traditional detection method may exist can have been solved to greatest extent, thus prevent the generation of external Introduced cases infectious disease to greatest extent.
Accompanying drawing explanation
The protein electrophoresis figure of purifying after the outer Lassa virus P-glycoprotein expression of wave carrier piece in Fig. 1 embodiment 1.
In Fig. 2 embodiment 1, Lassa virus detects negative and positive control fluoroscopic examination figure.
In figure, A is the visible ray figure of positive control, B is the fluorogram of positive control, and C is the visible ray figure of negative control, D is the fluorogram of negative control.
In Fig. 3 embodiment 1, Lassa virus detects positive analog sample different dilute concentration fluoroscopic examination result.
In figure, the fluorogram of A to be the visible ray figure of high titered positive analog sample (1:200), B be high titered positive analog sample (1:200); The fluorogram of C to be the visible ray figure of middle titered positive analog sample (1:500), D be middle titered positive analog sample (1:500); The fluorogram of E to be the visible ray figure of low titered positive analog sample (1:1000), F be low titered positive analog sample (1:1000).
In Fig. 4 embodiment 1, Lassa virus detects negative sample fluoroscopic examination result figure.
In figure, A, C, E are the visible ray figure of Healthy Human Serum sample, and B, D, F are corresponding Healthy Human Serum sample fluorescence figure.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
The indirect immunofluorescene assay method of embodiment 1 Lassa virus IgG antibody
One, experimental procedure
1. sample prepares
In the present embodiment, because positive sample source is limited, therefore by the different dilute concentration of rabbit positive serum of the husky VGP GP of tension as simulating positive sample and positive control, negative control sample is the serum of Healthy People.
2. material, reagent, instrument
Lab-TekIIChamberSlide chamber slides purchased from Nunc company, GInerator
tMexpression vector purchased from ImmuneTechnology company, Expi293F
tMthe all reagent of expression system is purchased from ThermoFisherScientific company, and photofulorography microscope AXIOSCOPEA1 is purchased from Zeiss company, and the mixing fluorescence two of goat-anti rabbit and people is anti-purchased from JacksonImmunoResearch company.
3. method
The preparation and purification of 3.1 Lassa virus Glycoprotein G P
The rabbit positive serum preparation of the husky VGP GP of 3.1 tensions
The full-length gene (Nig08-A19 strain) of expressing Lassa virus Glycoprotein G P is cloned into GInerator through the method for molecular cloning
tMafter carrier, with the method immunize New Zealand White Rabbit of genetic immunization, after first immunisation, secondary immunity, a small amount of antiserum is collected in ear vein blood sampling, method through indirect ELISA detects sero-fast tiring, after bioactivity is qualified, (>1:64000) carries out booster immunization (secondary immunity interval is after 3 weeks), collects antiserum after 10 days.Not immune new zealand white rabbit, as negative control, carries out duplicate operation and detection simultaneously.
The foundation of 3.2 Lassa virus antibody mediated immunity fluorescence detection methods
(1) preparation of the 293F cell wave carrier piece of Lassa virus glycoprotein is expressed:
By 293F cell when adhere-wall culture is good to cell state, cell count after trypsinization, is adjusted to 4X10 by cell density
5after individual/milliliter, ChamberSlide plate adds cell suspension with 250ul/ hole, softly rocks, be placed in cell culture incubator and cultivate 24 hours.Now cell is paved with at the bottom of hole substantially, then carries out cell transfecting with the full-length gene expression plasmid of Lassa virus glycoprotein.With liposome 2000 transfected plasmids, carry out with the ratio of 1ug plasmid/2ul liposome 2000 transfection reagent.Cell transfecting is after 48 hours, obtains the Lassa virus Glycoprotein G P expressed, carries out cell fix with paraformaldehyde, fixedly completes to carry out 5% lowlenthal serum again and close, and closes afterwards by 10% glycerine mounting 2-8 DEG C of stored refrigerated.
(2) configuration of sample diluting liquid and washing lotion: 5% lowlenthal serum is dissolved in 0.01MPBS damping fluid.Washing lotion be 0.05% polysorbas20 be dissolved in 0.01MPBS damping fluid.
(3) balance of the 293F cell wave carrier piece of Lassa virus glycoprotein is expressed: have the 293F cell wave carrier piece ChamberSlide of Lassa virus glycoprotein to be taken to room temperature from 2-8 DEG C expression, place 10 minutes.
(4) simulation positive sample, negative sample, positive control, negative control is added respectively: simulation positive sample is that rabbit anti-positive serum sample diluting liquid does 1:10 dilution, negative control sample is the serum of Healthy People, positive control is that rabbit anti-positive serum sample diluting liquid does 1:100 dilution, and negative control is the rabbit control serum doing 1:100 dilution with sample diluting liquid.Every hole adds 200ul.Cover ChamberSlide lid, room temperature places 60 minutes.
(5) sop up supernatant, every hole adds 300ul washing lotion, within about 10 seconds, sops up washing lotion, then repeats once.
(6) doubly dilute fluorescence labeling two with sample diluting liquid 1:2000 to resist, every hole adds 200ul, and room temperature lucifuge places 1 hour.All need after this step to do lucifuge process, prevent fluorescence from failing.Fluorescence labeling two resists for goat-anti rabbit and the anti-mixed liquor of goat-anti people two, and this two mixed liquors resisted can ensure that the husky VGP IgG of people's tension in rabbit anti-positive serum control and real positive sample all can show Positive fluorescence signal in conjunction with being labeled.
(7) sop up sample solution, every hole adds 300ul washing lotion, within about 10 seconds, sops up washing lotion, repeats.
(8) ChamberSlide is placed in fluorescence microscopy Microscopic observation fluorescence, result of determination, what have green fluorescence is positive reaction, and redgreen fluorescence does not then have immune response, represents in sample not containing Lassa virus antibody.
The checking that the husky VGP GP of 3.3293F cell wave carrier piece pull-up expresses:
In order to verify prepared expression Lassa virus glycoprotein 293F cell wave carrier piece on the expression of glycoprotein, the synchronous experiment of Lassa virus Glycoprotein G P preparation and purification has been carried out outside wave carrier piece, consider the problem of expression, experiment condition in experimentation slightly changes, but do not affect last expression of results, specific experiment is as follows:
By Expi293F
tMcell is at Expi293F
tMexpress in nutrient culture media and carry out suspension cultivation, when cell concentration rises to suitable concn, carry out cell transfection assays.Cell uses ExpiFectamine
tM293 transfection reagents carry out cell transfecting.When carrying out transfection with the cultivating system of 30ml, get the centrifuge tube A/B of two 1.5ml, add RPMI1640 cell culture fluid, GPDNA (Nig08-A19 strain, Genebank#AAT49008) 30ug in A pipe, cumulative volume 1.5ml, softly mixes.RPMI1640 cell culture fluid, ExpiFectamine is added in B pipe
tMtransfection reagent 80ul, cumulative volume 1.5ml, softly mixes, and incubated at room, after 5 minutes, adds A in B, softly mixes, and incubated at room adds in cell for 20 minutes.Albumen is collected after transfection after 96 hours.Without the need to changing nutrient culture media or adding nutrient culture media in albumen preparation process.The cell conditioned medium collected, through Ni post purifying, obtains the Lassa virus Glycoprotein G P expressed.
Two, experimental result
The result that the husky VGP GP of 1.293F cell wave carrier piece pull-up expresses:
In the synchronous experiment of the Lassa virus Glycoprotein G P preparation and purification carried out outside wave carrier piece, at the Expi293F of 30ml
tMlassa virus Glycoprotein G P is prepared in expression system, the cell conditioned medium collected is after Ni post purifying, and carry out the protein electrophoresis figure that protein electrophoresis obtains, the molecular weight of Lassa virus strain Nig08-A19 glycoprotein should be 32KD, protein electrophoresis figure is shown in obvious band between 25-37KD, specifically sees Fig. 1.
2. the rabbit positive serum of the husky VGP GP of tension is prepared and is detected
Prepare antiserum by the method for genetic immunization, use 1 new zealand white rabbit, the final antiserum collected has 50ml,
Its bioactivity the results are shown in Table 1.More than 1:1000000 can be reached by testing result this sero-fast tiring visible.
Table 1 antiserum titer determination result
3. the foundation of Lassa virus antibody mediated immunity fluorescence detection method
(1) positive and negative control test result
Utilize positive rabbit anti-serum 1:100 dilution as the positive control in this detection method, not immune rabbit anteserum 1:100 dilutes as negative control.Fluoroscopic examination that is negative and positive control the results are shown in Figure 2.A is the visible ray figure of positive control, B is the fluorogram of positive control, and C is the visible ray figure of negative control, D is the fluorogram of negative control.As can be seen from Figure 2, Positive control wells fluorescence signal is very strong, and the negative control hole of correspondence is without any fluorescence.
(2) positive analog sample testing result
Positive analog sample is carried out respectively 1:200,1:500 and 1:1000 dilution, obtain the positive analog sample of high, medium and low three gradient minuents respectively, obtain fluorescence signal figure after testing, specifically see Fig. 3.From fluorescence signal intensity, titre is higher, and fluorescence signal is stronger.
(3) husky negative sample testing result is drawn
Healthy Human Serum sample is that negative sample detects, and obtains fluorescence signal figure.All negative sample all do not detect fluorescence, and illustrate that detection specificity is better, no cross reaction, is specifically shown in Fig. 4.
Present invention uses the 293F cell of expression Lassa virus glycoprotein to provide detection target position, the glycoprotein of eukaryotic cell expression has the feature of native conformation, husky VGP (GP) antibody of tension in antiserum effectively can be detected.This method has strong (the human serum negative control 10 of detection different background of selectivity, no cross reaction problem), sensitivity higher (antiserum is still have stronger signal in 1:1000 dilution), use simple feature, for the infection detecting Lassa virus provides one simply, fast, method accurately.This shows that the detection perform of Lassa virus IgG antibody immunofluorescence method of the present invention is good, and can distinguish feminine gender and positive sample exactly, specificity is good, does not occur misjudging phenomenon.
Claims (9)
1. the indirect immunofluorescene assay method of a Lassa virus IgG antibody is characterized in that: comprise following detecting step:
(1) preparation of the 293F cell wave carrier piece of Lassa virus glycoprotein is expressed:
By Expi293F cell when adhere-wall culture is good to cell state, cell count after trypsinization, is adjusted to 4X10 by cell density
5after individual/milliliter, chamber slides ChamberSlide plate adds cell suspension with 250ul/ hole, softly rocks, be placed in cell culture incubator and cultivate 24 hours; Now cell is paved with at the bottom of hole substantially, then carries out cell transfecting with the full-length gene expression plasmid of Lassa virus glycoprotein; With liposome 2000 transfected plasmids, carry out with the ratio of 1ug plasmid/2ul liposome 2000 transfection reagent; Cell transfecting is after 48 hours, obtains the Lassa virus Glycoprotein G P expressed, carries out cell fix with paraformaldehyde, fixedly completes to carry out 5% lowlenthal serum again and close, and closes afterwards by 10% glycerine mounting 2-8 DEG C of stored refrigerated; Lassa virus used is Strain Nig08-A19;
(2) configuration of sample diluting liquid and washing lotion;
(3) balance of the 293F cell wave carrier piece of Lassa virus glycoprotein is expressed: have the 293F cell wave carrier piece ChamberSlide of Lassa virus glycoprotein to be taken to room temperature from 2-8 DEG C expression, place 10 minutes;
(4) testing sample, positive control, negative control is added;
(5) sop up supernatant, every hole adds 300ul washing lotion, within about 10 seconds, sops up washing lotion, then repeats once;
(6) add fluorescence labeling two to resist;
(7) sop up sample solution, every hole adds 300ul washing lotion, within about 10 seconds, sops up washing lotion, repeats;
(8) result judges: wave carrier piece ChamberSlide is placed in fluorescence microscopy Microscopic observation fluorescence, result of determination, and what have green fluorescence is positive reaction, and redgreen fluorescence does not then have immune response, represents in sample not containing Lassa virus antibody.
2. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, it is characterized in that: the diluent ingredient described in step (2) is that 5% lowlenthal serum is dissolved in the phosphate buffer PBS of 0.01M, and described washing lotion composition is that 0.05% polysorbas20 is dissolved in 0.01M phosphate buffer.
3. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, it is characterized in that: the positive control described in step (4) is the rabbit positive serum of the husky VGP GP of tension, its preparation comprises the steps: the full-length gene of expressing Lassa virus strain Nig08-A19 Glycoprotein G P to be cloned into Inerator through the method for molecular cloning
tMafter carrier, with the method immunize New Zealand White Rabbit of genetic immunization, after first immunisation, secondary immunity, a small amount of antiserum is collected in ear vein blood sampling, method through indirect ELISA detects sero-fast tiring, carry out booster immunization after bioactivity >1:64000, antiserum, after 3 weeks, is collected after 10 days in secondary immunity interval; Described negative control is not immune new zealand white rabbit, the same positive control of preparation process.
4. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, is characterized in that: step (4) described positive control dilution 1:100 dilutes, and negative control dilution 1:100 dilutes.
5. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, it is characterized in that: testing sample, positive control, negative control described in step (4), every hole adds 200ul, covers ChamberSlide lid, and room temperature places 60 minutes.
6. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, is characterized in that: the fluorescence labeling two described in step (6) resists for goat-anti rabbit and the anti-mixed liquor of goat-anti people two.
7. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, is characterized in that: doubly dilute with sample diluting liquid 1:2000 before the anti-mixed liquor of fluorescence two described in step (6) adds.
8. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, is characterized in that: the fluorescence two described in step (6) resists every hole to add 200ul, and room temperature lucifuge places 1 hour, does lucifuge process after this step.
9. the indirect immunofluorescene assay method of Lassa virus IgG antibody according to claim 1, it is characterized in that: with outside wave carrier piece, carry out the Lassa virus Glycoprotein G P preparation and purification confirmatory experiment synchronous with step (1), comprise following process:
By Expi293F
tMcell is at Expi293F
tMexpress in nutrient culture media and carry out suspension cultivation, when cell concentration rises to suitable concn, carry out cell transfection assays; Cell uses ExpiFectamine
tM293 transfection reagents carry out cell transfecting; When carrying out transfection with the cultivating system of 30ml, get the centrifuge tube A/B of two 1.5ml, add RPMI1640 cell culture fluid, Nig08-A19 strain GPDNA30ug in A pipe, cumulative volume 1.5ml, softly mixes; RPMI1640 cell culture fluid, ExpiFectamine is added in B pipe
tMtransfection reagent 80ul, cumulative volume 1.5ml, softly mixes, and incubated at room, after 5 minutes, adds A in B, softly mixes, and incubated at room adds in cell in 20 minutes; Albumen is collected after transfection after 96 hours; Without the need to changing nutrient culture media or adding nutrient culture media in albumen preparation process, the cell conditioned medium of collection, through Ni post purifying, obtains the Lassa virus Glycoprotein G P expressed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511005030.2A CN105548539B (en) | 2015-12-28 | 2015-12-28 | A kind of indirect immunofluorescene assay method of Lassa virus IgG antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511005030.2A CN105548539B (en) | 2015-12-28 | 2015-12-28 | A kind of indirect immunofluorescene assay method of Lassa virus IgG antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105548539A true CN105548539A (en) | 2016-05-04 |
| CN105548539B CN105548539B (en) | 2018-02-09 |
Family
ID=55827870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511005030.2A Active CN105548539B (en) | 2015-12-28 | 2015-12-28 | A kind of indirect immunofluorescene assay method of Lassa virus IgG antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105548539B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018106712A1 (en) * | 2016-12-05 | 2018-06-14 | The Administrators Of The Tulane Educational Fund | Arenavirus monoclonal antibodies and uses |
| CN110261620A (en) * | 2019-07-05 | 2019-09-20 | 江苏省人民医院（南京医科大学第一附属医院） | Cell immunofluorescence kit for determining human serum phosphotyrosine adaptor protein ShcA antibody and preparation method and application thereof |
| CN112945919A (en) * | 2021-01-29 | 2021-06-11 | 上海睿钰生物科技有限公司 | Detection method, system and application of virus neutralizing antibody |
| KR20210130049A (en) * | 2020-04-21 | 2021-10-29 | 대한민국(질병관리청장) | An antibody specific for nucleoprotein of lassa virus, hybridoma cell line producing the same, and a kit for detecting lassa virus using the same |
-
2015
- 2015-12-28 CN CN201511005030.2A patent/CN105548539B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| CHRISTIAN KLEWITZ ET AL: "Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity", 《EMBO REPORTS》 * |
| D. G. BAUSCH ET AL: "Diagnosis and Clinical Virology of Lassa Fever as Evaluated byEnzyme-Linked Immunosorbent Assay, Indirect Fluorescent-Antibody Test, and Virus Isolation", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018106712A1 (en) * | 2016-12-05 | 2018-06-14 | The Administrators Of The Tulane Educational Fund | Arenavirus monoclonal antibodies and uses |
| US11198723B2 (en) | 2016-12-05 | 2021-12-14 | The Administrators Of The Tulane Educational Fund | Arenavirus monoclonal antibodies and uses |
| CN110261620A (en) * | 2019-07-05 | 2019-09-20 | 江苏省人民医院（南京医科大学第一附属医院） | Cell immunofluorescence kit for determining human serum phosphotyrosine adaptor protein ShcA antibody and preparation method and application thereof |
| KR20210130049A (en) * | 2020-04-21 | 2021-10-29 | 대한민국(질병관리청장) | An antibody specific for nucleoprotein of lassa virus, hybridoma cell line producing the same, and a kit for detecting lassa virus using the same |
| KR102404143B1 (en) | 2020-04-21 | 2022-05-31 | 대한민국(질병관리청장) | An antibody specific for nucleoprotein of lassa virus, hybridoma cell line producing the same, and a kit for detecting lassa virus using the same |
| CN112945919A (en) * | 2021-01-29 | 2021-06-11 | 上海睿钰生物科技有限公司 | Detection method, system and application of virus neutralizing antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105548539B (en) | 2018-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gong et al. | Evaluation and comparison of serological methods for COVID-19 diagnosis | |
| CN101587043B (en) | Integrated method for enriching and detecting rare cell in biological fluid sample | |
| CN103323599B (en) | Time resolved immunofluorescence detection kit for rabies virus nucleoprotein, and preparation method thereof | |
| CN101196518B (en) | Hepatitis virus type C immune body chemiluminescence method diagnostic reagent kit and its producing method | |
| CN105548539A (en) | Indirect immunofluorescence detection method for Lassa Virus IgG antibody | |
| CN105259354B (en) | Kit for detecting tuberculosis T cell release gamma-interferon and use method of kit | |
| Granade et al. | Rapid detection and differentiation of antibodies to HIV-1 and HIV-2 using multivalent antigens and magnetic immunochromatography testing | |
| CN102731615A (en) | Detection reagent and detection method for PRRSV | |
| EP4113121A1 (en) | Antigen for 2019 novel coronavirus and detection use thereof | |
| CN105137072A (en) | Mycobacterium tuberculosis LAM (lipoarabinomannan) detection kit, preparation and use method thereof | |
| CN105738629B (en) | A kind of indirect immunofluorescene assay method of yellow fever virus IgG antibody | |
| CN106119415A (en) | A kind of test kit utilizing one-step method real-time fluorescent quantitative RT qPCR detection hepatitis C virus nucleic acid | |
| CN105738624A (en) | Indirect immuno-fluorescence detection method of rift valley fever virus IgG antibody | |
| CN110452921A (en) | The packaging and serum neutralizing antibody detection method of 10 type pseudovirus of Coxsackie virus A | |
| CN101403746B (en) | Conjugate used for immunity detection | |
| CN102313813B (en) | Integration method for enriching and detecting rare cells from biological fluid samples | |
| Major et al. | Detection of antibodies to HCV E1E2 by lectin-capture ELISA | |
| Chan et al. | Use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection | |
| CN108254555A (en) | A kind of fluorescence immunoassay for SFTSV detections detects card and its preparation method and application | |
| CN101762703A (en) | HCV (Hepatitis C Virus) antibody detection kit as well as preparation method and detection method thereof | |
| CN105330727B (en) | 1 type duck hepatitis A virus VP4 recombinant protein of one kind, ELISA kit and preparation method thereof | |
| CN109633163B (en) | procalcitonin/C reactive protein two-in-one detection kit | |
| CN106632618A (en) | Preparation method of swine hepatitis E virus ORF2 recombinant protein as well as ORF2 protein thereof and detection kit | |
| CN105403706A (en) | Heat shock protein 90 (HSP90) chemiluminescence detection kit | |
| Szeredi et al. | Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Tian Zhengan Inventor after: Li Shenwei Inventor after: Cha Qi Inventor after: Zhang Zilong Inventor after: Yang Liu Inventor after: Lou Yating Inventor after: Cao Min Inventor before: Tian Zhengan Inventor before: Zhang Zilong Inventor before: Yang Liu Inventor before: Li Shenwei Inventor before: Lou Yating Inventor before: Cao Min |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |