CN107543923A - Detect the kit and its detection method of avian leukosis virus A/B/J subgroup specific antibodies - Google Patents

Abstract

The present invention relates to the kit and its detection method of detection avian leukosis virus A/B/J subgroup specific antibodies, belong to biology field, the kit includes luminous microwell plate, sealed membrane, acceptor microballoon I, acceptor microsphere Ⅱ, donor microballoon, calibration object and analysis buffer；Avian leukosis virus A/B subgroup His cENV albumen is marked on the acceptor microballoon I, avian leukosis virus J subgroup His gp85 albumen is marked on acceptor microsphere Ⅱ；Donor microballoon marks rabbit-anti chicken secondary antibody.Detection is that the sample serum of collection is pre-processed first, and then acceptor microballoon I, acceptor microsphere Ⅱ, donor microballoon and sample serum are added on luminous microwell plate on request, optical signal value is detected under 680nm exciting lights.The detection to avian leukosis virus A/B/J specific antibodies is the method achieve, simple to operate without washing, detection is sensitive, and specificity and stability are stronger.

Description

Detect kit and its detection of avian leukosis virus A/B/J subgroup specific antibodies Method

Technical field

The invention belongs to biology field, is related to Animal diseases detection, in particular it relates to detect avian leukosis virus The kit and its detection method of A/B/J subgroup specific antibodies.

Background technology

Avian leukosis refers to as caused by Retroviridae A type retrovirus fowl c-type retrovirus group with fowl Various transmissible tumor diseases in class hematopoietic tissue based on some cell component hyperplasias.It is white with fowl lymphatic Blood disease, erythroblast property leukaemia and myeloblastosis are relatively conventional, pass through two kinds of sides of vertical transmission and horizontal transmission Formula is widely current in the chicken group of countries in the world, causes chicken death, becomes thin, anaemia, retarded growth, while causes body to be resisted Power declines and immunosupress, is to endanger one of principal disease of aviculture.Chicken infected avian leukosis virus (ALV) group is main It is divided into six subgroups of A, B, C, D, E, J, wherein A, B subgroup strain gene order has very high homology, has similar disease Malicious characteristic.A, B, C, D and J subgroup are exogenous that E subgroups mainly exist in the form of provirus, are endogenic.A, B is sub- Group is more typical in chicken group, and C, D subgroup are more rare, and J subgroups are a kind of new avian leukosis virus subgroups.

The generation of China's A/B/J subgroup avian leucosis is fairly common, and clinical case mass-sends exhibition from initial commercial broiler To commodity egg, local breeder flock and other poultry, and along with oncogenicity enhancing and the generation of tumor multiplicity.Research is aobvious Show, high infection rate, high incidence is presented in the A/B/J subgroup avian leucosis poison in China, and early infection is universal, morbidity age in days substantially carries Before, the features such as mixed infection is multiple/take place frequently, and host range extension, tumour spectrum expand, and pathological change complicates, to supporting for China Fowl industry causes huge loss.For this, foreign countries take strict cut-out vertical transmission and isolate the offspring being uninfected by and raise Foster control techniques, from 1987, the large-scale breeder company of international developed country was just announced exogenous avian leukosis later Poison purifies.Because purification cycle is long, cost is high, also fail to effectively implement at present in China, can only by early diagnosis, in time Superseded infected chicken purifies population, and therefore, fast and accurately diagnostic method is particularly important in the sick preventing and treating.Except root Outside according to tentative diagnosis such as clinical symptoms, pathological changes, making a definite diagnosis for avian leukosis typically relies primarily on pathogeny detection, conventional inspection Survey method has the detection methods such as Virus Isolation, indirect immunofluorescence assay and ELISA, wherein, Virus Isolation is uncomfortable Conjunction detects to a large amount of samples；The characteristics of indirect immunofluorescence assay is to need fluorescence microscope, is operationally had certain Limitation；Although ELISA is adapted to the detection of extensive sample, operating process is cumbersome, time-consuming, and testing result goes out frequently The situation that existing false positive or viral recessiveness can not detect, and not can determine that virus subtype.

AlophaScreen(Amplified Luminscent Proximity Homogeneous Assay Screen) Technology is a kind of homogeneous detection method for being applied to analyte based on effect close between particulate and particulate, and AlophaLISA It is then the homogeneous detection method for having similarity with classic ELISA technology in AlophaScreen technical foundation, we are referred to as For homogeneous chemistry luminescence technology.The core of AlophaLISA methods be donor (photosensitive particulate) and acceptor (luminous particle) both The particulate that diameter is about 200nm.Mutually it is coupled in two kinds of microparticle surfaces and biomolecule (antibody or antigen protein), each particulate table Face can cover hundreds and thousands of individual biomolecule, can capture testing molecule.After particulate is irradiated by 680nm laser, donor table Oxygen in solution (AlophaLISA reaction solutions) will be converted into singlet oxygen by the sensitising agent in face.Singlet oxygen is in the solution Propagation distance be about 200nm, if the distance between donor and acceptor are less than 200nm, singlet oxygen is with regard to that can diffuse to acceptor Particulate, light-sensitive compound in acceptor just can and singlet oxygen chemically react, wherein Eu³⁺Complex just produces high level It is luminous.On the contrary, if the distance between luminous particle and photosensitive particulate are more than 200nm, free oxygen can not diffuse to luminous particle, There will be no the generation of optical signal.AlophaLISA homogeneous reactions are namely based on principles above, if being coated on two kinds of particulate tables The biomolecule in face is the distance between the two kinds of particulates that just furthered in the presence of interaction, produces optical signal.By to optical signal Intensity detection, to judge object to be measured is whether there is in actually detected sample.Although being based on this principle, AlophaLISA technologies exist Lung cancer, hepatitis or hand-foot-and-mouth disease etc. many-side has applied, but on avian leukosis virus detection of specific antibody there has been no It is related to, in view of principle in itself and does not have actual operation, the otherness between detectable substance in addition can not between detection architecture Use is indiscriminately imitated, detection efficiency and detection quality how is improved on this basis, forms a set of effective standards system, be allowed to It can in practice be widely used, be still a very important problem.

The content of the invention

In view of the above-mentioned problems, the present invention provides a kind of reagent for detecting avian leukosis virus A/B/J subgroup specific antibodies Box, changing kit can realize to being detected while avian leukosis virus A/B/J specific antibodies, simple to operate without washing, Detect sensitive.

A kind of homogeneous chemistry luminescence reagent box for detecting avian leukosis virus A/B/J subgroup specific antibodies, including it is luminous Microwell plate, sealed membrane, acceptor microballoon I, acceptor microsphere Ⅱ, donor microballoon, calibration object and analysis buffer；

Avian leukosis virus A/B subgroup His-cENV albumen is marked on the acceptor microballoon I, the white blood of fowl is marked on acceptor microsphere Ⅱ The viral J subgroups His-gp85 albumen of disease；The donor microballoon marks rabbit-anti chicken secondary antibody；

The labeling process of the acceptor microballoon I, acceptor microsphere Ⅱ or donor microballoon is realized by the following method：Will be to be marked Thing is placed in centrifuge tube, is added phosphate buffer, centrifugation, washing and suspension, is added MES buffer solutions after blotting supernatant, then Sequentially add purified label, sodium cyanoborohydride solution and Tween-20,2-8 DEG C of 40~48h of lucifuge revolving reaction Afterwards, 37 DEG C of stirring 1h terminating reactions of CMO lucifuges are added；Supernatant is removed in centrifugation, adds HEPES buffer solution to be resuspended, and supernatant is removed in centrifugation, adds Liquid is preserved, acceptor microballoon I, acceptor microsphere Ⅱ or the donor microballoon of coupling label is made；

Calibration object is formulated by the sterling antibody extracted in the positive standard serum that is mixed into from more parts of high level serum；

The analysis buffer is 50mmol/L, pH7.4 Tris-HCl buffer solutions, wherein being 0.8% containing mass fraction Bovine serum albumin(BSA) that NaCl, mass fraction are 1%, the casein that mass fraction is 0.1%, 0.1% surfactant S9, The Qula that volume fraction is 0.1% leads to the Proclin-300 that -100, volume fraction is 0.05%.

Further, the donor microballoon is mark rabbit-anti chicken secondary antibodyDonor microballoon.

Further, the acceptor microballoon I is the AlphaPlex of mark His-cENV albumen^TM645 microballoons；Acceptor microballoon II is mark His-gp85 AlphaPlex^TM545 microballoons.

Further, the labeling process of the acceptor microballoon I, acceptor microsphere Ⅱ or donor microballoon is real by the following method Existing：20mg/mL thing to be marked is placed in centrifuge tube, 0.1mol/L, pH8.0 are added into every 0.2mL thing to be marked Phosphate buffer 1 mL, centrifugation, washing and suspend repeatedly for 3~5 times, blot the MES buffer solutions that supernatant adds 0.2mL, then The purified labels of 1~3mg/mL of 0.2mg~0.6mg concentration are added, it is 400~500mmol/L to add 8~12uL concentration Sodium cyanoborohydride solution and Tween-20 that 12.5uL volume fractions are 1%, after 2-8 DEG C of 40~48h of lucifuge revolving reaction, Add 37 DEG C of stirring 1h terminating reactions of 10uL CMO lucifuges；Supernatant is removed in centrifugation, adds 50mmol/L pH7.4 HEPES buffer solution 0.5mL is resuspended, and supernatant is removed in centrifugation, adds and preserves liquid, and the acceptor microballoon I, acceptor microsphere Ⅱ or donor that coupling label is made are micro- Ball；

Further, it is described to preserve the Tris-HCl buffer solutions that liquid is 50mmol/L, pH7.4, wherein including 6.05g/L's Tris, 10g/L trehalose, 10g/L glucose, 30g/L mannitol, 20g/L bovine serum albumin(BSA), 5g/L dried meat ammonia Acid and the Proclin-300 that volume fraction is 0.1%.

Further, the calibration object is made by following methods：50 parts of high level serum after testing is hybridly prepared into Positive standard serum, 10000rpm/min centrifugation 30min, then through affinitive layer purification, collect sterling antibody, sterling antibody is used Ampere bottle is dispensed and freezes to form freeze-dried powder, and freeze-dried powder is configured into 6 calibration object concentration with analysis buffer：0ng/mL、 10ng/mL, 40ng/mL, 160ng/mL, 640ng/mL and 2560ng/mL.

The present invention also provides a kind of side of the kit detection avian leukosis A/B/J subgroup specific antibodies described in utilization Method, comprise the following steps：

Step 1: the collection and preservation of clinical samples：Take without anti-coagulants vacuum blood collection tube collection chicken venous whole sample, 37 DEG C 2h is placed, then placed overnight in 2~8 DEG C, the serum of precipitation is suctioned out, 3000rpm/min centrifugation 5min, take supernatant to dispense In EP pipes and blood sampling date and identifier are indicated, sample serum is made；If the sample serum same day uses, 2~8 are placed directly in In DEG C refrigerator；If the next day use, -20 DEG C of freezen protectives；

Step 2: the acceptor microballoon I prepared, acceptor microsphere Ⅱ and donor microballoon are respectively added into 100 μ L in luminous microwell plate, Then the μ L of sample serum 20 are added, are well mixed, 37 DEG C of incubation 20min, then excite lower detection not in the light that wavelength is 680nm With the signal light value of subgroup specific antibody.

Further, the concentration of acceptor microballoon I is 0.015mg/mL；The concentration of acceptor microsphere Ⅱ is 0.020mg/mL；For The concentration of body microballoon is 0.030mg/mL.

Beneficial effect：

The present invention utilizes homogeneous chemistry luminescence technology, and envelope protein is coupled on acceptor microballoon, rabbit-anti chicken is marked on donor microballoon Secondary antibody, antigen or antibody protein are coupled on microballoon using the system of original creation, in the coupling mark system of albumen and microballoon, PH of buffer, solution concentration and dosage, reaction time and temperature etc. can all have an impact to coupling labeling process, and the present invention uses Different buffer solution washing microballoons, increases its surface group activity, in being reacted with label, sequentially adds sodium cyanoborohydride Solution and Tween-20, prevent the active group of microsphere surface from aoxidizing, or other chemical reactions occur, be passivated active group, Lose activity, or the serious reduction of activity；Coupling time and reaction condition are controlled, the colleague of reaction rate is being improved, is avoiding sending out Raw albumen autoimmunity syndrome, improve coupling amount.Washed again with buffer solution after completion of the reaction, reduce non-covalent adsorption, at utmost Coupling efficiency is improved, increases the homogeneity, sensitivity and specificity of coupled complex, and then determine microballoon in specific antibody High sensitivity and high efficiency in detection, furthermore, preserve trehalose, glucose, mannitol, the bovine serum albumin added in liquid In vain, coordinative role between proline and Proclin-300, the stability and specificity of coupling material are improved, can be fast exactly Speed detects target substance, the effect of having given full play of kit.The kit can also be realized to avian leukosis virus A/B/J Detected while specific antibody, and the antibody of these three subgroups, convenient and swift, efficiency can be distinguished according to the flashlight of detection Height, and without washing, simple to operate, detection is sensitive.Meanwhile open a kind of detection avian leukosis virus specific antibody and resist The new way of body, form a set of effective detection architecture.

Detected using the kit, avian leukosis A/B subgroups positive serum and the dilution of 256 times of dilution can be detected 512 times of J subgroup positive serums, sensitiveness are higher；15 parts of poultry diease positive serums are detected, the testing result of all samples It is all negative, shows the present invention and other poultry diease serum no cross reactions, there is good specificity, and to avian leukosis poison A/ The detection of B/J subgroup positive serums, luminous signal is very strong, illustrates there is good reactivity.In addition, carried out using the kit Detection, preferably, stability is strong for repeatability, can be preserved 12 months at 2-8 DEG C.The kit can be used for avian leukosis poison A/B/ J subgroups antibody carries out quantitative detection.

Brief description of the drawings

Fig. 1 is pUC19-cENV gene recombination plasmid double digestion qualification result electrophoretograms；Wherein, left side is using restricted The genetic fragment that restriction endonuclease EcoRI, XhoI double digestion pUC19-gp85 is obtained, right side DL10000Marker；

Fig. 2 is pUC19-gp85 gene recombination plasmid double digestion qualification result electrophoretograms；Wherein, 1 is to use restriction enzyme EcoRI, XhoI double digestion pUC19 empty plasmids, 2 be to be obtained using restriction enzyme EcoRI, XhoI double digestion pUC19-gp85 Genetic fragment, M is 1kb DNA Ladder；

Fig. 3 is recombinant expression protein cENV rear SDS-PAGEs before purification；Wherein, M is protein Marker in figure, and 1 is Induction bacterium bacterium solution precipitates, and 2 be supernatant, and 3 be great expression albumen precipitation, and 4 be result after great expression protein purification；

Fig. 4 is recombinant expression protein gp85 rear SDS-PAGEs before purification；Wherein, M is protein Marker in figure, and 1 is Induction bacterium bacterium solution precipitates, and 2 be supernatant, and 3 be great expression albumen precipitation, and 4 be result after great expression protein purification；

Fig. 5 is Western blot methods detection cENV recombinant protein result figures；

Fig. 6 is Western blot methods detection gp85 recombinant protein result figures.

Embodiment

Following examples or test example are used to illustrate the present invention, are not intended to limit protection scope of the present invention, are not carrying on the back In the case of from spirit of the invention and essence, the modifications or substitutions done to the inventive method, step or condition belong to this hair Bright protection domain.

Unless otherwise specified, chemical reagent used is conventional commercial reagent in embodiment or test example, technology hand used The conventional meanses that section is well known to those skilled in the art.

Main agents, instrument or consumptive material used and its source are as follows in the present invention：

Main agents：

MES (MES), cyaniding sodium borohydride (NaBH3CN), the salt of carboxymethyl azanol half (CMO), 4- hydroxyethyl piperazines Ethyl sulfonic acid (HEPES) and Prolcin-300 are purchased from Sigma-Aldrich companies；

Tween-20 is purchased from Pierce companies；

AlphaPlex^TM6 45 and AlphaPlex^TM545 are purchased from Perkin Elmer companies of the U.S.；

Albumen pre-dyed Marker, Taq polymerase, restriction enzyme Nco I and Xho I are purchased from Thermo companies；

T₄DNA ligase, dNTP, DNA Marker, IPTG, pUC19 carrier are purchased from doctor's biotechnology (Beijing) limited public affairs of precious day Department；

Plasmid extraction kit is purchased from OMEGA companies；

His tag antibodies are Beijing Quanshijin Biotechnology Co., Ltd's product.Main consumptive material and instrument：

Light excites Chemiluminescence Apparatus to be purchased from Perkin Elmer companies of the U.S.；

Ultrahigh speed freezing self-balancing centrifuge Osterode D-37520 are purchased from German Thermo fisher companies；

Ultraviolet specrophotometer TU-1900 is purchased from Beijing Puxi General Instrument Co., Ltd.

The expression and preparation of the avian leukosis poison A/B/J subgroup envelope proteins of test example 1

According to the gene order for the ALV-A/B/J subgroup envelope proteins delivered in NCBI GenBank, with reference to right both at home and abroad The progress of ALV-A/B/J subgroups, the envelope protein antigen epitope genes sequence for representing popular strain is filtered out, is recombinated A/B subgroup envelope protein cENV gene order 1244bp, its nucleotide sequence SEQ ID NO：Shown in 1；Recombinant J subgroup cyst membrane egg White gp85 gene order 1024bp, its nucleotide sequence SEQ ID NO：Shown in 3.For ease of follow-up test, expanded by PCR, Make the 5 of A/B subgroups envelope protein cENV gene orders and J subgroup envelope protein gp85 gene orders, the end position of digestion containing NcoI Point, 3 ' ends restriction enzyme site containing XhoI.Obtained cENV gene orders and gp85 gene orders are respectively connecting to pUC19 carriers On.Sequencing, after checking is correct, by pET32a (+) and the above-mentioned pUC-19 cloning vectors for being connected to target gene respectively with limitation Property restriction endonuclease Nco I and Xho I carry out double digestion 60min under 37 DEG C of water bath conditions, after 1% agarose gel electrophoresis, Glue reclaim large fragment after pET32a (+) double digestion, target gene fragment is reclaimed after pUC-19 cloning vector double digestions.Double digestion As a result as depicted in figs. 1 and 2.Will reclaim obtain large fragment and target gene fragment in 22 DEG C overnight connection, wherein with cENV The linked system of target gene fragment is：PET32a (+) carrier 1uL, cENV genetic fragments 1.5uL, ddH₂O 20uL, T₄DNA connects Enzyme 1.5uL, 10 × Green Buffer 3uL, the common 27uL of cumulative volume are met, obtains connection product pET32a-cENV recombinant plasmids； Linked system with gp85 target gene fragments is：PET32a (+) carrier 1uL, gp85 genetic fragments 1.5uL, ddH₂O 20uL, T₄DNA ligase 1.5uL, 10 × Green Buffer 3uL, the common 27uL of cumulative volume, obtain connection product pET32a-gp85 weights Group plasmid.

Connection product pET32a-cENV recombinant plasmids and pET32a-gp85 recombinant plasmids are conventionally turned respectively Enter 0.1%CaCl₂In the competence bacterium BL21 of preparation, it is coated in the LB solid mediums containing ampicillin, 37 DEG C It is incubated overnight, next day picking single bacterium colony, shakes bacterium, extraction plasmid sends to sequencing after carrying out double digestion identification, confirms wherein have such as SEQ ID NO：1 and SEQ ID NO：Sequence shown in 3, it is seen that the restructuring cENV genes and gp85 genes of acquisition respectively into Work(is cloned into pET32a-cENV recombinant plasmids and pET32a-gp85 recombinant plasmids.

Positive E. coli BL21 bacterium containing recombinant plasmid are inoculated in into the solid containing ampicillin (Amp) resistance to train Support in base, after 37 DEG C are incubated overnight, take 3~4 single bacterium colonies to be inoculated in the LB fluid nutrient mediums containing Amp, 37 DEG C of concussions (250rpm/min) is cultivated to OD₆₀₀=0.8, it is extremely final concentration of to add IPTG (isopropyl-beta D-thio galactopyranoside) 1mmol/L, 37 DEG C are continued culture 4 hours, are obtained fusion protein His-cENV and His-gp85 with HIS labels, are cultivated simultaneously The recombinant plasmid transformed BL21 bacterium not induced are as negative control.The amino acid sequence SEQ of the A/B subgroups His-cENV albumen ID NO:Shown in 02, the amino acid sequence SEQ ID NO of J subgroup His-gp85 albumen:Shown in 4.The albumen of expression is through SDS- PAGE and Western-blot identifications, as a result as shown in Figure 3 and Figure 4.

The engineering bacteria bacterium solution of culture is collected, 6000rpm/min centrifugation 10min, supernatant is abandoned, precipitates and weighed with PBS Suspension, GuNTA-0Buffer and PMSF is then added in re-suspension liquid and uses Ultrasonic Cell Disruptor ultrasonication BL21 bacteriums at low temperature, 120W, work 2s, interval 3s, 100 circulations of ultrasound；Then ultrasonication bacterium solution is transferred in 50ml centrifuge tubes, trim, 4 DEG C, 12000r/min is centrifuged 10 minutes, takes supernatant, -20 DEG C of preservations；Affinity chromatography is carried out to cracking thalline with Ni-NTA Purification Resins, Sample is added in chromatographic column, and flow velocity 12ml/h collects penetrating component.Chromatography is eluted with the GuNTA-60Buffer of 5 times of NTA volumes, Flow velocity 12ml/h, collect eluent.With the dialysis eluent of 0.5mol/LNaCl and 20mmol/L Tris-HCl solution 24 hours, Avian leukosis cENV albumen and gp85 albumen after purification carries out SDA-PAGE and Western-blot testing goal purity of protein Up to 97%.Obtained albumen is by Bradford methods measure protein content, and the packing of 2ml centrifuge tubes, -80 DEG C save backup.

The purification step of recombinant protein is as follows：

Precipitation is resuspended with the cleaning solution I of 9 times of volumes in the precipitation that above-mentioned centrifugation obtains, after standing 5min, 8000rpm, 4 DEG C, 15min, abandon supernatant.Precipitation is resuspended with the cleaning solution II of 9 times of volumes, after standing 5min, 8000rpm, 4 DEG C, 15min, abandons supernatant, Retain precipitation.Precipitation is resuspended with the cleaning solution II I of 9 times of volumes, after standing 5min, 8000rpm, 4 DEG C, 15min, abandons supernatant, protects Stay precipitation.Add 4-10mL lysates according to 100mL bacterium solutions, 4 DEG C overnight.8000rpm, 4 DEG C, 15min.Supernatant is taken, is as purified Albumen.

Preparation of reagents method described in inclusion body purification：

1st, bacterium re-suspension liquid (250mL)：PBS (NaCl 2g, KCl 0.05g, Na₂HPO₄0.36g, KH₂PO₄0.06g), 1mmol/L EDTA；

2nd, solution I (500mL)：500mmol/L Tris-HCl (PH=6.0), 50mmol/L NaCl, 50mmol/L EDTA；

3rd, solution II (250mL)：Solution I, 1mol/L urea；

4th, solution III (250mL)：Solution I, 2mol/L urea；

5th, solubilization of inclusion bodies liquid (250mL)：Solution I, 8mol/L urea.

The His-cENA albumen and His-gp85 albumen obtained using above-mentioned purifying is coated with elisa plate respectively, and use is enzyme-linked Immunosorbent adsorption test (ELISA) detects the biological activity of the albumen.Examined with His tag antibodies and IDEXX detection kits It is that positive chicken serum is positive control to survey avian leukosis poison A/B/J antibody, and healthy SFP chicken serums are negative control, and HRP is marked Sheep anti-mouse igg, sheep anti-chicken IgG ELIAS secondary antibody use to specifications.As a result positive control and the OD values with negative control are shown Ratio is big by 2.1, illustrates that the albumen has good biological activity and antigenicity.

Wherein, ELISA is comprised the following steps that：

1st, it is coated with：Take and purified recombinant protein, optium concentration, coating reaction are diluted to 0.05MpH9.6 carbonate buffer solution Plate, 100ul/ holes, 4 DEG C overnight；

2nd, wash：The coating buffer in ELISA Plate is got rid of, each hole is filled it up with PBST (PBS, 0.05%Tween-20), is stored at room temperature 3min, PBST is discarded, wash 3 times, 3min/ times, pat dry；

3rd, close：Each hole adds confining liquid (2%BSA), 200ul/ holes, puts 4 DEG C overnight, after closing, pats dry；

4th, it is loaded：Coating plate, 100ul/ holes are added after serum to be detected is pressed into gradient dilution, while sets up yin and yang attribute serum pair According to.Put 37 DEG C of incubators and be incubated 1h, wash 3 times, 3min/ times, pat dry；

5th, sheep anti-mouse igg, sheep anti-chicken IgG ELIAS secondary antibody is marked to press 1 HRP with PBST:2000 dilutions, 100ul/ holes, put 37 DEG C Incubator is incubated 1h, washs 3 times, 3min/ times, pats dry；6th, develop the color：Use TMB one-components nitrite ion (TIANGEN Products) Coating plate is added by 100ul/ holes, lucifuge colour developing 15min adds terminate liquid (2MH under the conditions of 37 DEG C₂SO₄) color development stopping；7th, with enzyme-linked Immune detector surveys its and detects the OD values in each hole in wavelength 450nm, and negative control OD450 values are N, positive control OD450 values For P.If P/N>2.1 are judged to the positive.

The preparation of the acceptor microballoon of test example 2 and donor microballoon

Take AlphaPlex^TM645 acceptor microballoon (20mg/mL) 0.2mL are placed in 1.5mL EP centrifuge tubes, add 0.1mol/L PH 8.0 phosphate buffer 1 mL, centrifuge washing are suspended 3 times, and each centrifugal force 16000g/min is centrifuged 15 minutes, is blotted Reset and add into 0.2mL MES buffer solutions, then add the recombinant protein that 1~3mg/mL of 0.3mg~0.6mg concentration has purified high activity Avian leukosis virus A/B subgroup His-cENV, it is molten to add the sodium cyanoborohydride that 8~12uL concentration is 400~500mmol/L Liquid and 12.5uL 1%Tween-20, after 2-8 DEG C of 40~48h of revolving reaction of lucifuge, it is anti-to add the 37 DEG C of stirrings of 10uL CMO lucifuges Answer 1h terminating reactions.By 4 DEG C of the acceptor microballoon marked, 13000g/min centrifugation 30min, supernatant is removed with pipettor, is added The HEPES buffer solution 0.5mL for entering 50mmol/L, pH7.4 is resuspended, and shakes suspension bottom of the tube acceptor microballoon；Repeat the above steps from The heart 2 times, the preservation liquid for adding 50mmol/L, pH7.4 are stand-by.

Take AlphaPlex^TM545 acceptor microballoon (20mg/mL) 0.2mL are placed in 1.5mL EP centrifuge tubes, are added 0.1mol/L, pH 8.0 phosphate buffer 1 mL, centrifuge washing suspend 3 times, and each centrifugal force 16000g/min centrifuges 15 points Clock, blots supernatant and adds 0.2mL MES buffer solutions, then adds 1~2mg/mL of 0.2mg~0.5mg concentration and has purified high activity Recombinant protein avian leukosis virus J subgroup His-gp85 albumen, add 8~12uL concentration be concentration be 400~ 500mmol/L sodium cyanoborohydride solution and 12.5uL 1%Tween-20, after 2-8 DEG C of 40~48h of revolving reaction of lucifuge, add Enter 37 DEG C of stirring reaction 1h terminating reactions of 10uL CMO lucifuges.4 DEG C of the acceptor microballoon marked, 13000g/min are centrifuged 30min, supernatant is removed with pipettor, the HEPES buffer solution 0.5mL for adding 50mmol/L, pH7.4 is resuspended, concussion suspension pipe Bottom acceptor microballoon；Repeat the above steps centrifugation 2 times, and it is stand-by to add 0.2mL 50mmol/L, pH7.4 preservation liquid.

Take aldehyde radicalDonor microballoon (20mg/mL) 0.2mL is placed in 1.5mL EP centrifuge tubes, is added 0.02mol/L pH7.6 phosphate buffer 1 mL, centrifuge washing suspend 3 times, and each centrifugal force 16000g/min centrifuges 15 points Clock, blots supernatant and adds 0.2mL MES buffer solutions, then adds 1~2mg/mL of 0.4mg~0.6mg concentration and has purified high activity Rabbit-anti chicken secondary antibody, add 8~12uL concentration be concentration be 400~500mmol/L sodium cyanoborohydride solution and After 12.5uL 1%Tween-20,2-8 DEG C of 40~48h of revolving reaction of lucifuge, 37 DEG C of stirring reaction 1h of 10uL CMO lucifuges are added Terminating reaction.By 4 DEG C of the acceptor microballoon marked, 13000g/min centrifugation 30min, supernatant is removed with pipettor, added 50mmol/L, pH7.4 HEPES buffer solution 0.5mL are resuspended, and shake suspension bottom of the tube acceptor microballoon；Repeat the above steps centrifugation 2 Secondary, the preservation liquid for adding 50mmol/L, pH7.4 is stand-by.

The preparation of the HEPES buffer solution：HEPES 5.96g and bovine serum albumin(BSA) (BSA) 5g are weighed, adds water 800mL Dissolving, adds 5mL Tween-20s, adds water to be settled to 1L.

The preservation liquid is 50m mol/L, pH7.4 Tris-HCl buffer solutions：Tris 6.05g are weighed, add water 800mL Dissolving, adjust pH to 7.4；Add trehalose 10g, glucose 10g, mannitol 30g, BSA 20g, proline 5g, Proclin-300 1mL, after dissolving plus water is settled to 1L.

The preparation of described 0.1mol/L, pH 8.0 phosphate buffer：Weigh Na₂HPO₄·12H₂O 33.9g and NaH₂PO₄0.731g, add water 800mL to dissolve, add 0.5mL Tween-20s, add water to be settled to 1L.

The preparation of described 0.02mol/L, pH7.6 phosphate buffer：Weigh Na₂HPO₄·12H₂O 5.8g、NaH₂PO₄ 0.496g and NaCl 8g, add water 800mL to dissolve, add 0.5mL Tween-20s, add water to be settled to 1L.

The preparation of the MES buffer solutions：MES 21.3g are weighed, add 800mL distilled water to dissolve, are adjusted with 10mol/L NaOH PH value is settled to 1L to 6.0.

The homogeneous chemistry luminescence detection kit of the avian leukosis A/B/J subgroup specific antibodies of test example 3 assembles

The homogeneous chemistry luminescence detection kit of avian leukosis A/B/J subgroup specific antibodies, including luminous microwell plate, sealing Film, acceptor microballoon, donor microballoon, calibration object and analysis buffer；It is sub- that the acceptor microballoon includes coupling avian leukosis virus A/B The AlphaPlex of group's His-cENV albumen^TM645 acceptor microballoons and coupling avian leukosis virus J subgroup His-gp85 albumen AlphaPlex^TM545 acceptor microballoons；The donor microballoon is mark rabbit-anti chicken secondary antibodyDonor microballoon；Institute State as the Tris-HCl buffer solutions that analysis buffer is 50mmol/L, pH7.4, wherein containing the NaCl that mass fraction is 0.8%, Casein that bovine serum albumin(BSA) that mass fraction is 1%, mass fraction are 0.1%, 0.1% surfactant S9, volume integral Number leads to the Proclin-300 that -100, volume fraction is 0.05% for 0.1% Qula；The luminous microwell plate is that 96 holes light Microwell plate (Perkin Elmer companies of the U.S.)；(U.S. PerkinElmer is public using TopSeal-A sealed membranes for the sealed membrane Department).

The calibration object is made by following methods：50 parts of high level serum after testing is hybridly prepared into positive criteria blood Clearly, 10000rpm/min centrifuges 30min, then through affinitive layer purification, collects sterling antibody, sterling antibody is dispensed with ampere bottle And freeze and form freeze-dried powder, freeze-dried powder is configured to 6 calibration object concentration with analysis buffer：0ng/mL、10ng/mL、40ng/ ML, 160ng/mL, 640ng/mL and 2560ng/mL.

Assemble and finish, kit saves backup in -20 DEG C.

The homogeneous chemistry luminescence detection kit detection avian leukosis A/B/J subgroup specificity of the present invention of test example 4 is anti- The condition optimizing of body

(1) acceptor microballoon and the optimal dilution ratio of donor microballoon

Using Checkerboard titration method, avian leukosis virus A/B subgroups His-cENV AlphaPlex^TM645 acceptor microballoons and fowl are white The viral J subgroups His-gp85 of blood disease AlphaPlex^TM545 acceptor microballoons all set respectively 0.005mg/mL, 0.010mg/mL, 0.015mg/mL, 0.020mg/mL, 0.025mg/mL, 0.030mg/mL totally 6 concentration, mark rabbit-anti chicken secondary antibodyDonor microballoon sets 0.010mg/mL, 0.020mg/mL, 0.030mg/mL, 0.040mg/mL totally 4 concentration, surveys Determine result and optimal dilution ratio is determined with signal/blank ratio (Signal/Background, S/B) and four parameter curves. The final AlphaPlex for determining labelled protein avian leukosis virus A/B subgroups His-cENV^TM645 acceptor microballoon optium concentrations are 0.015mg/ml, labelled protein avian leukosis virus J subgroups His-gp85 AlphaPlex^TM545 acceptor microballoon optium concentrations For 0.020mg/ml, rabbit-anti chicken secondary antibody is markedDonor microballoon optium concentration is 0.030mg/ml.

(2) reaction temperature and time

10min, 20min, 30min, 40min, 50min, 60min will be set as the reaction time, reaction temperature sets 25 DEG C and 37 DEG C, according to S/B values, selection S/B value highest times and most short temperature and time, the acceptor that will be prepared in luminous microwell plate Microballoon and donor microballoon respectively add 100 μ L, then add the μ L of sample serum 20, are well mixed, by the temperature that sets in advance and when Between dereaction, then light excite Chemiluminescence Apparatus read signal value.As a result select 37 DEG C incubation 20min, be optimum temperature and Incubation time.

(3) selection of most suitable analysis buffer

Choose following 4 kinds of buffer solutions：HEPES buffer solution, PBS, MES buffer solutions, Tris buffer solutions, using S/B as excellent Change standard.Final choice S/B value highests analysis buffer is 50mmol/L, pH7.4 Tris-HCl buffer solutions, wherein containing Casein that bovine serum albumin(BSA) that NaCl that mass fraction is 0.8%, mass fraction are 1%, mass fraction are 0.1%, The Qula that 0.1%S9, volume fraction are 0.1% leads to the Proclin-300 that -100, volume fraction is 0.05%

The method that 5 kit of the present invention of test example detects avian leukosis A/B/J subgroup specific antibodies

The method of kit detection avian leukosis A/B/J subgroup specific antibodies of the present invention, comprises the following steps：

Step 1: the collection and preservation of clinical samples：Take without anti-coagulants vacuum blood collection tube collection chicken venous whole sample, 37 DEG C 2h is placed, then placed overnight in 2~8 DEG C, the serum of precipitation is suctioned out, 3000rpm/min centrifugation 5min, take supernatant to dispense In EP pipes and blood sampling date and identifier are indicated, sample serum is made；If the sample serum same day uses, 2~8 are placed directly in In DEG C refrigerator；If the next day use, -20 DEG C of freezen protectives.

Step 2: the acceptor microballoon prepared and donor microballoon are respectively added into 100 μ L in luminous microwell plate, then add The μ L of sample serum 20, it is well mixed, 37 DEG C of incubation 20min, then excites Chemiluminescence Apparatus to read luminous value in light.

Step 3: the operating process detected according to homogeneous chemistry luminescence reagent box described in step 1 and step 2, will be prepared The calibration object of six kinds of concentration is measured, and the parameter fitting of acquired results four, which returns, to be calculated, and obtains regression equation and calibration object dosage Response curve.The luminous value and response curve of the sample serum determined according to step 2, calculate specific antibody in sample serum Dose value.

The master of the method for 6 kit of the present invention of test example detection avian leukosis virus A/B/J subgroup specific antibodies Want index

(1) sensitiveness

The strong positive serum specimen for by 3 parts being respectively avian leukosis A subgroups, B subgroups and J subgroups makees gradient dilution 1:8、1:16、1: 32、1:64、1:128、1:256、1:512、1:1024, detected with detection method of the present invention, as a result such as table 1 below institute Show.30 parts of positive sample serum is chosen, is tested with virus purification, detection method of the present invention and external import kit (IDEXX) determine, as a result understand simultaneously, the result that three kinds of detection methods detect positive serum sample known to 30 parts compares coincidence rate For 100%.

Table 1：Kit sensitivity Detection

Detection method of the present invention can detect the avian leukosis A subgroups and B subgroups of 256 times of dilution it can be seen from upper table 1 Strong positive serum, the lowest dose level value of the specific antibody of detectable A subgroups is 22ng/mL, detectable B subgroups The lowest dose level value of specific antibody is 26ng/mL；Detection method of the present invention can detect the avian leukosis of 512 times of dilution J subgroups positive serum by force, the lowest dose level value of the specific antibody of detectable J subgroups is 12ng/mL.

(2) it is specific

Buy Dutch 15 parts of poultry diease specific criteria positive serum samples of GD companies：Poultry infectious bursal disease (IBD), fowl are infected Property bronchitis (IBV, Beaudette strain), ewcastle disease (NDV), avian encephalomyelitis (AE), the poor blood factor of fowl (CAV), infectiousness Laryngotracheitis (ILT), synovia mycoplasma (MS), bird flu (AIV, 0126 plant), reovirus (REO), avian infectious branch gas Guan Yan (IBV, Mass strain), aviadenovirus (FAV, Phelp strain), aquatic bird/bird pox virus (FPV), fowl subtract egg virus (EDS-76), Frustrate blood and mycoplasma (MG) and paramyxovirus (APMV).It also have purchased 3 parts of avian leukosis A subgroups, B subgroups and J subgroup standards in addition Positive serum sample

Above-mentioned 15 kinds of poultry diease specific criteria positive serums and ALV-A/B/J subgroups standard positive serum are all carried out 1:8 dilutions, The method for quantitatively detecting antibody with homogeneous chemistry luminescence reagent box of the present invention is detected, specific detection data such as following table Shown in 2.

Table 2：Kit specific detection

There is upper table 2 to understand, 15 parts of poultry diease specific criteria blood serum samples of the detection method detection of kit of the present invention, institute The testing result for having sample is all negative, shows the present invention and other poultry diease serum no cross reactions, has good specificity. Avian leukosis poison A/B/J subgroup standard positive serums are detected, luminous signal is very strong, illustrates there is good reactivity, Neng Gouyong To carry out quantitative detection to avian leukosis poison A/B/J subgroups antibody.

(3) precision

Take avian leukosis A subgroups positive 1 part of serum specimen, B subgroups 1 part of positive serum specimen and J subgroups positive serum specimen 1 by force by force by force Part.By these three serum samples, everyone is repeated 20 times experiment to two laboratory technicians (A and B), calculates the measured result coefficient of variation (CV), As a result show：A laboratory technician measures result CV average percents for 20 times as 6.28%；B laboratory technician measures result CV average hundred for 20 times Point than being respectively less than 10% for 7.44%, CV percentages, illustrate it is reproducible, it is as a result reliable and stable.

(4) stability

Mark avian leukosis virus A/B subgroups His-cENV prepared by present invention AlphaPlex^TM645 acceptor microballoons, mark Remember avian leukosis virus J subgroups His-gp85 AlphaPlex^TM545 acceptor microballoons, mark rabbit-anti chicken secondary antibodyDonor microballoon, and analysis buffer are placed in 2-8 DEG C and preserved 0,3,6,9,12 month, are then established with the present invention The quality-control product (PC, IBD, IBV, NDV, REO, AIV, SPF) that is preserved to this laboratory of homogeneous chemistry luminescence method detected, see The Sensitivity and Specificity variation tendency of the homogeneous chemistry luminescence method is examined, assesses the stability of this method, as a result such as table 3 below institute Show.

Table 3：Stabilization of kit detects

There is upper table 3 to understand, the main agents used in kit of the present invention preserve 12 months at 2-8 DEG C, its sensitiveness, spy Different in nature result does not almost change, illustrates that the kit had very strong stability in 12 months.

SEQUENCE LISTING

<110>Heilongjiang Province's animal epidemic prevention and control centre, Luoyang Co., Ltd of modern biotechnology research institute

<120>Detect the kit and its detection method of avian leukosis virus A/B/J subgroup specific antibodies

<130> 1

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 1148

<212> DNA

<213>Avian leukosis virus

<400> 1

ccatgggtac acccttgctg ccaacgagag ttaattatat tctcattatt ggtgtcctgg 60

tcttgtgtga ggttacgggg gtgagagctg atgttcactt actcgagcag ccaggtggca 120

gcacggattt ctgcctctct acacagggcg gcagcacctc cccttttcag acatgtttga 180

taggtatccc gtcccctatt ggcggtagca aggggtacgt ctctggtaat tgcaccgcct 240

tgggtggcag ctatcggaag gtttcatgct tgttgttaaa gctgaatgtt tccctgttgg 300

acgagccagg tggcagcgaa ctacaactgt taggttccca gtctctccct ggaggcagct 360

acggtagtcc ggcgggcgtt tacggcggca gccaagttac acacatcctc ttgactgacg 420

gtggcagccc gtttacagta gtgacagcag gtggcagcgg aagtgaatat tgcggtgcat 480

atggctacgg aggcagcgtc agtggatgtt gcggggaatc aatcacgatt ctcccaccag 540

gggcgtgggt cgacggtggt agcaaaccaa aggcgctacc acccggaatt ttcctcattt 600

gtggggatgg cggaagcccc agtcgtccgg tagggggccc ctgctattta ggaaaactta 660

ccatgttagg cggcagcata ctcgccaatt cgggcggtag ccataaacga agcgtcacac 720

acctggatga tacaggcggc agctcagatg aagtaacgct ttggggtgga ggcagcgcaa 780

gaatctttgc atctatctta gctccggggg tagcagctgc gcaagcctta agaggtggca 840

gcagactagc ctgttggtcc gttaaacagg ctggcggtag caccacatca ctcctcgggg 900

acttattgga tgatgtcacg agtattcgac acgcggtcct gcagaaccga ggtggcagca 960

ttgacttctt gcttctggct cacggccatg gctgtgagga cattgctgga atgtgttgtt 1020

tcaatctcag tggcggcagc gaatgggccg ttcatttgct aaaaggacta cttttggggc 1080

tggtagttat cttgctgcta gtagtatgcc tgccttgctt tttgcaattc gtatctagta 1140

gcctcgag 1148

<210> 2

<211> 382

<212> PRT

<213>E. coli bl21

<400> 2

Met Gly Thr Pro Leu Leu Pro Thr Arg Val Asn Tyr Ile Leu Ile Ile

1 5 10 15

Gly Val Leu Val Leu Cys Glu Val Thr Gly Val Arg Ala Asp Val His

20 25 30

Leu Leu Glu Gln Pro Gly Gly Ser Thr Asp Phe Cys Leu Ser Thr Gln

35 40 45

Gly Gly Ser Thr Ser Pro Phe Gln Thr Cys Leu Ile Gly Ile Pro Ser

50 55 60

Pro Ile Gly Gly Ser Lys Gly Tyr Val Ser Gly Asn Cys Thr Ala Leu

65 70 75 80

Gly Gly Ser Tyr Arg Lys Val Ser Cys Leu Leu Leu Lys Leu Asn Val

85 90 95

Ser Leu Leu Asp Glu Pro Gly Gly Ser Glu Leu Gln Leu Leu Gly Ser

100 105 110

Gln Ser Leu Pro Gly Gly Ser Tyr Gly Ser Pro Ala Gly Val Tyr Gly

115 120 125

Gly Ser Gln Val Thr His Ile Leu Leu Thr Asp Gly Gly Ser Pro Phe

130 135 140

Thr Val Val Thr Ala Gly Gly Ser Gly Ser Glu Tyr Cys Gly Ala Tyr

145 150 155 160

Gly Tyr Gly Gly Ser Val Ser Gly Cys Cys Gly Glu Ser Ile Thr Ile

165 170 175

Leu Pro Pro Gly Ala Trp Val Asp Gly Gly Ser Lys Pro Lys Ala Leu

180 185 190

Pro Pro Gly Ile Phe Leu Ile Cys Gly Asp Gly Gly Ser Pro Ser Arg

195 200 205

Pro Val Gly Gly Pro Cys Tyr Leu Gly Lys Leu Thr Met Leu Gly Gly

210 215 220

Ser Ile Leu Ala Asn Ser Gly Gly Ser His Lys Arg Ser Val Thr His

225 230 235 240

Leu Asp Asp Thr Gly Gly Ser Ser Asp Glu Val Gln Leu Trp Gly Gly

245 250 255

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

260 265 270

Ala Gln Ala Leu Arg Gly Gly Ser Arg Leu Ala Cys Trp Ser Val Lys

275 280 285

Gln Ala Gly Gly Ser Thr Thr Ser Leu Leu Gly Asp Leu Leu Asp Asp

290 295 300

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

305 310 315 320

Asp Phe Leu Leu Leu Ala His Gly His Gly Cys Glu Asp Ile Ala Gly

325 330 335

Met Cys Cys Phe Asn Leu Ser Gly Gly Ser Glu Trp Ala Val His Leu

340 345 350

Leu Lys Gly Leu Leu Leu Gly Leu Val Val Ile Leu Leu Leu Val Val

355 360 365

Cys Leu Pro Cys Phe Leu Gln Phe Val Ser Ser Ser Leu Glu

370 375 380

<210> 3

<211> 1087

<212> DNA

<213>Avian leukosis virus

<400> 3

ccatgggtac acccttgctg ccaacgagag ttaattatat tctcattatt ggtgtcctgg 60

tcttgtgtga ggttacgggg gtgagagctg atgttcactt actcgagcag ccaggtggca 120

gcacggattt ctgcctctct acacagggcg gcagcacctc cccttttcag acatgtttga 180

taggtatccc gtcccctatt ggcggtagca aggggtacgt ctctggtaat tgcaccttgg 240

gtggcagcta tcggaagatt tcatgcttgt tgttaaagct gaatgtttcc ctgttggacg 300

agccaggtgg cagcgaacta caactgttag gttcccagtc tctccctgga ggcagctacg 360

gcagtccggc gggcgtttac ggcggcagcc aagttacaca catcctcttg actgacggtg 420

gcagcccgtt tacagtagtg acagcaggtg gcagcggaag tgaatattgc ggtgcatatg 480

gctacggagg cagcgtcagt ggatgttgcg gggaatcaat cacgattctc ccaccagggg 540

cgtgggtcga cggtggtagc aaaccaaagg cgctaccacc cggaattttc ctcatttgtg 600

gggatggcgg aagccccagt cgtccggtag ggggcccctg ctatttagga aaacttacca 660

tgttaggcgg cagcatactc gccaattcgg gcggtagcca taaacgaagc gtcacacacc 720

tggatgatac aggcggcagc tcagatgaag tacagctttg gggtggaggc agcgcaagaa 780

tctttgcatc tatcttagct ccgggggtag cagctggcgg tagcaccaca tcactcctcg 840

gggacttatt ggatgatgtc acgagtattc gacacgcggt cctgcagaac cgaggtggca 900

gcattgactt cttgcttctg gctcacggcc atggctgtga ggacattgct ggaatgtgtt 960

gtttcaatct cagtggcggc agcgaatggg ccgttcattt gctaaaagga ctacttttgg 1020

ggctggtagt tatcttgctg ctagtatgcc tgcttgcttt ttgcaattcg tatctagtag 1080

cctcgag 1087

<210> 4

<211> 383

<212> PRT

<213>E. coli bl21

<400> 4

Met Gly Thr Pro Leu Leu Pro Thr Arg Val Asn Tyr Ile Leu Ile Ile

1 5 10 15

Gly Val Leu Val Leu Cys Glu Val Thr Gly Val Arg Ala Asp Val Ile

20 25 30

Leu Leu Glu Gln Pro Gly Gly Ser Thr Asp Phe Cys Leu Ser Thr Gln

35 40 45

Gly Gly Ser Thr Ser Pro Phe Gln Thr Cys Leu Ile Gly Ile Pro Ser

50 55 60

Pro Ile Gly Gly Ser Lys Gly Tyr Val Ser Gly Asn Cys Thr Ala Leu

65 70 75 80

Gly Gly Ser Tyr Arg Lys Val Ser Cys Leu Leu Leu Lys Leu Asn Val

85 90 95

Ser Leu Leu Asp Glu Pro Gly Gly Ser Glu Leu Gln Leu Leu Gly Ser

100 105 110

Gln Ser Leu Pro Gly Gly Ser Tyr Gly Ser Pro Ala Gly Val Tyr Gly

115 120 125

Gly Ser Gln Val Thr His Ile Leu Leu Thr Asp Gly Gly Ser Pro Phe

130 135 140

Thr Val Val Thr Ala Gly Gly Ser Gly Ser Glu Tyr Cys Gly Ala Tyr

145 150 155 160

Gly Tyr Gly Gly Ser Val Ser Gly Cys Cys Gly Glu Ser Ile Thr Ile

165 170 175

Leu Pro Pro Gly Ala Trp Val Asp Gly Gly Ser Lys Pro Lys Ala Leu

180 185 190

Pro Pro Gly Ile Phe Leu Ile Cys Gly Asp Gly Gly Ser Pro Ser Arg

195 200 205

Pro Val Gly Gly Pro Cys Tyr Leu Gly Lys Leu Thr Met Leu Gly Gly

210 215 220

Ser Ile Leu Ala Asn Ser Gly Gly Ser His Lys Arg Ser Val Thr His

225 230 235 240

Leu Asp Asp Thr Gly Gly Ser Ser Asp Glu Val Gln Leu Trp Gly Gly

245 250 255

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

260 265 270

Ala Leu Gln Ala Leu Arg Gly Gly Ser Arg Leu Ala Cys Trp Ser Val

275 280 285

Lys Gln Ala Gly Gly Ser Thr Thr Ser Leu Leu Gly Asp Leu Leu Asp

290 295 300

Asp Val Thr Ser Ile Arg His Ala Val Leu Gln Asn Arg Gly Gly Ser

305 310 315 320

Ile Asp Phe Leu Leu Leu Ala His Gly His Gly Cys Glu Asp Ile Ala

325 330 335

Gly Met Cys Cys Phe Asn Leu Ser Gly Gly Ser Glu Trp Ala Val His

340 345 350

Leu Leu Lys Gly Leu Leu Leu Gly Leu Val Val Ile Leu Leu Leu Val

355 360 365

Val Cys Leu Pro Cys Phe Leu Gln Phe Val Ser Ser Ser Leu Glu

370 375 380

Claims (8)

1. A kind of 1. homogeneous chemistry luminescence reagent box for detecting avian leukosis virus A/B/J subgroup specific antibodies, it is characterised in that： Including microwell plate, sealed membrane, acceptor microballoon I, acceptor microsphere Ⅱ, donor microballoon, calibration object and the analysis buffer of lighting；

   Avian leukosis virus A/B subgroup His-cENV albumen is marked on the acceptor microballoon I, the white blood of fowl is marked on acceptor microsphere Ⅱ The viral J subgroups His-gp85 albumen of disease；The donor microballoon marks rabbit-anti chicken secondary antibody；

   The labeling process of the acceptor microballoon I, acceptor microsphere Ⅱ or donor microballoon is realized by the following method：Will be to be marked Thing is placed in centrifuge tube, is added phosphate buffer, centrifugation, washing and suspension, is added MES buffer solutions after blotting supernatant, then Sequentially add purified label, sodium cyanoborohydride solution and Tween-20,2-8 DEG C of 40~48h of lucifuge revolving reaction Afterwards, 37 DEG C of stirring 1h terminating reactions of CMO lucifuges are added；Supernatant is removed in centrifugation, adds HEPES buffer solution to be resuspended, and supernatant is removed in centrifugation, adds Liquid is preserved, acceptor microballoon I, acceptor microsphere Ⅱ or the donor microballoon of coupling label is made；

   Calibration object is formulated by the sterling antibody extracted in the positive standard serum that is mixed into from more parts of high level serum；

   The analysis buffer is 50mmol/L, pH7.4 Tris-HCl buffer solutions, wherein being 0.8% containing mass fraction Bovine serum albumin(BSA) that NaCl, mass fraction are 1%, the casein that mass fraction is 0.1%, 0.1% surfactant S9, body The Qula that fraction is 0.1% leads to the Proclin-300 that -100, volume fraction is 0.05%.
2. 2. kit as claimed in claim 1, it is characterised in that：The donor microballoon is mark rabbit-anti chicken secondary antibody AlphaScreen donor microballoons.
3. 3. kit as claimed in claim 1, it is characterised in that：The acceptor microballoon I is mark His-cENV albumen The microballoons of AlphaPlex 645；Acceptor microsphere Ⅱ is the mark His-gp85 microballoons of AlphaPlex 545.
4. 4. kit as claimed in claim 1, it is characterised in that：The acceptor microballoon I, acceptor microsphere Ⅱ or donor microballoon Labeling process is realized by the following method：20mg/mL thing to be marked is placed in centrifuge tube, waits to mark to every 0.2mL Remember the phosphate buffer 1 mL that 0.1mol/L, pH 8.0 is added in thing, centrifugation, wash and suspend 3~5 times repeatedly, blot supernatant 0.2mL MES buffer solutions are added, the purified labels of 1~3mg/mL of 0.2mg~0.6mg concentration is then added, adds 8 The Tween-20,2- that the sodium cyanoborohydride solution and 12.5uL volume fractions that~12uL concentration is 400~500mmol/L are 1% After 8 DEG C of 40~48h of lucifuge revolving reaction, 37 DEG C of stirring 1h terminating reactions of 10uL CMO lucifuges are added；Supernatant is removed in centrifugation, adds 50mmol/L pH7.4 HEPES buffer solution 0.5mL is resuspended, and supernatant is removed in centrifugation, adds and preserves liquid, and coupling label is made Acceptor microballoon I, acceptor microsphere Ⅱ or donor microballoon.
5. 5. the kit as described in claim 1 or 4, it is characterised in that：The liquid that preserves is 50mmol/L, pH7.4 Tris-HCl buffer solutions, wherein the trehalose of Tris, 10g/L comprising 6.05g/L, 10g/L glucose, 30g/L sweet dew Alcohol, 20g/L bovine serum albumin(BSA), 5g/L proline and volume fraction are 0.1% Proclin-300.
6. 6. kit as claimed in claim 1, it is characterised in that：The calibration object is made by following methods：By 50 parts of warps The high level serum of detection is hybridly prepared into positive standard serum, 10000rpm/min centrifugation 30min, then through affinitive layer purification, Sterling antibody is collected, sterling antibody ampere bottle is dispensed and freezed to form freeze-dried powder, is prepared freeze-dried powder with analysis buffer Into 6 calibration object concentration：0ng/mL, 10ng/mL, 40ng/mL, 160ng/mL, 640ng/mL and 2560ng/mL.
7. 7. utilize the kit detection avian leukosis A/B/J subgroup specific antibodies such as claim 1-4 or 6 as described in any one Method, it is characterised in that：Comprise the following steps：

   Step 1: the collection and preservation of clinical samples：Take without anti-coagulants vacuum blood collection tube collection chicken venous whole sample, 37 DEG C 2h is placed, then placed overnight in 2~8 DEG C, the serum of precipitation is suctioned out, 3000rpm/min centrifugation 5min, take supernatant to dispense In EP pipes and blood sampling date and identifier are indicated, sample serum is made；If the sample serum same day uses, 2~8 are placed directly in In DEG C refrigerator；If the next day use, -20 DEG C of freezen protectives；

   Step 2: the acceptor microballoon I prepared, acceptor microsphere Ⅱ and donor microballoon are respectively added into 100 μ L in luminous microwell plate, Then the μ L of sample serum 20 are added, are well mixed, 37 DEG C of incubation 20min, then excite lower detection not in the light that wavelength is 680nm With the signal light value of subgroup specific antibody.
8. 8. method as claimed in claim 7, it is characterised in that：The concentration of acceptor microballoon I is 0.015mg/mL；Acceptor microsphere Ⅱ Concentration be 0.020mg/mL；The concentration of donor microballoon is 0.030mg/mL.