CN108761077A

CN108761077A - Poultry diease viral disease protein chip antibody assay kit and its preparation method and application

Info

Publication number: CN108761077A
Application number: CN201810886569.0A
Authority: CN
Inventors: 周继勇; 闫丽萍; 胡建华; 肖倩; 雷静
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2018-11-06

Abstract

The invention discloses poultry diease viral disease protein chip antibody assay kits and its preparation method and application.The kit includes the protein chip reaction plate containing antigen, positive control sample, negative control sample.The antigen is at least one of following (a)~(c)：(a) include recombination AIV nucleoprotein (AIV-NP), NDV P albumen (NDV-P) and IBV Non structural protein 5s (IBV-nsp5)；(b) recombination AIV nucleoprotein (AIV-NP)；(c) NDV P albumen (NDV-P).The present invention is that iPDMS films is used to substantially reduce testing cost as the method for the protein chip of three kinds of poultry diease viral disease antibody of substrate while detection, improve the efficiency of detection for the first time both at home and abroad.The invention further includes that can individually detect a kind of virus or the kit of two kinds of antiviral antibodies.

Description

Poultry diease viral disease protein chip antibody assay kit and its preparation method and application

Technical field

The invention belongs to immunological detection method fields, and in particular to poultry diease viral disease protein chip antibody assay kit and Preparation method and application.

Background technology

Bird flu (AI), newcastle disease (ND) and infective bronchitis (IB) be endanger three kinds of world's poultry husbandry it is common Respiratory disease.Currently, vaccine immunity is to control outburst and the most common method of propagation of AI, ND and IB, and vaccine immunity We assess effect often through the height of the antibody level in Post-immunisation serum, and therefore, the level of antibody is directly related to Resistance of the vaccinated flock to disease.Therefore, antibody level in vaccinated flock is quickly and accurately detected, adjustment is immune in due course Program plays effective anti-AI, ND and IB processed vital.In terms of antibody test, common method is blood clotting suppression System experiment (HI) and enzyme-linked immunosorbent assay (ELISA).Hemagglutination-inhibition test is for large batch of sample detection, working strength Greatly；ELISA is widely applied clinically due to the characteristics of it is simple, quick, sensitive, economic and is suitable for large-scale use, But the commercialization detection reagent of the inadequate specification of the quality of existing ELISA kit on the market, the wherein U.S. IDEXX companies production Box is more stable reliable, is mainly used for the sampling observation of AI, ND and IB antibody level and research and development department in domestic large-scale poultry place The Comparability test of door exploitation same type new product.But it because import reagent box is expensive, limits these kits and supports at home Being widely used in chicken industry.Avian viral disease antibody detection is complicated, needs the measurement that a serum is carried out to multiple kits, no But it is cumbersome, and apply a large amount of manpower.High throughput detection has become a development of the following aquaculture disease surveillance Trend, protein chip antibody test technology can be detected simultaneously by three kinds of antibody with a serum, have detection efficiency high, quick The features such as perceptual height, high specificity, quick, cheap detection method.

Protein chip technology is that protein or polypeptide etc. are fixed on the carriers such as slide, microwell plate, gel, filter membrane, Reach in detection sample can have with the technology of the ingredient of the specific bonds such as albumen or polypeptide, this method detection quickly, operation letter The a series of advantages such as list, result high throughput.With the continuous development of the technology, its application field is also opened up constantly Exhibition, according to the principle and feature of protein chip, people also extend to it to examine food security, medical diagnosis on disease monitoring, screening In other multiple fields such as drug, livestock-raising.Currently, protein chip is widely used in terms of human tumor disease, dynamic Application is less in terms of object medical diagnosis on disease, has a extensive future.Wang Xiurong etc. are by 4 kinds of viruses of AIV, NDV, IBV and IBDV Proteantigen the protein chip that four kinds of antiviral antibodies of detection are established on the slide of aldehyde radicalization modification is fixed on by chemical bond Method has higher sensibility and specificity compared with agar gel precipitation method；Proteins and peptides microarray is to bioid Research is learned, drug development, clinical analysis etc. provides good platform.Cao Qiaoling etc. is by five kinds small point of nonyl phenol and chloramphenicol etc. Sub- substance is coupled with ovalbumin or bovine serum albumin(BSA), can be combined, be realized with respective antibody after conjugate is labeled Five kinds of small-molecule substances are carried out while being detected on same chip.

Nitrocellulose, polyvinylidene fluoride (PVDF) and glass are common chip materials in protein microarray.But It is the deficiency that these materials usually have following several respects：First, the reflecting point on chip is usually required with esters, aldehydes, ring The activation such as the polymer coatings such as oxygen resin, maleimide, hydrazine or poly-L-Lysine and functionalization are to reach ankyrin The purpose of matter, it is stringent that most methods are required for special chemical reagent and condition to require；Secondly, these chip materials are general There will be non-specific protein absorption, background signal is caused to increase, the sensitivity of protein microarray and stability decline, though So background area can be made to inactivate by closing the methods of background area, still, these methods would generally increase detection when Between；Often there is the form of coffee ring structure in microarray preparation process in third, is reduced so as to cause the accuracy of result.For Solve the problems, such as protein chip in application process it is existing these, we apply a kind of new chip material in our current research IPDMS (initiator integrated poly (dimethysiloxane), iPDMS), the chip made of this material With " ultralow " background and stable target protein signal can be generated.

The nucleoprotein (nucleocapsid protein, NP) of AIV is used as a kind of major structural protein, has highly conserved Property, and have high degree of immunogenicity, it can induce generation antibody.So NP albumen is the inspection of A subtype influenza virus specific serologicals The optimal selection of diagnostic antigen is surveyed, research reports that the ELISA diagnostic methods that useful NP albumen is established detect A subtype influenza virus. The P albumen of NDV is widely present in newcastle disease virus, and is played an important role in the reproduction process of virus, is had been reported that and is used NDV P albumen establish ELISA detection method detection chicken serum in NDV antibody.IBV Non structural protein 5s (non-structural Protein, nsp5) relative to structural proteins gene order have higher conservative, be related to the mutation probability of epitope It is relatively low.So detecting IBV serum antibodies using IBV non-structural proteins as antigen, have to different serotypes, or different groups The strain for knitting preferendum has the potentiality of versatility, this laboratory also establishes the ELISA antibody that IBV nsp5 are antigen and examine early period Disconnected method.Patent (the number of patent application for the protein chip method for individually detecting IBV antibody is applied in this laboratory： 201710918650.8).However, being protein chip film based on iPDMS, AIV, NDV antibody are detected respectively and is carried out at the same time The diagnostic method of AIV, NDV and IBV antibody test has not been reported.

A kind of modified silicon glued membrane (iPDMS) of this research application is used as substrate, using indirect method, establishes respectively and simultaneously Detect the protein chip detection method of AIV, NDV and IBV antibody in chicken serum.

Invention content

The technical problem to be solved by the present invention is to overcome deficiency in the prior art, provide a kind of for detecting avian viruses The antibody assay kit and its application method of disease, economy is provided for the diagnosis of clinic AI, ND and IB and the detection of antibody level Practical reliable detection instrument.

In order to solve the above technical problems, the solution of the present invention is：

A kind of antibody assay kit for detecting poultry diease viral disease, the kit include：

(1) the protein chip reaction plate containing antigen：The antigen is at least one of following (a)~(c)：

(a) include recombination AIV nucleoprotein (AIV-NP), NDV P albumen (NDV-P) and IBV Non structural protein 5s (IBV- nsp5)；

(b) recombination AIV nucleoprotein (AIV-NP)；

(c) recombination NDV P albumen (NDV-P)；

(2) positive control sample：Anti- IBV-nsp5 chicken serums, anti-AIV-NP chicken serums and anti-NDV-P chicken serums；

(3) negative control sample：Negative SPF chicken serums.

Antigen on the protein chip reaction plate preferably recombinates AIV nucleoprotein, NDV P albumen and IBV non-structural proteins White 5 three kinds；These three antigen coats are in the same reacting hole.

The working concentration of the antigen preferably 0.1~0.2mg/mL, further preferably 0.2mg/mL.

The kit further preferably includes following components:

(4) IgG enzyme labelled antibodies solution：The enzyme labelled antibody is the goat-anti chicken igg antibody of horseradish peroxidase-labeled；

(5) 20 × TBST concentrated cleaning solutions：The final concentration TBS containing 0.05%Tween-20, pH7.4；

(6) 10 × PBS serum dilutions：pH7.4；

(7) TMB developing solutions.

The extension rate preferably 1 of the IgG enzyme labelled antibodies solution:1000~1:2000, further preferred 1:2000.

The kit can be following six kinds of kits, be respectively：Avian influenza virus (AIV) protein chip antibody test tries Tri- kinds of agent box, newcastle disease virus (NDV) protein chip antibody assay kit, AIV-NDV-IBV protein chip antibody test examinations Agent box, tri- kinds of protein chip antibody assay kits of AIV-NDV, AIV-IBV tri- kinds of protein chip antibody assay kits, NDV- Tri- kinds of protein chip antibody assay kits of IBV.

The corresponding protein chip reaction plate containing antigen is respectively：(I) contain AIV NP protein chip reaction plates；(Ⅱ) Contain NDV P protein chip reaction plates；(III) contain the protein chip of recombination AIV NP albumen, NDV P albumen and IBV nsp5 Reaction plate；(IV) contain the protein chip reaction plate of recombination AIV NP albumen, NDV P albumen；(V) contain NDV P albumen and IBV The protein chip reaction plate of nsp5；(VI) contain the protein chip reaction plate of recombination AIV NP albumen and IBV nsp5.

With antigen recombination AIV nucleoprotein (AIV-NP), NDV P albumen (NDV-P) and IBV Non structural protein 5s (IBV- Nsp5 for), kit of the present invention most preferably includes the reagent of content in detail below：

(1) contain AIV nucleoprotein (AIV-NP), NDV P albumen (NDV-P) and IBV Non structural protein 5s (IBV-nsp5) 48 one piece of porin chip reaction plates；

(2) IgG enzyme labelled antibodies solution (1:2000 times of dilutions), volume 10mL.The enzyme labelled antibody is horseradish peroxidase The goat-anti chicken igg antibody of enzyme (HRP) label；

(3) 20 × TBST concentrated cleaning solutions：The final concentration TBS containing 0.05%Tween-20, pH7.4, volume 200mL；

(4) 10 × PBS serum dilutions：PH7.4, volume 200mL；

(5) positive control sample：Anti- IBV-nsp5 chicken serums, anti-AIV-NP chicken serums and anti-NDV-P chicken serums, volume are each 0.5mL；

(6) negative control sample：Negative SPF chicken serums, volume 0.5mL；

(7) TMB developing solutions, volume 3.5mL.

Preparation method of the present invention for detecting the antibody assay kit of poultry diease viral disease, including the albumen core The preparation of piece reaction plate and the preparation of other components；The preparation process of the wherein described protein chip reaction plate is：Antigen is prepared, point Not after BCA protein detection kits (BCA protein Assay kit) measured concentration, it is diluted to PBS buffer solution Final concentration of 1mg/mL；The micro- lattice point sample of albumen is carried out in 100,000 grades of clean rooms；After albumen vacuum drying, by the amount of 1mg/mL It is dissolved in containing in 30% acetonitrile solution, then dilutes antigen to working concentration, point sample to iPDMS films with spotting buffer On, protein chip, vacuum drying packaging is made；The PBS buffer solution is 0.01M PBS buffer solution (pH7.4)；The point sample The formula of buffer solution is：0.3M PB, 0.2%Glycerin, 0.01%Triton and 1.5%Mannitol.

The recombination IBV Non structural protein 5s are prepared by following methods：The gene of IBV nsp5 is through the side RT-PCR It after method amplification obtains, is cloned into prokaryotic expression carrier pEGX-4T-1, converts Escherichia coli；Screening carries IBV nsp5 The recombinant bacterium of gene, the nsp5 recombinant proteins that great expression is merged with GST under IPTG inductions；Utilize the affine layers of GSTrap FF Analyse column purification, the IBV nsp5 recombinant proteins after being purified, be stored in -20 DEG C it is spare；

The preparation process of the AIV nucleoprotein is：Design specific primer pET-32a-AIV-NP-F and pET-32a- AIV-NP-R, the nucleic acid extracted using the chick embryo allantoic liquid of AIV H3N2 virus strain infections are expanded through RT-PCR method and are obtained as template The gene of AIV NP is cloned into prokaryotic expression carrier pET32a, converts Escherichia coli, and screening carries the weight of target gene Group bacterium, the recombinant protein that great expression is merged with His under IPTG inductions obtain high-purity using His affinity chromatography column purifications Recombination AIV nucleoprotein be stored in -20 DEG C it is spare；

The preparation process of the NDV P albumen is：Design specific primer pColdI-NDV-P-F and pColdI-NDV-P- R, the nucleic acid extracted using the chick embryo allantoic liquid of NDV-Lasota virus strain infections are expanded through RT-PCR method as template and obtain NDV P Gene, it is cloned into respectively in prokaryotic expression carrier pColdI carriers, converts Escherichia coli, screening carries target gene Recombinant bacterium, the recombinant protein that great expression is merged with His under IPTG inductions are obtained high-purity using His affinity chromatography column purifications The recombination NDV P albumen of degree be stored in -20 DEG C it is spare.

The application method of the kit includes the following steps：

(1) protein chip reaction plate takes out from 4 DEG C and places room temperature, with cleaning solution rinse；The cleaning solution be containing The TBS of 0.05%Tween-20, i.e. TBST；

(2) serum is incubated：100 holes μ L/ PBS serum dilutions are added with 50~100 times of diluted serum to be checked, simultaneously The setting positive and negative serum control, 37 DEG C, 500rpm, concussion is incubated 5 minutes, after cleaning solution rinse, discards cleaning solution；

(3) secondary antibody is incubated：The diluted concentration of secondary antibody is 1:1000~1:2000,100 μ L 0.2M PBS bufferings are added per hole The sheep anti-chicken IgG secondary antibody of the diluted HRP labels of liquid (PH7.4), 37 DEG C, 500rpm, concussion is incubated 5 minutes, cleaning solution rinse 3 times Afterwards, cleaning solution is discarded；

(4) result is observed：60 μ L TMB developing solutions are added per hole, colour developing after five minutes, abandons developing solution, and cleaning solution rinses dry Only, testing result is visually observed；

(5) result judgement：Detect by an unaided eye colour developing situation, other than a positive quality control point is blue, if other points Colour developing, then be judged as the positive, be judged as feminine gender if not developing the color, if colour developing but color it is very light, be judged as weak sun.

Application of the above-mentioned kit in detecting poultry diease viral disease.The kit can detect bird flu, newcastle disease simultaneously And infectious bronchitis of chicken or in which two kinds arbitrary；Or any one in independent detection bird flu and newcastle disease.

Room temperature of the present invention is 25 ± 5 DEG C.

The kit of the present invention, range wide spectrum can detect A hypotype AIV antibody all in chicken group, NDV and IBV serum Antibody.It can singly examine while also being capable of joint inspection.One reaction system can detect three kinds of viral antibody simultaneously, improve inspection Efficiency is surveyed, testing cost is reduced.The protein chip detection method is a kind of method for visualizing, and the detection method used time does not surpass 20 minutes are spent, the detection used time is short, and has preferable sensibility and specificity.

Compared with prior art, the beneficial effects of the present invention are：

(1) AIV-NDV-IBV protein chips antibody joint inspection kit provided by the invention, with the structural proteins NP of AIV, The P albumen of NDV and the non-structural protein nsp5 of IBV as antigen, can be applied to it is clinical examined soon, detecting the reaction time is 15 minutes.It can provide with the diagnostic kit with the characteristic of the high coincidence rate of import kit.

(2) present invention is the albumen used iPDMS films as substrate for the first time both at home and abroad while detecting three kinds of poultry diease viral disease antibody The method of chip, substantially reduces testing cost, improves the efficiency of detection.The invention further includes that can individually detect a kind of disease The kit of malicious antibody.

(3) whether the present invention can infect AIV, NDV and IBV by the non-vaccinated flock of quick diagnosis in actual production, and Immune state or the chick maternal antibody for monitoring chicken group are horizontal.For the extensive antibody detection of clinically AIV, NDV and IBV, prevalence Disease learns investigation and the prevention and control of AI, ND and IB provide a kind of new effective detection means.

Description of the drawings

The induced expression of Fig. 1 pET-32a-AIV-NP recombinant expression carriers and purifying.

Wherein, M：Albumen Marker；1：Before zero load induction；2：After zero load induction；3：Before recombinant vector induction；4：Recombination carries After body induction；5：Purifying protein.

The induced expression of Fig. 2 pColdI-NDV-Lasota-P recombinant expression carriers and purifying.

Wherein, M：Albumen Marker；1：Before zero load induction；2：After zero load induction；3：Before recombinant vector induction；4：Recombination is again After proposing induction；5：Purifying protein.

The induced expression of Fig. 3 pGEX-4T-1-IBV-nsp5 recombinant expression carriers and purifying.

Tri- kinds of antigens of Fig. 4 are verified with the reactive Western blot of chicken positive serum.

Wherein, a：AIV-NP is reacted with chicken AIV positive serum Western blot.Wherein M：Albumen Marker；1：Recombination Albumen His-AIV-NP；2：After pET-32a zero loads induction

b：NDV-P is reacted with chicken positive serum Western blot；Wherein M：Albumen Marker；1：Recombinant protein His- NDV-P；2：After pColdI- zero loads induction

c：IBV-nsp5 is reacted with chicken positive serum Western blot, wherein M：Albumen Marker；1：Recombinant protein GST-

Nsp5；2：After pGEX-4T-1 zero loads induction.

Fig. 5 AIV, three kinds of antigen point sample concentration of NDV and IBV antibody joint inspections protein chip and serum diluting multiple it is excellent Change.

Wherein, a：The variation of colored intensity when AIV-NP difference point sample concentration；b：It develops the color when NDV-P difference point sample concentration strong The variation of degree；c：The variation of colored intensity when IBV-nsp5 difference point sample concentration；d：The optimization of serum diluting multiple.

The determination of Fig. 6 AIV, NDV and IBV antibody joint inspection protein chip secondary antibody extension rates.

The optimization of Fig. 7 AIV, NDV and IBV antibody joint inspection protein chip sealing conditions.

The specific test result of Fig. 8 AIV, NDV and IBV antibody joint inspection protein chips.

Wherein, 1：IBDV positive serums；2：ALV-J positive serums；3：CAV positive serums；4：AIV positive serums；5：IBV Positive serum；6：NDV positive serums；7：AIV-IBV-NDV positive serums；8：SPF chicken serums.

Specific implementation mode

It is described further with reference to embodiment and attached drawing.It should be appreciated that these embodiments are for illustration purposes only, and It is not used in and limits the scope of the invention.

Material therefor, reagent, instrument and method in following embodiment are the routine in this field without specified otherwise Material, reagent, instrument and method can be obtained by commercial channel.

The preparation and identification of [embodiment 1] antigen

Using AIV NP and NDV P specific primers, primer sequence is as follows,

Table 1 expands AIV-NP and NDV-P aligning primers

The chick embryo allantois infected with AIV H3N2 and NDV-Lasota strain (offer of virology key lab of the Ministry of Agriculture) The nucleic acid of liquid extracting is template, and the gene for obtaining AIV NP and NDV P is expanded through RT-PCR method, it is cloned into protokaryon respectively In expression vector pET32a and pColdI carrier, Escherichia coli are converted, screening carries the recombinant bacterium of target gene, is induced in IPTG The recombinant protein that lower great expression is merged with His utilizes His affinity chromatography column purifications, recombinant protein such as Fig. 1 and Fig. 2 of purifying It is shown, separately win high-purity recombinant protein be stored in -20 DEG C it is spare；The nucleotide sequence and its coding of the AIV NP genes Amino acid sequence such as SEQ ID NO：1 and SEQ ID NO：Shown in 2；The amino acid sequence of the gene of NDV P and its coding Amino acid sequence such as SEQ ID NO：3 and SEQ ID NO：Shown in 4.

The gene of IBV nsp5 is cloned into prokaryotic expression carrier pEGX-4T-1 after RT-PCR method amplification obtains In, convert Escherichia coli；Screening carries the recombinant bacterium of IBV nsp5 genes, and great expression is merged with GST under IPTG inductions Nsp5 recombinant proteins；Using GSTrap FF affinity chromatography column purifications, the IBV nsp5 recombinant proteins after being purified, after elution The albumen sampling of acquisition carries out SDS-PAGE analyses (Fig. 3).The recombinant protein GST-nsp5 molecular size ranges of purifying are 60KD, tool There is very high purity.Be stored in -20 DEG C it is spare.(specific preparation method, referring to avian infectious bronchitis virus nsp5 ELISA Antibody assay kit and its application, application number：201610239101.3 application publication number：105785049 A of CN, application are public Cloth day：2016.07.20).

SDS-PAGE is run with AIV-NP the and NDV-P albumen of purifying respectively, chicken AIV, NDV and IBV are used after transferring film, closing Positive serum as primary antibody identify recombinant protein and corresponding positive serum reactivity.Western blot detection knots Fruit shows that AIV-NP, NDV-P and IBV-nsp5 albumen energy positive serum corresponding with its of purifying react, and mesh Band size and expected albumen it is in the same size.AIV-NP, NDV-P and IBV-nsp5 albumen and corresponding empty carrier induced expression Product does not react, and feminine gender SPF chicken serums discord AIV-NP, NDV-P and IBV-nsp5 albumen generates nonspecific reaction (Fig. 4) shows that three kinds of albumen of purifying have preferable reactivity.

The preparation and screening of [embodiment 2] yin and yang attribute serum

1. the preparation of positive and negative serum

3 monthly age SPF chickens 8, stochastic averagina are taken to be divided into four groups, one of which in addition three groups, is distinguished as negative control group His-NP albumen, His-P albumen and the GST-nsp5 albumen purified with embodiment 1 is immunized, and positive serum is prepared.Specifically exempt from Epidemic disease process is as follows：

The His-NP albumen, His-P albumen and GST-nsp5 albumen of purifying are mixed with isometric Freund's complete adjuvant, After fully emulsified, head is subcutaneously carried out in chicken neck according to the dosage of 1mg/ only and exempted from.Later, it at interval of two weeks, takes and exempts from equivalent with head His-NP albumen, His-P albumen and GST-nsp5 albumen with after isometric incomplete Freund's adjuvant mixing and emulsifying, carry out two Exempt to exempt from three.Three exempt from 10 days afterwards, use NP-ELISA, P-ELISA and nsp5-ELISA to measure antibody level of serum respectively, reach and want Rear neck artery bloodletting, the blood of collection is asked to place 1h or 4 DEG C in 37 DEG C and stand overnight, 3000rpm centrifuges 10 minutes separation serum, - 70 DEG C of preservations, as AIV-NP positive serums, NDV-P positive serums and IBV-nsp5 positive serums after packing.Feminine gender group chicken, It takes same method to take a blood sample, detaches serum, AIV-NP, NDV-P are verified as with NP-ELISA, P-ELISA and nsp5-ELISA After IBV-nsp5 negative antibodies, -70 DEG C of preservations, as negative serum are dispensed.

2. the preparation of mono-specific antiserum

The strains of influenza viruses and NDV-Lasota strains provided according to virology key lab of the Ministry of Agriculture, prepares single-factor Serum is respectively：H1,H2,H3,H4,H5,H6,H7,H8,H9.3 monthly age SPF chickens are taken, are randomly divided into nine groups, every group of two chickens. It is immunized respectively with H1, H2, H3, H4, H5, H6, H7, H8, H9 subtype influenza virus allantoic fluid of inactivation, prepares positive blood Clearly.Specific immunologic process is as described above.

3. the screening of clinical serum

With the kit of the AIV of IDEXX, the screening Antibody of Influenza positive and negative blood serum sample.With IDEXX's IBV antibody assay kits screen the clinic IBV positives and negative serum sample.Using hemagglutination-inhibition test screening the NDV positives and Negative serum sample, specific practice are carried out fully according to kit operational manual.As a result in 24 parts of clinical serums, 18 parts are AIV, IBV are positive, and 17 parts positive for NDV.

[embodiment 3] for bird flu, newcastle disease and avian infectious bronchitis virus antibody test kit and its Application method 1, kit specifically include following reagent：

(1) contain AIV nucleoprotein (AIV-NP), NDV P albumen (NDV-P) and IBV Non structural protein 5s (IBV-nsp5) 48 five pieces of porin chip reaction plates；

(2) IgG enzyme labelled antibodies solution, volume 10mL.The enzyme labelled antibody is the sheep of horseradish peroxidase (HRP) label Anti-chicken IgG antibody；

(3) 20 × TBST concentrated cleaning solutions：Final concentration contains the TBS of 0.05% (v/v) Tween-20, pH7.4, volume 200mL；

(4) 10 × PBS serum dilutions：PH7.4, volume 200mL；

(6) negative control sample：Negative SPF chicken serums, volume 0.5mL；

(7) TMB developing solutions, volume 3.5mL.

The preparation of protein chip reaction plate：

AIV nucleoprotein, NDV P albumen and the IBV Non structural protein 5s of Prepare restructuring, respectively through BCA protein detection kits After (BCA protein Assay kit) measured concentration, it is diluted to final concentration of 1mg/mL with PBS buffer solution ,-

20 DEG C save backup；The micro- lattice point sample of albumen is carried out in 100,000 grades of clean rooms；After albumen vacuum drying, by 1mg/ The amount of mL is dissolved in containing in 30% (v/v) acetonitrile solution, then dilutes antigen to working concentration, point sample with spotting buffer Onto iPDMS films, protein chip is made, vacuum drying packaging, 4 DEG C save backup；The PBS buffer solution is 0.01M The PBS buffer solution of pH7.4；The formula of the spotting buffer is：0.3M PB, 0.2% (v/v) Glycerin, 0.01% (v/ V) Triton and 1.5% (g/100ml) Mannitol.

The application method of kit is as follows：

(1) protein chip reaction plate takes out from 4 DEG C and places room temperature, with cleaning solution rinse；The cleaning solution：Containing 0.05% (v/v) TBS of Tween-20, i.e. TBST；

The optimization of [embodiment 4] AIV-NDV-IBV antibody protein chip testing conditions

Based on above-mentioned basic program, the reaction condition of AIV, NDV and IBV antibody joint inspection protein chip is carried out excellent Change.

(1) optimization of antigen point sample concentration and serum diluting multiple

Point 0.0125mg/mL, 0.025mg/mL, 0.05mg/mL, 0.1mg/mL respectively in each hole of protein chip, Protein chip is made in AIV-NP, NDV-P and IBV-nsp5 albumen of 0.2mg/mL, 0.4mg/mL, and origin is fought using square formation method Sample concentration optimizes, and positive and negative serum is made 1 by 48 orifice plate tandems respectively:50,1:100,1:200 and 1:400 times of dilutions. After protein chip reaction, analyzing processing is carried out with Image J software collection data, and to data, determine antigen point sample concentration and Serum diluting multiple.As a result it shows when the point sample concentration of three kinds of albumen is more than 0.2mg/mL, the colored intensity that positive blood is checked There is no significant changes, so finally determining a concentration of 0.2mg/mL of point sample (Fig. 5 a, b, c) of three kinds of antigens.It is dilute for serum The optimum results for releasing multiple are shown, after serum diluting multiple is more than 100 times, the colored intensity range of decrease that positive blood is checked increases, So finally determining that best serum diluting multiple is 1:100 times (Fig. 5 d).

(2) optimization of enzyme labelled antibody working concentration

Fixed fixed best point sample concentration and serum diluting multiple, make 1 respectively by ELIAS secondary antibody:1000,l:2000, 1:4000,1:8000,1:16000 and 1:32000 times of dilutions, after carrying out protein chip detection, with Image J software collection numbers According to, and analyzing processing is carried out to data, determine best secondary antibody extension rate.The results show that the extension rate when secondary antibody is more than At 2000 times, (Fig. 6) is greatly lowered in the colored intensity of positive serum reflecting point, so final choice 1:2000 are used as best two Anti- extension rate.

(3) optimization of sealing condition

Confining liquid chooses 1%BSA and two kinds of 5% defatted milk is compared with PBS control, utilizes the good item of above-mentioned optimization Part detects with a positive and negative serum, eight repetitions is arranged.The results show that using 1%BSA, 5% defatted milk closing with The result difference that PBS acts on 30 minutes is not notable (Fig. 7), so in order to shorten detection time, improves detection efficiency, we are final Selection is not closed.

Therefore, finally determining optimum reaction condition is：The best point sample concentration of three kinds of proteantigens is 0.2mg/mL, is waited for It is 1 to examine serum diluting multiple:100, enzyme labelled antibody extension rate is 1:2000；Serum and ELIAS secondary antibody reaction time difference to be checked It is 5 minutes.

The specificity of [embodiment 5] AIV, NDV and IBV antibody joint inspection protein chips

Three protein chips are prepared using the three kinds of antigen point sample concentration determined, respectively by IBDV, ALV-J and CAV The positive serum of chicken carries out protein chip detection according to the condition optimized, while the positive serum pair of AIV, NDV and IBV is arranged It is compareed according to negative SPF chicken serums, the specificity of protein chip is determined with this.The results show that above-mentioned serum does not develop the color, and (Fig. 8) is set up in yin and yang attribute control.

The clinical application of [embodiment 6] AIV, NDV and IBV antibody joint inspection protein chips

24 parts of clinical serum samples are taken, use the blood clotting of AIV, NDV and IBV antibody joint inspection protein chip and AIV, NDV respectively Inhibit experiment and IBV ELISA kits (production of IDEXX companies of the U.S.) Parallel testing, compares AIV, NDV and IBV antibody joint inspection Sensibility, specificity and the coincidence rate of protein chip and import ELISA kit.Import AIV and IBV ELISA kit detects Method is carried out with reference to its specification.The result shows that：For AIV antibody tests, this method testing result and hemagglutination-inhibition test knot Fruit complies fully with (table 2,3)；For IBV antibody tests, this method testing result is detected with IDEXX IBV antibody assay kits As a result it is also fully consistent with；For IBV antibody tests, compared with hemagglutination-inhibition test result, sensibility is this method testing result 94.4%, specificity is 100%, and overall coincidence rate is 95.8% (table 4).Data above show this research institute establish AIV, NDV and IBV antibody joint inspection protein chips have good specificity and sensibility.

2 AIV, NDV and IBV antibody joint inspection protein chip of table is compared with AIV hemagglutination-inhibition tests

3 AIV, NDV and IBV antibody joint inspection protein chip of table is compared with NDV hemagglutination-inhibition tests

4 AIV, NDV and IBV antibody joint inspection protein chip of table is compared with IDEXX IBV antibody assay kits

[embodiment 7] is used for the kit and its application method of avian influenza virus antibody detection

1, the specific following reagent of kit：

(1) contain 48 one piece of porin chip reaction plates of AIV nucleoprotein (AIV-NP)；

(2) IgG enzyme labelled antibodies solution (1:2000 dilutions), volume 1mL.The enzyme labelled antibody is horseradish peroxidase (HRP) the goat-anti chicken igg antibody marked；

(4) 10 × PBS serum dilutions：PH7.4, volume 200mL；

(5) positive control sample：Anti- AIV-NP chicken serums, volume 0.5mL；

(6) negative control sample：Negative SPF chicken serums, volume 0.5mL；

(7) TMB developing solutions, volume 3.5mL.

The preparation of protein chip reaction plate：

The AIV nucleoprotein of Prepare restructuring measures dense through BCA protein detection kits (BCA protein Assay kit) After degree, it is diluted to final concentration of 1mg/mL with PBS buffer solution, -20 DEG C save backup；It is carried out in 100,000 grades of clean rooms The micro- lattice point sample of albumen；After albumen vacuum drying, it is dissolved in containing in 30% acetonitrile solution by the amount of 1mg/mL, then uses point sample Buffer solution dilutes antigen to working concentration 0.2mg/mL, on point sample to iPDMS films, is made protein chip, vacuum drying packaging, and 4 It DEG C saves backup；The PBS buffer solution is the PBS buffer solution of 0.01M pH7.4；The formula of the spotting buffer is： 0.3M PB, 0.2%Glycerin (v/v), 0.01% (v/v) Triton and 1.5% (g/100ml) Mannitol.

Kit application method includes the following steps：

(2) serum is incubated：100 holes μ L/ PBS serum dilutions are added with 100 times of diluted serum to be checked, are arranged simultaneously Positive and negative serum control, 37 DEG C, 500rpm, concussion is incubated 5 minutes, after cleaning solution rinse, discards cleaning solution；

(3) secondary antibody is incubated：The diluted concentration of secondary antibody is 1:2000,100 μ L 0.2M PBS buffer solution are added per hole (PH7.4) the sheep anti-chicken IgG secondary antibody of diluted HRP labels, 37 DEG C, 500rpm, concussion is incubated 5 minutes, cleaning solution rinse 3 times Afterwards, cleaning solution is discarded；

[embodiment 8] is used for the kit and its application method of newcastle disease virus antibody test

1, the specific following reagent of kit：

(1) contain 48 one piece of porin chip reaction plates of NDV P albumen (NDV-P)；

(4) 10 × PBS serum dilutions：PH7.4, volume 200mL；

(5) positive control sample：Anti- NDV-P chicken serums, volume 0.5mL；

(6) negative control sample：Negative SPF chicken serums, volume 0.5mL；

(7) TMB developing solutions, volume 3.5mL.

The preparation of protein chip reaction plate：

The NDV P albumen of Prepare restructuring is surveyed through BCA protein detection kits (BCA protein Assay kit) respectively After determining concentration, it is diluted to final concentration of 1mg/mL with PBS buffer solution, -20 DEG C save backup；In 100,000 grades of clean rooms Carry out the micro- lattice point sample of albumen；After albumen vacuum drying, it is dissolved in containing in 30% acetonitrile solution by the amount of 1mg/mL, then uses Spotting buffer dilutes antigen to working concentration 0.2mg/mL, and on point sample to iPDMS films, protein chip, vacuum drying packet is made Dress, 4 DEG C save backup；The PBS buffer solution is the PBS buffer solution of 0.01M pH7.4；The formula of the spotting buffer For：0.3M PB, 0.2%Glycerin (v/v), 0.01% (v/v) Triton and 1.5% (g/100ml) Mannitol.

Kit application method includes the following steps：

Sequence table

<110>Agricultural University Of Nanjing

<120>Poultry diease viral disease protein chip antibody assay kit and its preparation method and application

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1497

<212> DNA

<213>Avian influenza virus AIV H3N2 (AIV H3N2)

<400> 1

atggcgtccc aaggcaccaa acggtcttat gaacagatgg aaactgatgg ggatcgccag 60

aatgcaaccg agattagggc atccgtcggg aagatgattg atggaattgg gagattctac 120

atccaaatgt gcactgaact taaactcagt gattatgaag ggcggttgat ccagaacagc 180

ttgacaatag agaaaatggt gctctctgct tttgatgaga gaaggaataa atatctggaa 240

gaacacccca gcgcggggaa agatcctaag aaaactggag ggcccatata caggagagta 300

gatggaaaat ggattaggga actcgtcctt tatgacaaag aagaaataag gcgaatctgg 360

cgccaagcca acaatggtga ggatgcgaca gctggtctaa ctcacatgat gatctggcat 420

tccaatttga atgatgcaac ataccagagg acaagagctc ttgttcgaac cggaatggat 480

cccaggatgt gctctctgat gcagggctcg actctcccta gaaggtccgg agctgcaggt 540

gctgcagtca aaggaatcgg gacaatggtg atggagctga tcagaatggt caaacggggg 600

atcaacgatc gaaatttctg gagaggtgag aatgggcgga aaacaagaag tgcttatgag 660

aggatgtgca acattcttaa agggaaattt caaacagctg cacaaagagc aatggtggat 720

caagtgagag aaagtcggaa cccaggaaat gctgagatcg aagatctcat atttttggca 780

agatctgcat tgatattgag agggtcagtt gctcacaaat cttgcctacc tgcctgtgtg 840

tatggacctg cagtatccag tgggtacaac ttcgaaaaag agggatattc cttggtggga 900

atagaccctt tcaaactact tcaaaatagc caagtataca gcctaatcag acctaacgag 960

aatccagcac acaagagtca gctggtgtgg atggcatgcc attctgctgc atttgaagat 1020

ttaagattgt taagcttcat cggagggacc aaagtatctc cgcgggggag actttcaact 1080

agaggagtac aaattgcttc aaatgagaac atggataata tgggatcgag tactcttgaa 1140

ctgagaagcg ggtactgggc cataaggacc aggagtggag gaaacactaa tcaacagagg 1200

gcctccgcag gccaaatcag tgtgcaacct acgttttctg tacaaagaaa cctcccattt 1260

gaaaagtcaa ccgtcatggc cgcattcact ggaaatacgg agggaagaac ctcagacatg 1320

agggcagaaa tcataagaat gatggaaggt gcaaaaccag aagaagtgtc gttccggggg 1380

aggggagttt tcgagctctc ggacgagaag gcgacgaacc cgatcgtgcc ctcttttgac 1440

atgagtaatg aaggatctta tttcttcgga gacaatgcag aagagtacga caattaa 1497

<210> 2

<211> 498

<212> PRT

<213>Avian influenza virus AIV H3N2 (AIV H3N2)

<400> 2

Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Asp

1 5 10 15

Gly Asp Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Lys Met

20 25 30

Ile Asp Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys

35 40 45

Leu Ser Asp Tyr Glu Gly Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu

50 55 60

Lys Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu

65 70 75 80

Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile

85 90 95

Tyr Arg Arg Val Asp Gly Lys Trp Ile Arg Glu Leu Val Leu Tyr Asp

100 105 110

Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp

115 120 125

Ala Thr Ala Gly Leu Thr His Met Met Ile Trp His Ser Asn Leu Asn

130 135 140

Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp

145 150 155 160

Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser

165 170 175

Gly Ala Ala Gly Ala Ala Val Lys Gly Ile Gly Thr Met Val Met Glu

180 185 190

Leu Ile Arg Met Val Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg

195 200 205

Gly Glu Asn Gly Arg Lys Thr Arg Ser Ala Tyr Glu Arg Met Cys Asn

210 215 220

Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Val Asp

225 230 235 240

Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu

245 250 255

Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His

260 265 270

Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Pro Ala Val Ser Ser Gly

275 280 285

Tyr Asn Phe Glu Lys Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe

290 295 300

Lys Leu Leu Gln Asn Ser Gln Val Tyr Ser Leu Ile Arg Pro Asn Glu

305 310 315 320

Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala

325 330 335

Ala Phe Glu Asp Leu Arg Leu Leu Ser Phe Ile Gly Gly Thr Lys Val

340 345 350

Ser Pro Arg Gly Arg Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn

355 360 365

Glu Asn Met Asp Asn Met Gly Ser Ser Thr Leu Glu Leu Arg Ser Gly

370 375 380

Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln Gln Arg

385 390 395 400

Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg

405 410 415

Asn Leu Pro Phe Glu Lys Ser Thr Val Met Ala Ala Phe Thr Gly Asn

420 425 430

Thr Glu Gly Arg Thr Ser Asp Met Arg Ala Glu Ile Ile Arg Met Met

435 440 445

Glu Gly Ala Lys Pro Glu Glu Val Ser Phe Arg Gly Arg Gly Val Phe

450 455 460

Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp

465 470 475 480

Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr

485 490 495

Asp Asn

<210> 3

<211> 1188

<212> DNA

<213> NDV-Lasota(NDV-Lasota)

<400> 3

atggccacct ttacagatgc agagatcgac gagctatttg agacaagtgg aactgtcatt 60

gacaacataa ttacagccca gggtaaacca gcagagactg ttggaaggag tgcaatccca 120

caaggcaaga ccaaggtgct gagcgcagca tgggagaagc atgggagcat ccagccaccg 180

gccagtcaag acaaccccga tcgacaggac agatctgaca aacaaccatc cacacccgag 240

caaacgaccc cgcatgacag cccgccggcc acatccgccg accagccccc cacccaggcc 300

acagacgaag ccgtcgacac acagctcagg accggagcaa gcaactctct gctgttgatg 360

cttgacaagc tcagcaataa atcgtccaat gctaaaaagg gcccatggtc gagcccccaa 420

gaggggaatc accaacgtcc gactcaacag caggggagtc aacccagtcg cggaaacagt 480

caggaaagac cgcagaacca agtcaaggcc gcccctggaa accagggcac agacgtgaac 540

acagcatatc atggacaatg ggaggagtca caactatcag ctggtgcaac ccctcatgct 600

ctccgatcaa ggcagagcca agacaatacc cttgtatctg cggatcatgt ccagccacct 660

gtagactttg tgcaagcgat gatgtctatg atggaggcga tatcacagag agtaagtaag 720

gtcgactatc agctagatct tgtcttgaaa cagacatcct ccatccctat gatgcggtcc 780

gaaatccaac agctgaaaac atctgttgca gtcatggaag ccaacttggg aatgatgaag 840

attctggatc ccggttgtgc caacatttca tctctgagtg atctacgggc agttgcccga 900

tctcacccgg ttttagtttc aggccctgga gacccctctc cctatgtgac acaaggaggc 960

gaaatggcac ttaataaact ttcgcaacca gtgccacatc catctgaatt gattaaaccc 1020

gccactgcat gcgggcctga tataggagtg gaaaaggaca ctgtccgtgc attgatcatg 1080

tcacgcccaa tgcacccgag ttcttcagcc aagctcctaa gcaagttaga tgcagccggg 1140

tcgatcgagg aaatcaggaa aatcaagcgc cttgctctaa atggctaa 1188

<210> 4

<211> 395

<212> PRT

<213> NDV-Lasota(NDV-Lasota)

<400> 4

Met Ala Thr Phe Thr Asp Ala Glu Ile Asp Glu Leu Phe Glu Thr Ser

1 5 10 15

Gly Thr Val Ile Asp Asn Ile Ile Thr Ala Gln Gly Lys Pro Ala Glu

20 25 30

Thr Val Gly Arg Ser Ala Ile Pro Gln Gly Lys Thr Lys Val Leu Ser

35 40 45

Ala Ala Trp Glu Lys His Gly Ser Ile Gln Pro Pro Ala Ser Gln Asp

50 55 60

Asn Pro Asp Arg Gln Asp Arg Ser Asp Lys Gln Pro Ser Thr Pro Glu

65 70 75 80

Gln Thr Thr Pro His Asp Ser Pro Pro Ala Thr Ser Ala Asp Gln Pro

85 90 95

Pro Thr Gln Ala Thr Asp Glu Ala Val Asp Thr Gln Leu Arg Thr Gly

100 105 110

Ala Ser Asn Ser Leu Leu Leu Met Leu Asp Lys Leu Ser Asn Lys Ser

115 120 125

Ser Asn Ala Lys Lys Gly Pro Trp Ser Ser Pro Gln Glu Gly Asn His

130 135 140

Gln Arg Pro Thr Gln Gln Gln Gly Ser Gln Pro Ser Arg Gly Asn Ser

145 150 155 160

Gln Glu Arg Pro Gln Asn Gln Val Lys Ala Ala Pro Gly Asn Gln Gly

165 170 175

Thr Asp Val Asn Thr Ala Tyr His Gly Gln Trp Glu Glu Ser Gln Leu

180 185 190

Ser Ala Gly Ala Thr Pro His Ala Leu Arg Ser Arg Gln Ser Gln Asp

195 200 205

Asn Thr Leu Val Ser Ala Asp His Val Gln Pro Pro Val Asp Phe Val

210 215 220

Gln Ala Met Met Ser Met Met Glu Ala Ile Ser Gln Arg Val Ser Lys

225 230 235 240

Val Asp Tyr Gln Leu Asp Leu Val Leu Lys Gln Thr Ser Ser Ile Pro

245 250 255

Met Met Arg Ser Glu Ile Gln Gln Leu Lys Thr Ser Val Ala Val Met

260 265 270

Glu Ala Asn Leu Gly Met Met Lys Ile Leu Asp Pro Gly Cys Ala Asn

275 280 285

Ile Ser Ser Leu Ser Asp Leu Arg Ala Val Ala Arg Ser His Pro Val

290 295 300

Leu Val Ser Gly Pro Gly Asp Pro Ser Pro Tyr Val Thr Gln Gly Gly

305 310 315 320

Glu Met Ala Leu Asn Lys Leu Ser Gln Pro Val Pro His Pro Ser Glu

325 330 335

Leu Ile Lys Pro Ala Thr Ala Cys Gly Pro Asp Ile Gly Val Glu Lys

340 345 350

Asp Thr Val Arg Ala Leu Ile Met Ser Arg Pro Met His Pro Ser Ser

355 360 365

Ser Ala Lys Leu Leu Ser Lys Leu Asp Ala Ala Gly Ser Ile Glu Glu

370 375 380

Ile Arg Lys Ile Lys Arg Leu Ala Leu Asn Gly

385 390 395

<210> 5

<211> 34

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 5

ggatccgaat tcatggcgtc ccaaggcacc aaac 34

<210> 6

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 6

cggcgaagct tttaattgtc gtactcttct gc 32

<210> 7

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 7

aaaaagagct catggccacc tttacagatg ca 32

<210> 8

<211> 32

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 8

aaattaagct tttagccatt tagagcaagg cg 32

Claims

1. a kind of for detecting the antibody assay kit of poultry diease viral disease, which is characterized in that the kit includes：

(a) include arbitrary two or three recombinated in AIV nucleoprotein, NDV P albumen and IBV Non structural protein 5s；

(b) include recombination AIV nucleoprotein；

(c) include recombination NDV P albumen；

(3) negative control sample：Negative SPF chicken serums；

2. kit according to claim 1, which is characterized in that the antigen on the protein chip reaction plate is recombination Three kinds of AIV nucleoprotein, NDV P albumen and IBV Non structural protein 5s；These three antigen coats are in the same reacting hole.

3. kit according to claim 2, which is characterized in that the working concentration of the antigen is 0.1~0.2mg/mL, Preferably 0.2mg/mL.

4. kit according to claim 1, which is characterized in that the kit further includes following components:

(6) 10 × PBS serum dilutions：pH7.4；

(7) TMB developing solutions.

5. kit according to claim 4, which is characterized in that the extension rate of the IgG enzyme labelled antibodies solution is 1: 1000~1:2000, preferably 1:2000.

6. the preparation method of the antibody assay kit for detecting poultry diease viral disease described in any one of claim 1-5, It is characterized in that the preparation method of the wherein protein chip reaction plate containing antigen includes：Antigen is prepared, respectively after measured concentration, is used PBS buffer solution is diluted to final concentration of 1mg/mL；The micro- lattice point sample of albumen is carried out in 100,000 grades of clean rooms；Albumen vacuum After drying, it is dissolved in containing in 30% acetonitrile solution by the amount of 1mg/mL, is then extremely worked with spotting buffer dilution antigen On point sample to iPDMS films, protein chip, vacuum drying packaging is made in concentration；The PBS buffer solution is 0.01M pH7.4's PBS buffer solution；The formula of the spotting buffer is：0.3M PB, 0.2%Glycerin, 0.01%Triton and 1.5% Mannitol。

7. the application method of any kit in Claims 1 to 5, it is characterised in that include the following steps：

(1) protein chip reaction plate takes out from 4 DEG C and places room temperature, with cleaning solution rinse；The cleaning solution is containing 0.05% The TBS of Tween-20, i.e. TBST；

(2) serum is incubated：100 holes μ L/ PBS serum dilutions are added with 50~100 times of diluted serum to be checked, are arranged simultaneously Positive and negative serum control, 37 DEG C, 500rpm, concussion is incubated 5min and discards cleaning solution after cleaning solution rinse；

(3) secondary antibody is incubated：The diluted concentration of secondary antibody is 1:1000~1:2000, it is slow that 100 μ L pH7.4,0.2M PBS are added per hole The sheep anti-chicken IgG secondary antibody of fliud flushing diluted HRP label, 37 DEG C, 500rpm, concussion is incubated 5min, after cleaning solution rinse 3 times, abandons Fall cleaning solution；

(4) result is observed：60 μ L TMB developing solutions are added per hole, colour developing after five minutes, abandons developing solution, cleaning solution is rinsed well, meat Eye observation testing result；

(5) result judgement：Detect by an unaided eye colour developing situation, other than a positive quality control point is blue, if other points develop the color, Then be judged as the positive, be judged as feminine gender if not developing the color, if colour developing but color it is very light, be judged as weak sun.

8. application of any kit in detecting poultry diease viral disease in Claims 1 to 5.

9. application according to claim 8, which is characterized in that the kit can detect simultaneously bird flu, newcastle disease and Infectious bronchitis of chicken, or in which two kinds, or individually detection bird flu and newcastle disease in any one.