CN104215762A - Indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody - Google Patents
Indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody Download PDFInfo
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Abstract
The invention discloses an indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody and belongs to the technical field of serum antibody detection. The indirect ELISA method includes that serum 3-type duck hepatitis virus VP1 protein is utilized as an envelope antigen with the peridium quantity as 0.1-1mug per hole, HRP-mice anti-duck IgY is utilized as the HRP with the use concentration as 0.2-2mug/mL, and the coloration time is 10 minutes. The kit comprises a VP1 protein antigen envelope board, the HRP-mice anti-duck IgY, sample diluent, a scrubbing solution, TMB solutions A and B and a stop solution. The method and the kit can be used for detecting the serum 3-type duck hepatitis virus a antibody in duck serum and duck egg yolk, and the serum does not have cross reaction with positive serum of other viruses. Compared with a traditional neutral reaction detection method, the method has the advantage of being convenient and accurate.
Description
Technical field
The invention belongs to Serum Antibody Detection technical field, relate to a kind of indirect ELISA method and the kit that detect Serotype-3 DHAV antibody.
Background technology
DHAV (Duck hepatitis A virus, DHAV) be a kind of cause duck virus hepatitis main pathogenic former, all have popular in the world, huge economic loss caused to duck aquaculture.DHAV is according to gene evolution analysis, and be divided into 3 different genotype, Gene A type, Type B, C type, owing to there is no serological cross reaction between each genotype, therefore, also can be referred to as 3 different serotypes, serum 1 type, 2 types and 3 types.
It should be noted that, serum 2 type and 3 type DHAVs unconventional 2 type DHVs and 3 type DHVs, because according to the council of international virology classification in recent years, 2 traditional type DHVs and 3 type DHVs are classified as Astroviridae, this two-strain Epidemic Scope and harmfulness all very little, generally do not arouse attention; By traditional 1 type DHV and so-called New Type Duck Hepatitis Virus called after hepatitis A virus popular in recent years, classify as Picornaviridae.Still serum 1 type and the Serotype-3 DHAV of current Major Epidemic clinically, but because 1 type DHAV finds comparatively early, relevant detection means is more complete, and the duck virus hepatitis that the scorching virus of prevention and corntrol 1 type that the is applied as duck A type of the biological products such as live vaccine and Yolk antibody causes plays active and effective effect, and Serotype-3 DHAV was just found gradually and reports after 2000, and along with the widespread use of serum 1 type duck Hepatitis A Vaccine and Yolk antibody, this immune pressure causes Serotype-3 duck hepatitis A to infect in popular expansion trend.Current 3 type DHAV antibody tests mainly still depend on traditional neutralization test method, and neutralization test is time-consuming, effort, and testing result affects comparatively large by the subjectivity that embryo source quality and operation population judge, unstable result.
Summary of the invention
The object of the present invention is to provide a kind of indirect ELISA method detecting Serotype-3 DHAV antibody, the present invention also aims to provide a kind of indirect ELISA reagent kit detecting Serotype-3 DHAV antibody.
Object of the present invention is achieved through the following technical solutions:
Detect an indirect ELISA method for Serotype-3 DHAV antibody, using Serotype-3 DHAV VP1 albumen as envelope antigen; The little mouse-anti duck IgY of HRP mark is as ELIAS secondary antibody.
Preferably, the package amount of described VP1 albumen is every hole 0.1 ~ 1 μ g; Confining liquid is BSA or skim milk, and concentration is 1% ~ 10%.The working concentration of the little mouse-anti duck IgY of described HRP mark is 0.2 ~ 2 μ g/mL; The time of colour developing is 10 minutes; Criterion is: microplate reader measures each hole light absorption value under 450nm wavelength, Cut off(CO value) the average OD of=negative control
450nmbe worth × 2.1 times, sample average OD
450nmvalue is greater than CO value and is judged to the positive; Sample average OD
450nmvalue is less than CO value and is judged to feminine gender.
Detect an indirect ELISA reagent kit for Serotype-3 DHAV antibody, comprise VP1 proteantigen bag by plate,
The little mouse-anti duck IgY that HRP marks, sample diluting liquid, cleansing solution, tmb substrate solution A, tmb substrate solution B and stop buffer.
The indirect ELISA reagent kit of described detection Serotype-3 DHAV antibody also comprises anti-DHAV-3 duck positive serum, anti-DHAV-3 duck negative serum.
Described VP1 albumen carries out expression and purification with the pGEX carrier prokaryotic expression system of GST label obtain preferably by using.In protein expression Induction Process, IPTG induced concentration is 1.0mmol/L, and cultivation temperature is 28 ~ 30 DEG C, can obtain more solubility VP1 albumen with this understanding.
Preferably, described pGEX carrier is pGEX-4T-1; The Escherichia coli of expressing VP1 albumen are built by the method comprising following steps and obtain: the RNA extracting Serotype-3 DHAV, template cDNA is prepared in reverse transcription, carry out the amplification of VP1 gene, amplimer is DHAV-3 VP1FP and DHAV-3 VP1RP, the VP1 gene of amplification and pGEX-4T-1 carrier EcoR I and Xho I are carried out double digestion, being connected by T4 DNA ligase, then transform BL21(DE3) competent cell obtains expressing the Escherichia coli of VP1 albumen;
DHAV-3 VP1FP：GA
GAATTCGGTGATTCCAATCAGCTTGGTG，（EcoR I），
DHAV-3 VP1RP：GG
CTCGAGTTCAATTTCCAAATGGAGCTC，（Xho I）。
In described antigen coated microplate, the package amount of VP1 albumen is preferably every hole 0.1 ~ 1 μ g; Described HRP-little mouse-anti duck IgY ELIAS secondary antibody is commercial reagents, is preferably and is diluted to 0.2 ~ 2 μ g/mL with the PBS containing 1% BSA.
The indirect ELISA method of described detection Serotype-3 DHAV antibody or kit can be used for the detection of Serotype-3 DHAV antibody in duck serum and duck's egg yolk.
The using method of the indirect ELISA reagent kit of described detection Serotype-3 DHAV antibody, comprises the steps:
(1) with sample diluting liquid dilution, measuring samples is diluted, add in antigen coated plate hole, establish positive control and negative control simultaneously.
(2) after antigen and antibody combination form antigen antibody complex, wash plate with cleansing solution, pat dry.
(3) the little mouse-anti duck IgY adding HRP mark is combined with antigen antibody complex, washes plate, pat dry with cleansing solution.
(4) add tmb substrate solution A and the colour developing of B equal-volume mixed liquor, then add stop buffer.
(5) its absorption value is read at wavelength 450nm place.
Advantage of the present invention:
(1) wrap in the present invention by the preparation with VP1 albumen, adopt pGEX prokaryotic expression carrier, relative to other expression vector, more easily form soluble protein, more easily obtain purity and active higher fusion through affinity chromatography.
(2) specificity of the present invention is good, cross reaction is not produced with the positive serum of other viruses, although envelope antigen derives from escherichia expression system, but do not produce cross reaction with enteropathogenic E．Coli, Riemerlla anatipestifer positive serum, also with serum 1 type DHAV antibody no cross reaction, susceptibility is high, testing result good stability.
(3) the present invention not only can be used for detecting 3 type DHAV antibody in duck serum, also can detect the Yolk antibody in duck's egg, prepare Yolk antibody, and duck embryo makes live vaccine, can be used as practical detection method for employing duck's egg.
The present invention is not only the Epidemiology monitor important means of clinical duck virus hepatitis epidemic disease, also for the research and development of the biological products such as duck virus hepatitis vaccine provide important technological means, as the detection for antibody rule of proliferation after vaccine immunity, the maternal antibody level monitoring of newborn duckling, plant the Yolk antibody detection etc. of egg, the present invention is subject to the neutralization reaction detection method of embryo source impact relative to traditional lengthy and tedious, complicated, consuming time and testing result, has convenient, advantage accurately.
Accompanying drawing explanation
Fig. 1 is the electrophoretogram of VP1 protein expression and purifying, M: albumen Maker; 1: the albumen that prokaryotic expression is not purified, foreign protein is more; 2: the albumen after affinity purification, band is single, purity and concentration better.
Embodiment
Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The expression and purification of embodiment 1 envelope antigen VP1 albumen
(1) amplification of object fragment and purifying
Adopt commercialization RNA to extract kit (MiniBEST Universal RNA Extraction Kit, TaKaRa), concrete operations refer to instructions, and carry out the extraction of Serotype-3 DHAV RNA, template cDNA is prepared in reverse transcription.
Reverse transcription system is: RNA 4.0 μ L, random primer (20 μMs), Oligod (T) (20 μMs) each 0.5 μ L, dNTPs(2.5 μM) 4.0 μ L, Rnase Inhibitor 0.5 μ L, reverse transcriptase M-MLV(TaKaRa) 0.5 μ L, 5 × M-MLV Buffer 4.0 μ L.
After reaction system is mixed, wink from.Reaction conditions is: 42 DEG C of insulation 1h, 70 DEG C of effect 10min.
The cDNA obtained with reverse transcription carries out the amplification of VP1 gene for template, and amplimer is:
DHAV-3 VP1FP：GA
GAATTCGGTGATTCCAATCAGCTTGGTG，（EcoR I），
DHAV-3 VP1RP：GG
CTCGAGTTCAATTTCCAAATGGAGCTC，（Xho I）。
Reaction system is (overall reaction system is 50 μ L): 10 × PCR buffer 5.0 μ L, dNTP mixture(2.5mM) 4.0 μ L, each 1 μ L(10 μM of upstream and downstream primer), high-fidelity Ex-Taq polymerase (TaKaRa) 0.5 μ L, cDNA template is 4.0 μ L, and remainder supplies DEPC-H
2o to 50 μ L system.Reaction is carried out in ABI PCR instrument, the first step: 94 DEG C, 5min, 1cycle; Second step: 94 DEG C, 30sec, 58 DEG C, 30sec, 72 DEG C, 30sec, 30cycles; 72 DEG C, 10min, overall elongation.Adopt commercialization glue to reclaim purification kit and carry out purifying recovery (MiniBEST DNA Fragment Purification Kit, TaKaRa) to amplified fragments, concrete operations refer to instructions.
(2) structure of expression vector
Amplified fragments is carried out glue and reclaim purifying, the fragment that purifying reclaims and empty vectors pGEX-4T-1 EcoR I and Xho I carry out double digestion, endonuclease bamhi carries out glue equally and reclaims purifying, then T4 DNA ligase is used, object fragment is connected with carrier, connect and transform BL21(DE3) competent cell, obtain positive plasmid clone.By the positive colony bacterium liquid obtained, send to order-checking, after order-checking is correct, carry out protein expression.
(3) abduction delivering of VP1 gene and purifying
Protein expression condition is: cultivated at 28 ~ 30 DEG C by positive plasmid bacterium, is cultured to bacterial concentration OD600 when reaching 0.8, adds IPTG(1M) to induce, the IPTG final concentration added is 1.0mM, and 28 ~ 30 DEG C are continued cultivation 3 hours.By the bacterium liquid after induction, centrifugal collecting precipitation, with GST post affinity chromatography sample-loading buffer (120mM NaCl, 2.5mM KCl, 10mM Na
2hPO
4, 1.5mM KH
2pO
410mM DTT, pH 7.3) resuspended, ultrasonication, gets supernatant and identifies after centrifugal, after destination protein obtains expression, the supernatant of collection is crossed GST post and carry out purifying (GSTrap FF, GE Healthcare), BCA method measures the concentration of purifying protein, the purity (Fig. 1) of PAGE electroresis appraisal purifying protein.
The preparation method of the indirect ELISA reagent kit of embodiment 2 Serotype-3 DHAV antibody
(1) be coated on ELISA Plate hole uniformly by VP1 proteantigen, it is phosphate buffer (PBS, 0.01M pH7.2) that bag is buffered liquid, namely contains NaCl 8.0g, KCl 0.2g, Na in 1 liter of solution
2hPO
412H
2o 3.6g, KH
2pO
40.24g, VP1 albumen package amount is every hole 0.1 ~ 1 μ g; Confining liquid is BSA or skim milk, and concentration is 1% ~ 10%.
(2) HRP-little mouse-anti duck IgY ELIAS secondary antibody is commercial reagents, is diluted to 0.2 ~ 2 μ g/mL with the PBS containing 1% BSA.
(3) other solution preparations of kit: 1. sample diluting liquid: 1% BSA, 0.05% NaN
3, PBS(0.01M, pH7.2).2. 10 × cleansing solution: add Tween-20 (Tween-20) in 0.1mol/L PBS solution, makes the concentration of Tween-20 be 0.5%(V/V).3. zymolyte is 3,3', 5,5'-tetramethyl benzidine (TMB) solution: I) substrate solution A: containing 0.01 ~ 0.1% hydrogen peroxide (H
2o
2) 1% sodium acetate solution, pH5.0; II) substrate solution B: be dissolved with 0.2 ~ 1g 3,3', 10mL dimethyl sulfoxide (DMSO) and the 1000mL water mixed solution being dissolved with 10g sodium acetate, pH5.0 of 5,5'-tetramethyl benzidine.4. stop buffer: getting 54.3mL concentration is that 95% concentrated sulphuric acid adding distil water is to 1000mL.5. positive control: by formulated for the duck positive serum sample diluting liquid 10 times dilution containing DHAV-3 antibody.6. negative control: by formulated for the duck negative serum sample diluting liquid 10 times dilution not containing DHAV-3 antibody.
The method of operating of the indirect ELISA reagent kit of embodiment 3 Serotype-3 DHAV antibody
(1) by measuring samples sample diluting liquid 100 times dilution, every hole adds 100 μ L, adds the positive and negative controls simultaneously, if blank.Each sample and contrast parallel application of sample 3 holes.Put 37 DEG C and hatch 1 hour, cleansing solution washes plate 5 times, pats dry, and every hole adds HRP-little mouse-anti duck IgY ELIAS secondary antibody 100 μ L, hatches 1 hour for 37 DEG C; Cleansing solution washes plate 5 times, pats dry; Add substrate colour developing by after tmb substrate solution A and the mixing of B equal-volume, every hole 100 μ L, lucifuge develops the color 10 minutes, adds stop buffer, every hole 50 μ L.Microplate reader is utilized to measure absorbance value (OD at 450nm
450nmvalue).
(2) result judges: Cut off(CO value) the average OD of=negative control
450nmbe worth × 2.1 times, sample average OD
450nmvalue is greater than CO value and is judged to the positive; Sample average OD
450nmvalue is less than CO value and is judged to feminine gender.
The preparation of embodiment 4 Serotype-3 duck hepatitis A attenuated vaccine
Duck embryo is caused the malicious DHAV-3(10 of weak kind
6.5eLD
50/ 0.1mL) carry out 1000 times of dilutions by stroke-physiological saline solution, inoculate well-developed 10 age in days SPF duck embryos, every embryo 0.1mL, the duck embryo allantoic liquid of results 72h ~ 96h death, chorioallantoic membrane, embryo.
By the duck embryo tissue of above-mentioned results, after tissue mashing machine's homogenate, filter with 0.5 millimeter of copper gauze, gained liquid adds equivalent freeze drying protectant, carries out freeze-drying, is Serotype-3 duck hepatitis A attenuated vaccine.
The indirect ELISA reagent kit of embodiment 5 Serotype-3 DHAV antibody is applied the detection of clinical sample
Following sample is detected: after Serotype-3 duck hepatitis A attenuated vaccine immunity prepared by 1 age in days duckling inoculation embodiment 4,1 week (1W), 2 weeks (2W), 4 weeks (4W) blood sampling are separated the serum obtained according to detection method described in embodiment 3; Use Serotype-3 duck hepatitis A attenuated vaccine immunity kind duck, gather in the crops respectively at 1 week (E1W), 2 weeks (E2W), 3 weeks (E3W) after immunity middle Yolk antibody of laying eggs; Enteropathogenic E．Coli positive serum (E.coli, fine jade expands tires 1: 128), Riemerlla anatipestifer positive serum (RA, fine jade expands tires 1: 64), serum 1 type DHAV positive serum (DHAV-1, Neutralizing titer 1: 32).Wherein, the process of (1) serum to be detected: by centrifugal for serum 10000r/min to be checked 10 minutes, get supernatant as measuring samples.(2) process of Yolk antibody: disinfect kind of an egg sample, sample thief opens shell membrane successively, extracts yolk 1mL, adds in the centrifuge tube that 1mL physiological saline is housed, and make it mix in physiological saline.Extracting methenyl choloride 2mL successively adds in above-mentioned suspension respectively, adds physiological saline 4mL again, then low-speed centrifugal (4000r/min) 10 minutes after mixing.After centrifugal, be divided into three layers, the superiors are like the same transparency liquid of water, and middle is White Flocculus to bottom, bottommost yellow.The superiors' transparency liquid is Yolk antibody, gets supernatant transparent liquid as measuring samples.
Table 1 clinical sample detection experiment
The above results shows; this kit can realize the detection to clinical duck Serotype-3 DHAV antibody, and after vaccine immunity 1 age in days duckling, after 1 week, serum just has antibody to produce; about 2 weeks antibody peaks value, and after 4 weeks, serum antibody still can protect higher titre.This kit also can detect Yolk antibody in duck's egg, and the titre of Yolk antibody is basic consistent with the antibody horizontal in duck serum.This kit detects the positive serum of serum 1 type DHAV positive serum, enteropathogenic E．Coli positive serum (E.coli), Riemerlla anatipestifer positive serum (RA) and is feminine gender.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Chopper Biology Co., Ltd.
<120> mono-kind detects indirect ELISA method and the kit of Serotype-3 DHAV antibody
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> DHAV-3 VP1FP
<400> 1
gagaattcgg tgattccaat cagcttggtg 30
<210> 2
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> DHAV-3 VP1RP
<400> 2
ggctcgagtt caatttccaa atggagctc 29
Claims (10)
1. detect an indirect ELISA method for Serotype-3 DHAV antibody, it is characterized in that: envelope antigen
Be Serotype-3 DHAV VP1 albumen, ELIAS secondary antibody is the little mouse-anti duck IgY of HRP mark.
2. the indirect ELISA method of detection Serotype-3 DHAV antibody according to claim 1, is characterized in that: the package amount of described VP1 albumen is every hole 0.1 ~ 1 μ g; The working concentration of the little mouse-anti duck IgY of described HRP mark is 0.2 ~ 2 μ g/mL; The time of colour developing is 10 minutes.
3. the indirect ELISA method of detection Serotype-3 DHAV antibody according to claim 1, is characterized in that: criterion is: microplate reader measures each hole light absorption value under 450nm wavelength, the average OD of CO value=negative control
450nmbe worth × 2.1 times, sample average OD
450nmvalue is greater than CO value and is judged to the positive; Sample average OD
450nmvalue is less than CO value and is judged to feminine gender.
4. detect an indirect ELISA reagent kit for Serotype-3 DHAV antibody, it is characterized in that: comprise VP1 proteantigen bag by the little mouse-anti duck IgY of plate, HRP mark, sample diluting liquid, cleansing solution, tmb substrate solution A, tmb substrate solution B and stop buffer.
5. the indirect ELISA reagent kit of detection Serotype-3 DHAV antibody according to claim 4, is characterized in that: comprise anti-DHAV-3 duck positive serum, anti-DHAV-3 duck negative serum.
6. the indirect ELISA reagent kit of detection Serotype-3 DHAV antibody according to claim 4, is characterized in that: described VP1 albumen carries out expression and purification by pGEX carrier prokaryotic expression system and obtains; In protein expression Induction Process, IPTG induced concentration is 1.0mmol/L, and cultivation temperature is 28 ~ 30 DEG C.
7. the indirect ELISA reagent kit of detection Serotype-3 DHAV antibody according to claim 6, is characterized in that: described pGEX carrier is pGEX-4T-1; The Escherichia coli of expressing VP1 albumen are built by the method comprising following steps and obtain: the RNA extracting Serotype-3 DHAV, template cDNA is prepared in reverse transcription, carry out the amplification of VP1 gene, amplimer is DHAV-3 VP1FP and DHAV-3 VP1RP, the VP1 gene of amplification and pGEX-4T-1 carrier EcoR I and Xho I are carried out double digestion, connected by T4 DNA ligase, then transform the Escherichia coli that BL21 competent cell obtains expressing VP1 albumen;
DHAV-3 VP1FP：GAGAATTCGGTGATTCCAATCAGCTTGGTG，
DHAV-3 VP1RP：GGCTCGAGTTCAATTTCCAAATGGAGCTC。
8. the indirect ELISA reagent kit of detection Serotype-3 DHAV antibody according to claim 4, is characterized in that: in described antigen coated microplate, the package amount of VP1 albumen is every hole 0.1 ~ 1 μ g; The concentration of the little mouse-anti duck IgY of described HRP mark is 0.2 ~ 2 μ g/mL.
9. the indirect ELISA method described in any one of claim 1-3 or the application of the kit described in any one of claim 4-8 in duck serum and duck's egg yolk in the antibody test of Serotype-3 DHAV.
10. the using method of the indirect ELISA reagent kit of the detection Serotype-3 DHAV antibody described in any one of claim 4-8, is characterized in that comprising the steps:
(1) with sample diluting liquid dilution, measuring samples is diluted, add in antigen coated plate hole, establish positive control and negative control simultaneously;
(2) after antigen and antibody combination form antigen antibody complex, wash plate with cleansing solution, pat dry;
(3) the little mouse-anti duck IgY adding HRP mark is combined with antigen antibody complex, washes plate, pat dry with cleansing solution;
(4) add tmb substrate solution A and the colour developing of B equal-volume mixed liquor, then add stop buffer;
(5) its absorption value is read at wavelength 450nm place.
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| CN106771237A (en) * | 2016-12-13 | 2017-05-31 | 湖南农业大学 | A kind of ELISA kit for detecting porcine sapelo virus antibody |
| CN108169496A (en) * | 2018-03-14 | 2018-06-15 | 山东省农业科学院家禽研究所 | A kind of DHAV-3 types polypeptide indirect ELISA antibody assay kit and its application |
| CN108445208A (en) * | 2018-03-14 | 2018-08-24 | 山东省农业科学院家禽研究所 | A kind of universal DHAV polypeptides indirect ELISA antibody assay kit and its application |
| CN108169496B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | DHAV-3 type polypeptide indirect ELISA antibody detection kit and application thereof |
| CN108445208B (en) * | 2018-03-14 | 2022-06-28 | 山东省农业科学院家禽研究所 | Universal DHAV polypeptide indirect ELISA antibody detection kit and application thereof |
| CN108845127A (en) * | 2018-04-13 | 2018-11-20 | 四川农业大学 | The indirect ELISA detection method of DHAV-3 antibody based on VP2 or VP4 recombinant protein antigen and application |
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