CN102778568B - Preparation and application of total antibody ELISA (enzyme linked immunosorbent assay) kit for detecting fever accompanied by thrombocytopenia syndrome virus - Google Patents
Preparation and application of total antibody ELISA (enzyme linked immunosorbent assay) kit for detecting fever accompanied by thrombocytopenia syndrome virus Download PDFInfo
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- CN102778568B CN102778568B CN201210121490.1A CN201210121490A CN102778568B CN 102778568 B CN102778568 B CN 102778568B CN 201210121490 A CN201210121490 A CN 201210121490A CN 102778568 B CN102778568 B CN 102778568B
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Abstract
The invention relates to preparation and application of a total antibody ELISA (enzyme linked immunosorbent assay) kit for detecting fever accompanied by thrombocytopenia syndrome virus. The kit comprises a gene recombination NP (nucleoprotein)-coated ELISA reaction plate, a horseradish peroxidase (HRP)-coupled NP (HRP-rNP) and a tetramethyl benzidine (TMB) color development reagent. The kit can be used for state evaluation after novel bunyavirus infection, epidemiological investigation of novel bunyavirus infection of people and immune effect evaluation of novel bunyavirus inactivated vaccines.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation and application that detects the total antibody ELISA kit of heating companion's decrease of platelet syndrome virus.
background technology
New bunyavirus (Novel Bunyavirus, SFTS Bunyavirus), heating companion decrease of platelet syndrome (Fever with Throbocytopenia Associated Syndrome, SFTS) cause of disease, for a kind of new virus of bunyaviridae phlebotomus fever virus genus, first finding in China in 2009, is also the first new virus of finding of China.Patient occurs, taking high heat, decrease of platelet, white blood cell reduction, multiple organ dysfunction disorder, the serious acute infectious disease as feature such as hemorrhage, having a strong impact on the people's health and lives safety.Up to the present, 16 provinces such as existing Henan, the whole nation, Jiangsu, Hubei, Shandong, Anhui and Liaoning find 300 multiple-cases, cause more than 40 people's death.Disease Control and Prevention Center of country, Disease Control and Prevention Center of Jiangsu Province are successively successfully separated to this disease virus, and it have been carried out to the researchs such as aetology, epidemiology, clinical medicine.This virus sequence is illustrated at present; examination criteria based on RT-PCR is set up; but it is not also perfect to the immunology detection reagent of SFTS; make to evaluate this virus infections and lack a confirmation method to PCR result, also cannot assess the previous infection level of colony and the protective effect of vaccine.
Summary of the invention
One of object of the present invention is to provide a strain new, for detection of the new bunyavirus JS-2007-001 of heating companion decrease of platelet syndrome virus, the new bunyavirus of this virus taxis called after (Severe fever with thrombocytopenia syndrome virus; Novel Bunyaviridae), its preserving number is CCTCCV201211; The preservation time is on March 1st, 2012; Depositary institution is: Chinese Typical Representative culture collection center; Preservation address is: Luo Jia Shan, Wuhan University's Life Science College wuchang, wuhan.
Two of object of the present invention has been to provide utilizes above-mentioned new bunyavirus JS-2007-001 preparation to detect total antibody EISA kit of heating companion decrease of platelet syndrome virus.
The invention has the advantages that; find through adaptability cultivation, Immunity identification and immanoprotection action qualification; the JS-2007-001 virus seed culture of viruses screening in the present invention has best growth characteristics and adaptive character; reach summit of growth within a short period of time; can stablize and obtain compared with infectious titer; it is higher that immune animal obtains antibody horizontal, becomes a kind of stable detection that can commercial applications and generate heat and accompany the kit of decrease of platelet syndrome virus thereby can develop.
Preparation and the application of the open a kind of ELISA kit that detects new bunyavirus NP albumen (being new bunyavirus nucleoprotein) antibody of the present invention, kit comprises the coated ELISA reaction plate of rNP (ribonucleoprotein (RNP)), HRP-rNP, tetramethyl benzidine (TMB) developer.The present invention openly adopts reverse transcription PCR method new bunyavirus (JS-2007-001 Strain) the NP albumen open reading frame sequence that increases to insert pQE30 and build pQE30-NP expression plasmid, with pQE30-NP transfection M15 bacterial strain, conventional with IPTG abduction delivering 6His-NP recombinant protein, Hitrap purifying 6His-NP recombinant protein, and be coated in ELISA reaction plate, conventional the hatching with sample to be tested of this substrate can be in conjunction with the anti-NP antibody in sample, subsequent detection is used HRP-rNP albumen, with zymolyte TMB colour developing, and draw antibody titer with microplate reader analysis.For the evaluation of the early stage and happy phase infection conditions to Xin Buni virus.For the evaluation of antibody horizontal after the epidemiologic observation of population infection and vaccine immunity.
Further, the present invention relates to one for detection of the new total antibody ELISA kit of bunyavirus, described kit comprises following auxiliary reagent: reaction plate, HRP-rNP albumen, zymolyte tetramethyl benzidine (TMB) developer that rNP albumen is coated; The wherein said coated reaction plate of rNP albumen is prepared by the following method: adopt SEQ ID No.1 and SEQ ID No.2 as amplimer, be the NP albumen open reading frame sequence of the new bunyavirus JS-2007-001 of CCTCC V201211 by reverse transcription PCR method amplification preserving number, subsequently the sequence after amplification is inserted to pQE30 and build pQE30-NP expression plasmid, use subsequently pQE30-NP transfection M15 bacterial strain, adopt again conventional IPTG abduction delivering 6His-NP recombinant protein, Hitrap purifying 6His-NP recombinant protein, and be coated in ELISA reaction plate.
Kit of the present invention, it is 5mIU/mL to new bunyavirus NP specific antibody limit of identification.
The invention still further relates to the application of kit in the diagnostic reagent for the preparation of the infection conditions of Xin Buni virus is assessed.
The invention still further relates to the application of the described kit diagnostic reagent that antibody horizontal is assessed after the epidemiologic observation for the preparation of for population infection and vaccine immunity.
Preparation method of the present invention, the main quality index of kit is: reagent quality index reaches 2010 editions " Chinese Pharmacopoeias " the 3rd, the requirement in in-vitro diagnosis class code.Comprising sensitivity, specificity, accuracy, stability.
Its detection sensitivity of the reagent of preparing taking the present invention is 5mIU/mL; Specificity checking: with other equal bunyavirus antibody no cross reaction; Accuracy checking: reagent differences between batches (CV%) are not higher than 15%.
Brief description of the drawings
Fig. 1 is the preparation flow figure of new bunyavirus antibody ELISA detection kit.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described.The present embodiment only, for to explanation of the present invention, is not construed as limiting the invention.
Expression and the purifying of embodiment 1:rNP albumen
The preserving number of getting deactivation is the new bunyavirus JS-2007-001 strain of CCTCC V201211, adopts conventional QIAamp RNA viral RNA mini kit (Qiagen, Germany) to prepare viral RNA template.
Adopt following primer as being amplimer:
SEQ?ID?No.1：
Primer 1 5`-ggatccgcatgcATGTCAGAGTGGTCCAGGATTG-3`;
SEQ?ID?No.2：
Primer 2 5`-gccaagcTTACAGATTCCTGTAAGCAGCAGCAG-3`;
Utilize conventional Invitrogen One step RT-PCR kit (Invitrogen, USA) the new bunyavirus NP albumen open reading frame that increases; Adopt conventional molecular biology method that this reading frame is inserted between the site SphI and HindIII of bacterial expression plasmid pQE30 (Qiagen, Germany), obtain bacterial expression plasmid pQE30-NP.By the conventional Transformed E .Coli of this plasmid M15 bacterial strain (Qiagen, Germany), the method abduction delivering 6His-NP recombinant protein of recommending by manufacturer, and with Hitrap (Qiagen, Germany) conventional purifying 6His-NP recombinant protein, SDS-PAGE detects protein molecular weight and purity, and conventional protein quantification is for subsequent use.
The coated ELISA reaction plate preparation of embodiment 2:rNP albumen.
Purifying NP albumen is diluted to (10 μ g/mL) with the coated damping fluid of 0.05M pH9.6 carbonate.Routine adds the NP protein liquid having diluted in reaction plate hole, and every hole 100 μ L, put 2～8 DEG C of coated 18-20 hour, discard liquid in hole, wash plate 3～5 times, and every hole adds enzyme stabilizers (Shandong, safe day and biological) 150～200 μ L.2～8 DEG C are reacted 4～6 hours.Discard enzyme stabilizers in hole, adopt the dry coated plate of desivac, with sealing bag encapsulated reaction plate, put 2～8 DEG C and save backup.
The preparation of embodiment 3:HRP-rNP albumen.
Adopt improvement sodium periodate method (basic medical immunological experiment, Jin Baiquan, Li Enshan.Beijing world book publishing house, 1990) mark rNP.5mg HRP is dissolved in 0.5mL distilled water, add the NalO4 aqueous solution 0.5mL of freshly prepared 0.06M, mix and put 4 DEG C of refrigerators 30 minutes, taking-up adds the glycol water 0.5mL of 0.16M, room temperature is placed after 30 minutes and is added the aqueous solution 1mL containing purifying NP albumen, mix and fill bag filter, with 0.05M, the carbonate buffer solution of pH9.5 slowly stirs dialysis and within 6 hours, makes it combination in 4 DEG C of refrigerators, then sucking-off, add NaBH4 solution (5mg/mL) 0.2mL, put 4 DEG C of refrigerators 2 hours, above-mentioned bond mixed liquor is added to equal-volume saturated ammonium sulfate solution, put 4 DEG C of refrigerators centrifugal after 30 minutes, gained sediment is dissolved in to a little 0.02M, in pH7.4PBS, and 4 DEG C of dialysed overnight, next day the centrifugal insolubles of removing, obtain HRP-rNP albumen, with 0.02M, pH7.4PBS is diluted to 5mL, add the glycerine that contains stabilizing agent, final concentration 50%, mixing rearmounted low temperature saves backup.
Embodiment 4: Quality Control reference material, auxiliary reagent preparation and packing
HRP-rNP albumen working fluid: see embodiment 3.
Antibody positive contrast: with the damping fluid that contains enzymatic protective reagent, the anti-rNP protein antibodies after purifying is quantitatively diluted, by marking of kit of the present invention, determine antibody content.By the accurate packing of reference material after quantitative, 4 DEG C of preservations are put in the sealing of freeze drying rear pressing cover.
Negative antibody contrast: normal human serum.
Lavation buffer solution (0.15M KH
2pO
4pH7.4 PBS): 0.2 gram, Na
2hPO
412H
2o 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5mL, adding distil water is to 1000mL.
Sample diluting liquid: BSA 0.1g, adds lavation buffer solution to 100mL.
Substrate nitrite ion A1: substrate buffer solution (pH 5.0 phosphoric acid Jujube citric acids): 0.2M Na
2hPO
425.7mL, 0.1M citric acid 24.3mL, adding distil water 50mL.
Substrate nitrite ion B1:TMB (10mg/5mL absolute ethyl alcohol) 0.5mL, substrate buffer solution (pH 5.5) 10mL, 0.75% H
2o 32 μ L.
2M H
2sO
4stop buffer: distilled water 178.3mL, dropwise adds the concentrated sulphuric acid (98%) 21.7mL.
1 of the enzyme-linked reaction plate (96 hole) that packing: rNP is coated; 1 bottle of HRP-rNP working fluid (10mL); 1 bottle (0.5mL) of antibody positive contrast; 1 bottle (0.5mL) of negative antibody contrast; 1 bottle of 10mL of sample diluting liquid (containing 0.1%BSA 0.15M PBS damping fluid (pH7.4)); 1 bottle of 200mL of 10 times of cleansing solutions (1.5M PBS T damping fluid, pH7.4); Substrate nitrite ion A1 bottle 6mL; Substrate nitrite ion B1 bottle 6mL; 2M H
2sO
41 bottle of 6mL of stop buffer.
Embodiment 5: kit detects new bunyavirus specific total antibodies
Qualitative detection: every hole adds after sample diluting liquid 50 μ L, adds negative control, positive control and sample to be checked respectively in respective aperture, 50 μ L/ holes.
Quantitatively detect: sample does serial dilution with sample diluting liquid in clean small test tube, adds in respective aperture 50 μ L/ holes.Blank 1 hole (only adding developer and stop buffer) is established in each experiment, by reaction plate shrouding membrane closure, puts in 37 DEG C of water-baths and hatches 30min.
Hatch after termination and wash plate 4～5 times with cleansing solution, remaining liquid in clean thieving paper arsis dry hole.
Except blank well, in each hole, add HRP-rNP working fluid 100 μ L/ holes, in 37 DEG C of water-baths, hatch 30min.
Hatch after termination and wash plate 4～5 times with cleansing solution, remaining liquid in clean thieving paper arsis dry hole.
In each hole, add developer A, the each 50 μ L/ holes of B liquid, in 37 DEG C of water-baths, hatch 10min.
Every hole adds stop buffer 50 μ L, softly shakes 20s and mixes.
ELISA Plate is put under microplate reader 450nm wavelength, with blank well zeroing, measure each hole light absorption A value.
Experimental result must meet following actual parameter, and experiment can be set up: positive control A value >=1.5; Negative control A value≤0.1.If test figure does not meet above-mentioned test validity standard, need revision test.
Qualitatively judge: if negative control average A value < 0.05 calculates by 0.05; If negative control average A value > 0.05, calculates by actual A value.The calculating of cutoff value: cutoff=negative control average A value × 2.1.Testing result >=cutoff value is judged to the positive, and testing result < cutoff value is judged to feminine gender.
Quantitatively judge: sample, after serial dilution, is measured each dilutability A value, be judged to antibody titer terminal, the quantitative unit using sample dilutability as antibody horizontal with the high dilution A value >=cutoff value of sample.
Embodiment 6: kit quality control.
Its detection sensitivity of the reagent of preparing taking the present invention is 5mIU/mL; Specificity checking, with other equal bunyavirus antibody no cross reaction; Accuracy checking, reagent differences between batches (CV%) should be higher than 15%.
Claims (5)
1. for detection of the new total antibody ELISA kit of bunyavirus, described kit comprises following auxiliary reagent: reaction plate, HRP-rNP albumen, zymolyte tetramethyl benzidine (TMB) developer that rNP albumen is coated; The wherein said coated reaction plate of rNP albumen is prepared by the following method: adopt SEQ ID No.1 and SEQ ID No.2 as amplimer, be the NP albumen open reading frame sequence of the new bunyavirus JS-2007-001 of CCTCC V201211 by reverse transcription PCR method amplification preserving number, subsequently the sequence after amplification is inserted to pQE30 and build pQE30-NP expression plasmid, use subsequently pQE30-NP transfection M15 bacterial strain, adopt again conventional IPTG abduction delivering 6His-NP recombinant protein, Hitrap purifying 6His-NP recombinant protein, and be coated in ELISA reaction plate.
2. kit as claimed in claim 1, it is 5mIU/mL to new bunyavirus NP specific antibody limit of identification.
3. the application of kit claimed in claim 1 in the diagnostic reagent for the preparation of the infection conditions of Xin Buni virus is assessed.
4. the application in the kit claimed in claim 1 diagnostic reagent that antibody horizontal is assessed after the epidemiologic observation for the preparation of population infection and vaccine immunity.
5. the preparation method for detection of the new total antibody ELISA kit of bunyavirus as claimed in claim 1.
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| CN103954756B (en) * | 2014-05-12 | 2015-11-18 | 郑州伊美诺生物技术有限公司 | A kind of people of detection generates heat the kit of companion's thrombocytopenic syndromes antiviral antibody IgG and application thereof |
| CN105510580B (en) * | 2015-12-01 | 2017-03-22 | 浙江普康生物技术股份有限公司 | Polypeptide-ELISA (enzyme-linked immunosorbent assay) kit for detecting specific antibody of N (nucleocapsid) protein of SFTSV (severe fever with thrombocytopenia syndrome virus) |
| CN107727848A (en) * | 2017-09-25 | 2018-02-23 | 贵州省人民医院 | A kind of colloidal gold immuno-chromatography test paper strip for detecting people and the anti-SFTS antibody of animal blood serum |
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| WO2005054854A1 (en) * | 2003-11-05 | 2005-06-16 | The United States Of America As Represented By The Secretary Of The Navy Naval Medical Research Center | Fluorescence polarization instruments and methods for detection of exposure to biological materials by fluorescence polarization immunoassay of saliva, oral or bodily fluids |
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| CA2775655A1 (en) * | 2005-01-20 | 2006-07-20 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Di | Multiplexed analysis for determining a serodiagnosis of viral infection |
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