CN101710120A - Application of stabilizer trehalose in pig hepatitis E diagnostic kit - Google Patents
Application of stabilizer trehalose in pig hepatitis E diagnostic kit Download PDFInfo
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- CN101710120A CN101710120A CN200910201070A CN200910201070A CN101710120A CN 101710120 A CN101710120 A CN 101710120A CN 200910201070 A CN200910201070 A CN 200910201070A CN 200910201070 A CN200910201070 A CN 200910201070A CN 101710120 A CN101710120 A CN 101710120A
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Abstract
The invention discloses an application of stabilizer trehalose in a pig hepatitis E diagnostic kit; 8-12wt percent of trehalose is added in enzyme combination diluent of the pig hepatitis E diagnostic kit, the function of the enzyme combination diluent is kept to be stable by utilizing the stabilizer trehalose, so as to keep the stabilization of the pig hepatitis E diagnostic kit.
Description
Technical field
The present invention relates to the application of a kind of stabilizing agent trehalose in pig viral hepatitis type E diagnostic kit.
Background technology
Hepatitis E virus (Hepatitis E virus, HEV) be a kind of non-first-non-hepatitis b disease poison of propagating through fecal-oral route, can cause outbreak of epidemic the crowd, main infringement is between twenty and fifty, can cause 20% case fatality rate the pregnant woman, 1986-1988 Xinjiang of China southern areas cause Hepatitis E popular because of contaminated drinking water, 119280 examples of falling ill altogether, dead 707 examples are that a Hepatitis E of maximum is popular in the world so far.The Hepatitis E incidence of disease has the trend that increases year by year in recent years, and according to China Ministry of Public Health statistics, viral hepatitis type E incidence of disease ratio in 2003 rose 40.4% in 2002.Therefore, the work schedule of NSFs at different levels has been listed in the viral hepatitis type E prevention and control in.
Recent study is found: HEV can infected pigs, ox, sheep, mouse, chicken etc., is higher than 50% in the antibody positive rate of pig and mouse, infers that thus animal is the important intermediate host that HEV propagates.Gene sequencing shows: hepatitis E virus of separating in the animal body and people's HEV have high dna homolog, and edible brawn of existing people and venison and infect the report of viral hepatitis type E, prove viral hepatitis type E be a kind of can be by food-borne infecting both domestic animals and human eqpidemic disease.Ge Shengxiang etc. (2003) have carried out the HEV serosurvey to 8626 adult pigs on 120 pig farms of 20 provinces of China, the result shows that average infection positive rate is 83.4%, wherein the highest province is the Inner Mongol (100%), and minimum is Hubei (68%).In view of the high infection rate of China HEV, epi-position to pig viral hepatitis type E virus is studied, set up special, effective pig viral hepatitis type E detection method, develop safe and effective pig viral hepatitis type E vaccine, the popular occurrence regularity of understanding, block this disease by the propagation of pig, for guaranteeing that the people's is healthy and safe significant to the people.
In recent years, increasing Hepatitis E diagnostic kit is developed and develops.The standard of estimating diagnostic kit should comprise aspects such as sensitivity, specificity, accuracy and stability.Wherein stability promptly after the reagent preparation, is stored and is transported through after a while for estimating the important indicator of this diagnostic reagent, and whether activity changes, and is the importance of reagent inherent quality.Advanced elisa kit antigen is coated on the microwell plate in advance, and enzyme conjugates is diluted to working concentration, and developer is made into liquid, more than three kinds of composition activity easily lose.How making it stable is an important subject, and the report of relevant this respect is very few.[Rong Guangya such as Rong Guangya; Sun Jie; Zhou Jiwen etc.; the development of hepatitis E diagnostic kit and application thereof; China's virology, 1998,13 (1): 64-69] stability of confining liquid in people's Hepatitis E diagnostic kit and enzyme conjugates dilution is studied; the result proves: trehalose adds in the confining liquid can protect the viral hepatitis type E antigen active effectively, and trehalose adds in the enzyme conjugates can make the HRP-goat anti-human igg who is diluted to working concentration activity stabilized.But, up to the present, also do not have the application report of stabilizing agent trehalose at pig viral hepatitis type E diagnostic kit.
Though the research of pig viral hepatitis type E more and more obtains the attention of scientific circles, but at present both at home and abroad the research of pig viral hepatitis type E is limited to epidemiology survey and some simple zoogenetic infections tests of regional area more, followingly remains to be answered a question and technical bottleneck still having aspect the pig viral hepatitis type E prevention and control research.The epi-position research report that does not promptly also have at present pig viral hepatitis type E virus, the kit that is used for the detection of pig viral hepatitis type E on the market is to be used for the kit that people's viral hepatitis type E detects, though pig viral hepatitis type E virus is very high with people's viral hepatitis type E virus sequence homology, but between them still be some difference after all, it is just special not high that the viral hepatitis type E virus epitopes of personnel selection detects pig viral hepatitis type E virus, can not specially reflect pig viral hepatitis type E regularty of epidemic.Therefore stable, quick, the special pig viral hepatitis type E diagnostic kit of development can be used for the popular monitoring of pig viral hepatitis type E on the one hand; Also can control the popular of pig viral hepatitis type E on the other hand, the blocking-up viral hepatitis type E is propagated to the people by pig and is laid a solid foundation, for guaranteeing that the people's is healthy and safe significant.
Summary of the invention
Technical matters to be solved by this invention provides the application of a kind of stabilizing agent trehalose in pig viral hepatitis type E diagnostic kit, utilizes the stabilizing agent trehalose to keep stable function at the enzyme conjugates dilution, keeps the stability of pig viral hepatitis type E virus kit.
Pig viral hepatitis type E virus kit of the present invention is with [Zhao Kai such as Zhao Kai, Liu Qiwen, Yu Ruisong, et al.Screening of specific diagnostic peptides of swine hepatitis Evirus-Virol J, 2009,6:186-doi:10.1186/1743-422X-6-186.] the synthetic polypeptide swHEV11 from pig HEV swCH25 that screens is made as envelope antigen, its preparation method is as follows:
Antigen diluent: get polypeptide that 10ul concentration is 10mg/ml (polypeptide that 5mg is synthetic is dissolved in 0.5ml dimethyl sulfoxide (DMSO) (DMSO) and gets) and 90ul PBS liquid mixing, the antigenic dilution solubility that obtains is 1ug/ul.Get 15ul antigenic dilution and 5ml coating buffer mixing again, the antigen diluent solubility that obtains is 3ug/ml.Add antigen diluent solubility 100ul/ hole on ELISA Plate, 4 ℃ of bags are by 12h.
Sealing: inhale and to go bag by night, every hole adds the 200ul confining liquid behind 37 ℃ of sealing 2h, wash plate 3 times with TBST liquid after, pat dry, ELISA Plate is sealed is put under 4 ℃, for future use.
The principal ingredient of described pig viral hepatitis type E virus kit is:
Pig HEV positive control serum, pig HEV negative control sera, HRP mark IgG enzyme conjugates dilution, sample diluting liquid, 0.1%TBST (cleansing solution), substrate solution, stop buffer.
Described stabilizing agent trehalose can keep the stability of this pig viral hepatitis type E diagnostic kit at diagnosis pig viral hepatitis type E antiviral antibody.
Wherein, be added with trehalose in the enzyme conjugates dilution of this pig viral hepatitis type E diagnostic kit, its addition is the 8-12wt% of enzyme conjugates dilution.Its concrete application is as follows:
After enzymic-labelled antibody was diluted to working concentration, activity was very unstable.The present invention adds the trehalose stabilizing agent of enzyme conjugates dilution 10wt% in dilution, observe the goat-anti pig IgG protective effect of stabilizing agent to horseradish peroxidase (HRP) mark.After adding stabilizing agent, behind enzyme labelled antibody to 37 ℃, the 4d, active no significant change is not almost completely lost (referring to table 1) and do not add stabilizing agent enzyme labelled antibody activity.Illustrate that the stabilizing agent trehalose is to two anti-effects with stable protection.
Table 1 enzyme conjugates dilution is to the protective effect of antigen active
Wherein: 1 is not contain trehalose in the enzyme conjugates dilution, and 2 is to contain trehalose in the enzyme conjugates dilution.
Beneficial effect:
The present invention utilizes the stabilizing agent trehalose can make the enzyme conjugates dilution keep stable function, has kept the stability of pig viral hepatitis type E virus kit effectively.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is described in further detail.
One, experiment reagent and material
Polypeptide swHEV11 (synthetic) by GL-Biochem (shanghai) Ltd. Peptide synthesizer, Na
2HPO
4(Shanghai Xinhua chemical plant), NaH2PO
4(Shanghai Xinhua chemical plant), Na
2CO
3(Shanghai reagent two factories), NaHCO
3(Shang Hai Wen And-Yan biochemical technology company limited); NaCl (Chemical Reagent Co., Ltd., Sinopharm Group); KCl (a chemical reagent company limited is tried in Shanghai); Tris-Base (the two-way Bath development in science and technology in Shanghai company limited); Tween-20 (Farco Chemical Supplies); dimethyl sulfoxide (DMSO) (Shanghai Ling Feng chemical reagent company limited); 3; 3; 5,5-tetramethyl benzidine (TMB) (Shanghai Si Gaole bio tech ltd), citric acid (Shanghai chemical reagent company limited); trisodium citrate (Chemical Reagent Co., Ltd., Sinopharm Group), H
2O
2(Shanghai chemical reagent company limited); concentrated hydrochloric acid (Chemical Reagent Co., Ltd., Sinopharm Group); BSA (Shanghai Si Gaole bio tech ltd); two anti-(Immunology ConsultantsLaboratory; Inc.); D-trehalose (Shanghai Hu Feng bio tech ltd), ten thousand safe kits (Beijing Tso Biological Pharmaceutical Co).ELISA Plate (Xiamen City cloud roc development in science and technology company limited), electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), wash plate machine (Wellwash 4 MK2) and microplate reader (Multiskan MK3) (Thermo electron corporation), PH detector (Mettler Toledo), micro oscillator and eddy mixer (its woods Bel instrument Manufacturing Co., Ltd of Haimen City).
Two, the preparation of detected materials
Gather pig blood from the slaughterhouse, pig blood is put 37 ℃ of incubator 1h again in the room temperature natural coagulation, puts 4 ℃ of refrigerator overnight then, treats separation of serum after the clot contraction, and serum is light yellow transparent liquid.With ten thousand safe kits detect institute take a blood sample clear, its detected positive serum that comes as positive control, its detected negative serum as negative control.
Three, enzyme linked immunological absorption (ELISA) method is set up
With [Zhao Kai such as Zhao Kai, Liu Qiwen, Yu Ruisong, et al.Screening ofspecific diagnostic peptides of swine hepatitis E virus.Virol J, 2009,6:186.doi:10.1186/1743-422X-6-186.] the synthetic polypeptide swHEV11 from pig HEV swCH25 that screens is made into pig viral hepatitis type E diagnostic kit as envelope antigen.
Embodiment 1
In the enzyme conjugates dilution, add the stabilizing agent trehalose and do not add the mensuration of the ELISA Plate stability of stabilizing agent trehalose
(1) antigen diluent
Get polypeptide that 10 μ l concentration are 10mg/ml (polypeptide that 5mg is synthetic is dissolved in 0.5ml dimethyl sulfoxide (DMSO) DMSO and gets) and 90 μ l PBS liquid mixings, the antigenic dilution solubility that obtains is 1ug/ μ l.Get 15 μ l antigenic dilutions and 5ml coating buffer mixing again, the antigen diluent solubility that obtains is 3 μ g/ml.Add antigen diluent solubility 100 μ l/ holes on ELISA Plate, 4 ℃ of bags are by 12h.
(2) sealing
Coating buffer is removed in suction, and every hole adds 200 μ l confining liquids in 37 ℃ of sealing 2h.
(3) adding one resists
Wash plate 3 times with TBST liquid, every hole adds 100 μ l sample diluting liquids and 10 μ l serum, and the concussion mixing is put 37 ℃ of incubation 1h.
(4) added for two anti-(contain the stabilizing agent trehalose of 8wt%, 10wt%, 12wt% in the enzyme conjugates dilution respectively and do not contain trehalose, the enzyme conjugates dilution is placed 37 ℃ of following 4d).
Wash plate 5 times with TBST liquid, it is anti-that every hole adds 100 μ l two, puts 37 ℃ of incubation 1h.The goat-anti pig IgG of two anti-middle enzyme conjugates dilutions and horseradish peroxidase (HRP) mark is with dilution in 1: 10000.
(5) colour developing
Wash plate 5 times with TBST liquid, every hole adds substrate solution 100ul, 37 degree colour developing 10min.
(6) stop
The stop buffer that every hole adds 50 μ l 1mol/l HCl comes cessation reaction, surveys the OD450 value with microplate reader, and its result is referring to table 1.From this result as can be seen: after adding stabilizing agent, behind enzyme labelled antibody to 37 ℃, the 4d, active no significant change is not almost completely lost and do not add stabilizing agent enzyme labelled antibody activity, illustrate the stabilizing agent trehalose to two anti-have stablize the effects of protecting.
Annotate: the prescription of above-mentioned each solution:
PBS liquid (pH7.4): Na
2HPO
40.575g, NaH
2PO
40.114g, distilled water 500ml.
Coating buffer (pH9.6): Na
2CO
31.59g, NaHCO
32.93g, distilled water 1000ml.
Confining liquid: BSA (bovine serum albumin(BSA)) 3g, TBS (TBS solution) liquid (pH8.6) 100ml.
TBS liquid (pH8.6): NaCl 16g, KCl 0.4g, Trizma-Base (tromethamine alkali) 2.42g, distilled water 2000ml.
TBST (triethanolamine polysorbas20 buffer salt solution) liquid (pH8.6): TBS liquid (pH8.6) 1000ml, Tween-20 1.0ml.
Sample diluting liquid: BSA 1g, TBST liquid 100ml.
Enzyme conjugates dilution: BSA 1g, TBST liquid 100ml.
Citrate buffer solution (pH3.5-4.0): citric acid 0.84g, sodium citrate 0.294g, distilled water 100ml.
Stop buffer (1M HCl): concentrated hydrochloric acid (content 36-38wt%) 40.88ml, distilled water 459.12ml.
Substrate solution: 10ml citrate buffer solution
TMB faces with preceding adding 40mg
30%H
2O
2Face with preceding adding 3 μ l.
Should be noted that at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.
Claims (3)
1. the application of stabilizing agent trehalose in pig viral hepatitis type E diagnostic kit is characterized in that, the application of stabilizing agent trehalose when keeping described pig viral hepatitis type E diagnostic kit diagnosis pig viral hepatitis type E antiviral antibody stable.
2. the application of stabilizing agent trehalose according to claim 1 in pig viral hepatitis type E diagnostic kit is characterized in that, adds trehalose in the enzyme conjugates dilution of described pig viral hepatitis type E diagnostic kit.
3. the application of stabilizing agent trehalose according to claim 2 in pig viral hepatitis type E diagnostic kit is characterized in that the addition of described trehalose is the 8-12wt% of enzyme conjugates dilution.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375056A (en) * | 2010-08-27 | 2012-03-14 | 烟台赛尔斯生物技术有限公司 | Immobilized bio-macromolecular stabilizer as well as preparation method and application thereof |
| CN103823057A (en) * | 2014-03-07 | 2014-05-28 | 中国农业科学院兰州兽医研究所 | Colloidal gold test strip for quick diagnosis of total swine HEV (Hepatitis E Virus) antibody |
| CN103837680A (en) * | 2014-03-07 | 2014-06-04 | 中国农业科学院兰州兽医研究所 | Method for preparing porcine hepatitis E virus (HEV) total antibody enzyme-linked immuno sorbent assay (ELISA) detection kit |
| CN104792981A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Enzyme-labeled antibody conjugate stabilizer and application thereof |
| CN111896744A (en) * | 2020-07-28 | 2020-11-06 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
| CN112684177A (en) * | 2020-12-17 | 2021-04-20 | 北京维德维康生物技术有限公司 | Dairy multi-factor rapid detection kit and detection method thereof |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN112684177B (en) * | 2020-12-17 | 2024-05-28 | 北京维德维康生物技术有限公司 | Milk product multi-factor rapid detection kit and detection method thereof |
-
2009
- 2009-12-14 CN CN200910201070A patent/CN101710120A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| ZHAO KAI等: "Screening of specific diagnostic peptides of swine hepatitis E virus", 《VIROLOGY JOURNAL》 * |
| 戎广亚等: "新型戊型肝炎诊断试剂盒的研制及其应用", 《中国病毒学》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102375056A (en) * | 2010-08-27 | 2012-03-14 | 烟台赛尔斯生物技术有限公司 | Immobilized bio-macromolecular stabilizer as well as preparation method and application thereof |
| CN104792981A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Enzyme-labeled antibody conjugate stabilizer and application thereof |
| CN103823057A (en) * | 2014-03-07 | 2014-05-28 | 中国农业科学院兰州兽医研究所 | Colloidal gold test strip for quick diagnosis of total swine HEV (Hepatitis E Virus) antibody |
| CN103837680A (en) * | 2014-03-07 | 2014-06-04 | 中国农业科学院兰州兽医研究所 | Method for preparing porcine hepatitis E virus (HEV) total antibody enzyme-linked immuno sorbent assay (ELISA) detection kit |
| CN103823057B (en) * | 2014-03-07 | 2016-01-20 | 中国农业科学院兰州兽医研究所 | Total antibody colloidal gold fast diagnose test paper bar of one boar HEV and preparation method thereof |
| CN111896744A (en) * | 2020-07-28 | 2020-11-06 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
| CN111896744B (en) * | 2020-07-28 | 2022-02-22 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
| CN112684177A (en) * | 2020-12-17 | 2021-04-20 | 北京维德维康生物技术有限公司 | Dairy multi-factor rapid detection kit and detection method thereof |
| CN112684177B (en) * | 2020-12-17 | 2024-05-28 | 北京维德维康生物技术有限公司 | Milk product multi-factor rapid detection kit and detection method thereof |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN113267621B (en) * | 2021-05-14 | 2021-12-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
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Application publication date: 20100519 |