CN107543923B - Kit for detecting avian leukosis virus A/B/J subgroup specific antibody and detection method thereof - Google Patents
Kit for detecting avian leukosis virus A/B/J subgroup specific antibody and detection method thereof Download PDFInfo
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Abstract
The invention relates to a kit for detecting avian leukosis virus subgroup A/B/J specific antibody and a detection method thereof, belonging to the field of molecular biology, wherein the kit comprises a luminous microporous plate, a sealing film, an acceptor microsphere I, an acceptor microsphere II, a donor microsphere, a calibrator and an analysis buffer solution; the receptor microsphere I is marked with avian leukosis virus subgroup A/B His-cenV protein, and the receptor microsphere II is marked with avian leukosis virus subgroup J His-gp85 protein; the donor microsphere is marked with rabbit anti-chicken secondary antibody. The detection is that firstly, the collected sample serum is pretreated, then the acceptor microsphere I, the acceptor microsphere II, the donor microsphere and the sample serum are added to a luminous micropore plate according to requirements, and an optical signal value is detected under 680nm exciting light. The method realizes detection of avian leukosis virus A/B/J specific antibody, and has the advantages of no need of washing, simple operation, sensitive detection, and strong specificity and stability.
Description
Technical Field
The invention belongs to the field of molecular biology, relates to animal disease detection, and particularly relates to a kit for detecting avian leukosis virus A/B/J subgroup specific antibodies and a detection method thereof.
Background
Avian leukemia refers to various transmissible neoplastic diseases caused by retroviruses A, avian C, and the group of retroviruses, mainly the excessive proliferation of certain cell components in the hematopoietic tissues of birds. Avian lymphocytic leukemia, erythroblastic leukemia and myeloblastic leukemia are common, and are widely spread in chicken flocks of various countries in the world through two modes of vertical transmission and horizontal transmission, so that the diseases of chicken death, emaciation, anemia and poor growth and development are caused, and the resistance of an organism is reduced and immune suppression is caused, so that the avian lymphocytic leukemia, the erythroblastic leukemia and the myeloblastic leukemia are one of main diseases which endanger the poultry industry. The Avian Leukosis Virus (ALV) group infected with chicken flock is mainly divided into A, B, C, D, E, J subgroups, wherein the gene sequence of the strain of A, B subgroup has high homology and similar virus characteristics. A. B, C, D and subgroup J are exogenous, and subgroup E is predominantly in the form of a provirus and endogenous. A. Subgroup B is more common in chicken flocks, while subgroup C, D is less common, subgroup J is a new subgroup of avian leukosis viruses.
The A/B/J subgroup avian leukosis in China is quite common, and clinical cases develop from the initial commercial broiler group to commercial laying hens, local breeding hens and other poultry, and are accompanied with tumorigenicity enhancement and tumor diversity. Research shows that the A/B/J subgroup avian leukosis virus in China has the characteristics of high infection rate, high morbidity, common early infection, obviously advanced disease day and age, multiple/frequent mixed infection, expanded host range, expanded tumor spectrum, complicated pathological changes and the like, and causes great loss to the poultry industry in China. In this regard, foreign countries have adopted strict measures to shut down vertical transmission and separately breed uninfected offspring, and the large breeder companies in internationally developed countries have announced the decontamination of exogenous chicken leukemia viruses since 1987. Because the purification period is long, the cost is high, the method cannot be effectively implemented at present in China, and only the infected chickens can be diagnosed at an early stage and eliminated in time to purify the population, so that a rapid and accurate diagnosis method is particularly important in the prevention and treatment of the disease. The definitive diagnosis of the avian leukemia generally depends on pathogenic detection except for primary diagnosis according to clinical symptoms, pathological changes and the like, and common detection methods include virus separation identification, indirect immunofluorescence assay, ELISA and the like, wherein the virus separation identification is not suitable for detecting a large number of samples; the indirect immunofluorescence test is characterized by needing a fluorescence microscope and having certain limitation on operation; although ELISA is suitable for detection of large-scale samples, the operation process is complicated and long-time, false positive or virus recessive can not be detected occasionally, and virus subtype can not be determined.
The AlophaScreen (Amplified luminescence reagent homogeneity analysis Screen) technology is a Homogeneous detection method applied to an analyte based on the effect of Proximity between particles, and AlophaLISA is a Homogeneous detection method which is similar to the classical ELISA technology on the basis of the AlophaScreen technology and is called Homogeneous chemiluminescence technology. The core of the AlophaLISA method is two particles with a diameter of about 200nm, namely a donor (photosensitive particle) and an acceptor (luminescent particle). The surface of two kinds of particles is coupled with biological molecules (antibodies or antigen proteins), and each particle surface can be covered with hundreds of biological molecules to capture molecules to be detected. When the particle is fineAfter being irradiated by laser of 680nm, the photosensitizer on the surface of the donor can convert oxygen in the solution (AlophaLISA reaction solution) into singlet oxygen. The singlet oxygen has a propagation distance of about 200nm in the solution, and if the distance between the donor and the acceptor is less than 200nm, the singlet oxygen can diffuse into the acceptor particles, and the photosensitive compound in the acceptor can chemically react with the singlet oxygen, wherein Eu3+The complex produces high-level luminescence. In contrast, if the distance between the light-emitting particles and the photosensitive particles is greater than 200nm, the monomer oxygen cannot diffuse into the light-emitting particles, and no optical signal is generated. The AlophaLISA homogeneous reaction is based on the principle that if biomolecules coated on the surfaces of two particles interact, the distance between the two particles is shortened, and an optical signal is generated. And (3) judging whether the target object to be detected exists in the actual detection sample or not by detecting the intensity of the optical signal. Although the AlophaLISA technology is applied to lung cancer, hepatitis or hand-foot-and-mouth disease and other aspects based on the principle, the detection of the avian leukosis virus specific antibody is not related, and how to improve the detection efficiency and the detection quality on the basis of the fact that the principle does not have practical operability and the difference between detection objects and detection systems cannot be used for carrying, so that a set of effective standard system is formed, and the AlophaLISA technology can be widely applied in practice and still is a very important subject.
Disclosure of Invention
Aiming at the problems, the invention provides a kit for detecting avian leukosis virus A/B/J subgroup specific antibodies, and the kit can realize simultaneous detection of the avian leukosis virus A/B/J specific antibodies, does not need washing, and has simple operation and sensitive detection.
A homogeneous phase chemiluminescence kit for detecting avian leukosis virus A/B/J subgroup specific antibodies comprises a luminescence microporous plate, a sealing film, an acceptor microsphere I, an acceptor microsphere II, a donor microsphere, a calibrator and an analysis buffer solution;
the receptor microsphere I is marked with avian leukosis virus subgroup A/B His-cenV protein, and the receptor microsphere II is marked with avian leukosis virus subgroup J His-gp85 protein; the donor microsphere marks a rabbit anti-chicken secondary antibody;
the marking process of the acceptor microsphere I, the acceptor microsphere II or the donor microsphere is realized by the following method: placing a to-be-marked object in a centrifuge tube, adding a phosphate buffer solution, centrifuging, washing and suspending, sucking up a supernatant, adding an MES buffer solution, then sequentially adding the purified marked object, a sodium cyanoborohydride solution and Tween-20, carrying out a light-shielding rotary reaction at 2-8 ℃ for 40-48 h, adding CMO, and stirring at 37 ℃ for 1h to terminate the reaction; centrifuging to remove supernatant, adding HEPES buffer solution for heavy suspension, centrifuging to remove supernatant, and adding preservation solution to obtain receptor microsphere I, receptor microsphere II or donor microsphere of the coupled marker;
the calibrator is prepared by pure antibodies extracted from positive standard serum mixed by multiple high-value sera;
the analysis buffer solution is 50mmol/L Tris-HCl buffer solution with pH7.4, wherein the analysis buffer solution comprises 0.8% of NaCl by mass fraction, 1% of bovine serum albumin by mass fraction, 0.1% of casein by mass fraction, 0.1% of surfactant S9 by mass fraction, 0.1% of triton-100 by volume fraction and 0.05% of Proclin-300 by volume fraction.
Further, the donor microspheres are AlphaScreen @ donor microspheres which mark rabbit anti-chicken secondary antibodies.
Further, the receptor microsphere I is an AlphaPlex-645 microsphere marked with His-cENV protein; the receptor microsphere II is an AlphaPlex 545 microsphere marked with His-gp 85.
Further, the marking process of the acceptor microsphere I, the acceptor microsphere II or the donor microsphere is realized by the following method: placing 20mg/mL of a substance to be marked in a centrifuge tube, adding 1mL of 0.1mol/L, pH 8.0.0 phosphate buffer solution into every 0.2mL of the substance to be marked, repeatedly centrifuging, washing and suspending for 3-5 times, sucking out supernatant, adding 0.2mL of MES buffer solution, then adding 0.2-0.6 mg of purified marker with the concentration of 1-3 mg/mL, adding 8-12 uL of 400-500 mmol/L sodium cyanoborohydride solution and 12.5uL of 1% Tween-20 in volume fraction, carrying out lucifugal rotation reaction at the temperature of 2-8 ℃ for 40-48 h, adding 10uL of CMO, and stirring for 1h at the temperature of 37 ℃ to terminate the reaction; centrifuging to remove supernatant, adding 0.5mL HEPES buffer solution with the concentration of 50mmol/L and the pH value of 7.4 for resuspension, centrifuging to remove supernatant, and adding preservation solution to prepare receptor microspheres I, receptor microspheres II or donor microspheres of the coupled marker;
further, the preservation solution is 50mmol/L Tris-HCl buffer solution with pH7.4, which comprises 6.05g/L Tris, 10g/L trehalose, 10g/L glucose, 30g/L mannitol, 20g/L bovine serum albumin, 5g/L proline and 0.1% Proclin-300 by volume fraction.
Further, the calibrator is prepared by the following method: mixing 50 parts of detected high-value serum to prepare positive standard serum, centrifuging at 10000rpm/min for 30min, purifying by affinity chromatography, collecting pure antibody, subpackaging the pure antibody with an ampoule and freeze-drying to form freeze-dried powder, and preparing the freeze-dried powder into 6 calibrator concentrations by using an analysis buffer solution: 0ng/mL, 10ng/mL, 40ng/mL, 160ng/mL, 640ng/mL, and 2560 ng/mL.
Further, the method for detecting the specific antibody of the avian leukosis subgroup A/B/J by using the kit comprises the following steps:
step one, collecting and storing clinical specimens: collecting a chicken vein whole blood sample by using a vacuum blood collection tube without an anticoagulant, standing for 2 hours at 37 ℃, standing overnight at 2-8 ℃, sucking out precipitated serum, centrifuging for 5 minutes at 3000rpm/min, taking supernatant, and subpackaging in an EP tube, and marking a blood collection date and an identification number to obtain sample serum; if the sample serum is used on the same day, directly placing the sample serum in a refrigerator at the temperature of 2-8 ℃; if the product is used every other day, freezing and storing at-20 deg.C;
and step two, adding 100 mu L of prepared receptor microspheres I, receptor microspheres II and donor microspheres into a luminescent microplate, then adding 20 mu L of sample serum, uniformly mixing, incubating for 20min at 37 ℃, and then detecting the signal light values of different subgroup specific antibodies under the excitation of light with the wavelength of 680 nm.
Further, the concentration of the receptor microsphere I is 0.015 mg/mL; the concentration of the receptor microsphere II is 0.020 mg/mL; the concentration of donor microspheres was 0.030 mg/mL.
Has the advantages that:
according to the invention, a homogeneous chemiluminescence technology is utilized, capsular membrane protein is coupled on a receptor microsphere, rabbit-anti-chicken secondary antibody is marked on a donor microsphere, antigen or antibody protein is coupled to the microsphere by adopting an original system, and in the coupling marking system of protein and microsphere, the pH value, solution concentration and dosage, reaction time, temperature and the like of a buffer solution can all influence the coupling marking process; the coupling time and the reaction condition are controlled, the self coupling of the protein is avoided and the coupling amount is improved at the same time of improving the reaction rate. After the reaction is finished, the reaction solution is washed again, the non-covalent adsorption is reduced, the coupling efficiency is improved to the maximum extent, the uniformity, the sensitivity and the specificity of the coupling compound are increased, the high sensitivity and the high efficiency of the microsphere on the specific antibody detection are further determined, and in addition, the trehalose, the glucose, the mannitol, the bovine serum albumin, the proline and the Proclin-300 added into the preservation solution are coordinated, so that the stability and the specificity of the coupling substance are improved, the target substance can be accurately and rapidly detected, and the effect of the kit is fully exerted. The kit can also realize the simultaneous detection of the avian leukosis virus A/B/J specific antibodies, can distinguish the antibodies of the three subgroups according to the detected signal light, is convenient and quick, has high efficiency, does not need washing, and is simple to operate and sensitive in detection. Meanwhile, a new way for detecting the specific antibody and antibody of the avian leukosis virus is developed, and a set of effective detection system is formed.
The kit is used for detection, and can detect avian leukosis A/B subgroup positive serum diluted by 256 times and J subgroup positive serum diluted by 512 times, and has higher sensitivity; the detection of 15 parts of poultry positive serum shows that the detection results of all samples are negative, which shows that the invention has no cross reaction with other poultry positive serum and has good specificity, and the detection of the poultry leukemia virus A/B/J subgroup positive serum shows that the invention has strong luminescent signal and good reactivity. In addition, the kit is used for detection, has good repeatability and strong stability, and can be stored for 12 months at the temperature of 2-8 ℃. The kit can be used for quantitatively detecting the avian leukosis virus A/B/J subgroup antibody.
Drawings
FIG. 1 is an electrophoresis chart showing the results of double restriction enzyme identification of pUC19-cENV gene recombinant plasmid; wherein, the left side is a gene fragment obtained by double enzyme digestion of pUC19-gp85 by using restriction enzymes EcoRI and XhoI, and the right side is DL10000 Marker;
FIG. 2 is an electrophoresis chart showing the result of double restriction enzyme identification of pUC19-gp85 gene recombinant plasmid; wherein, 1 is a pUC19 empty plasmid subjected to double digestion by using restriction enzymes EcoRI and XhoI, 2 is a gene fragment obtained by pUC19-gp85 subjected to double digestion by using restriction enzymes EcoRI and XhoI, and M is 1kb DNA Ladder;
FIG. 3 is an SDS-PAGE electrophoresis of the recombinant expression protein cENV before and after purification; wherein, M in the figure is a protein Marker, 1 is induced bacteria liquid sediment, 2 is supernatant, 3 is large-amount expression protein sediment, and 4 is a result obtained after the large-amount expression protein is purified;
FIG. 4 is an SDS-PAGE electrophoresis of the recombinant expressed protein gp85 before and after purification; wherein, M in the figure is a protein Marker, 1 is induced bacteria liquid sediment, 2 is supernatant, 3 is large-amount expression protein sediment, and 4 is a result obtained after the large-amount expression protein is purified;
FIG. 5 is a graph showing the results of detection of a cENV recombinant protein by Western blot;
FIG. 6 is a graph showing the results of detection of gp85 recombinant protein by Western blot.
Detailed Description
The following examples or experimental examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention, and modifications or substitutions of the methods, procedures or conditions of the present invention may be made without departing from the spirit and spirit of the present invention.
Unless otherwise indicated, the chemical reagents used in the examples or experimental examples are conventional commercially available reagents and the techniques used are conventional techniques well known to those skilled in the art.
The main reagents, instruments or consumables used in the invention and the sources thereof are as follows:
the main reagents are as follows:
2-Morpholinoethanesulfonic acid (MES), sodium cyanoborohydride (NaBH 3 CN), carboxymethylhydroxylamine hemisalt (CMO), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and Prolcin-300 were purchased from Sigma-Aldrich;
tween-20 was purchased from Pierce;
AlphaScreen ™, Alphaplex 645 and Alphaplex 545 are commercially available from Perkin Elmer, USA;
protein prestainer, Taq polymerase, restriction enzymes Nco I and Xho I were purchased from Thermo corporation;
T4DNA ligase, dNTP, DNA Marker, IPTG, pUC19 vectors were purchased from Baozi medical technology (Beijing) Co.Ltd;
the plasmid extraction kit is purchased from OMEGA;
the His tag antibody is a product of Beijing Quanjin Biotechnology Limited. Main consumables and instrument:
light activated chemiluminescence apparatus is available from Perkin Elmer, USA;
osterode D-37520, an ultra-high speed freezing self-balancing centrifuge, was purchased from Thermo fisher, Germany;
the UV spectrophotometer TU-1900 was purchased from Beijing Pujingyo general instruments, Inc.
Test example 1 expression and preparation of avian leukosis Virus subgroup A/B/J envelope protein
According to the published ALV-A/B/J subgroup cyst membrane protein gene sequence in NCBI GenBank, combining with the research progress of ALV-A/B/J subgroup at home and abroad, screening the cyst membrane protein antigen epitope gene sequence representing the epidemic strain, obtaining the recombinant A/B subgroup cyst membrane protein cENV gene sequence 1244bp, the nucleotide sequence SEQ ID NO: 1 is shown in the specification; the gene sequence of recombinant subgroup J envelope protein gp85 is 1024bp, and the nucleotide sequence is SEQ ID NO: 3, respectively. For subsequent experiments, the envelope protein cENV gene sequence of subgroup A/B and the envelope protein gp85 gene sequence of subgroup J are amplified by PCR，The ends all contain NcoI cleavage sites, 3，Both ends contain XhoI cleavage sites. The resulting sequence of the cENV gene and the sequence of the gp85 gene were ligated to a pUC19 vector, respectively. Sequencing and verifying correctnessThen, pET32a (+) and the pUC-19 cloning vector to which the target gene had been ligated were subjected to double digestion with restriction enzymes Nco I and Xho I, respectively, in a water bath at 37 ℃ for 60min, followed by electrophoresis on 1% agarose gel, followed by double digestion with pET32a (+) to recover a large fragment, and double digestion with the pUC-19 cloning vector to recover the target gene fragment. The results of the double digestion are shown in FIGS. 1 and 2. Ligating the recovered large fragment and the target gene fragment at 22 ℃ overnight, wherein the ligation system to the target gene fragment of cENV is: pET32a (+) vector 1uL, cENV Gene fragment 1.5uL, ddH2O 20uL，T4DNA ligase 1.5uL, 10 × Green Buffer 3uL, total volume totally 27uL, obtain ligation product pET32a-cENV recombinant plasmid; the linker system with the gp85 target gene fragment is: pET32a (+) vector 1uL, gp85 Gene fragment 1.5uL, ddH2O 20uL，T4DNA ligase 1.5uL, 10 XGreen Buffer 3uL, total volume totally 27uL, obtain the ligation product pET32a-gp85 recombinant plasmid.
The ligation products pET32a-cENV recombinant plasmid and pET32a-gp85 recombinant plasmid are respectively transferred into 0.1 percent CaCl according to a conventional method2The prepared competent bacteria BL21 are coated in LB solid culture medium containing ampicillin, cultured overnight at 37 ℃, single colony is picked the next day, shaken, plasmid is extracted for double enzyme digestion identification and then sent for sequencing, and the competent bacteria BL21 are confirmed to have the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, and the obtained recombinant cENV gene and gp85 gene have been successfully cloned into pET32a-cENV recombinant plasmid and pET32a-gp85 recombinant plasmid, respectively.
Inoculating positive escherichia coli BL21 bacteria containing recombinant plasmids into a solid culture medium containing ampicillin (Amp) resistance, performing overnight culture at 37 ℃, inoculating 3-4 single colonies into an LB liquid culture medium containing Amp, and performing shake culture at 37 ℃ (250 rpm/min) to OD600=0.8, adding IPTG (isopropyl- β -D-thiogalactopyranoside) to the final concentration of 1mmol/L, continuing culturing for 4 hours at 37 ℃ to obtain fusion proteins His-cenV and His-gp85 with HIS labels, simultaneously culturing non-induced recombinant plasmid transformation BL21 bacteria as a negative control, wherein the amino acid sequence of the His-cenV protein of the A/B subgroup is shown as SEQ ID NO:02, and the amino acid sequence of the His-gp85 protein of the J subgroup is shown asSEQ ID NO. 4. The expressed protein was identified by SDS-PAGE and Western-blot, and the results are shown in FIGS. 3 and 4.
Collecting the cultured engineering bacteria liquid, centrifuging at 6000rpm/min for 10min, discarding the supernatant, re-suspending the precipitate with PBS to obtain a re-suspension, adding GuNTA-0 Buffer and PMSF into the re-suspension, and ultrasonically crushing BL21 bacteria, 120W, working for 2s, intermittent 3s and ultrasonic for 100 cycles at low temperature by using an ultrasonic crusher; then transferring the ultrasonic disruption bacterial liquid into a 50ml centrifuge tube, balancing, centrifuging for 10 minutes at 4 ℃ and 12000r/min, and taking the supernatant and storing at-20 ℃; the lysed cells were subjected to affinity chromatography using Ni-NTA purification resin, and the sample was applied to a column and the permeated fraction was collected at a flow rate of 12 ml/h. The chromatography was performed with GuNTA-60 Buffer in a volume of 5 times the volume of NTA at a flow rate of 12ml/h, and the eluate was collected. And dialyzing the eluent by using 0.5mol/L NaCl and 20mmol/L Tris-HCl solution for 24 hours, and detecting the purity of the target protein by SDA-PAGE and Western-blot on the purified avian leukosis CENV protein and gp85 protein to reach 97 percent. The protein content of the obtained protein is determined by a Bradford method, and the protein is subpackaged in a 2ml centrifuge tube and stored at the temperature of 80 ℃ for later use.
The purification steps of the recombinant protein were as follows:
the precipitate obtained by the above centrifugation was washed with 9 times the volume of the washing solution
Resuspending the precipitate, standing for 5min at 8000rpm at 4 deg.C for 15min, and discarding the supernatant. With 9 volumes of washing solution
Resuspending the precipitate, standing for 5min at 8000rpm at 4 deg.C for 15min, discarding the supernatant, and retaining the precipitate. With 9 volumes of washing solution
Resuspending the precipitate, standing for 5min at 8000rpm at 4 deg.C for 15min, discarding the supernatant, and retaining the precipitate. The volume of the lysate was adjusted to 100mL of the lysate to 4-10mL of the lysate, and the mixture was left overnight at 4 ℃. 8000rpm, 4 ℃, 15 min. And taking the supernatant to obtain the purified protein.
The preparation method of the reagent for inclusion body purification comprises the following steps:
1. bacterial resuspension (250 mL): PBS (NaCl 2g, KCl 0.05g, Na)2HPO40.36g，KH2PO40.06g），1mmol/L EDTA；
2. Solutions of
（500mL）：500mmol/L Tris-HCl（PH=6.0），50mmol/L NaCl，50mmol/LEDTA；
3. Solutions of
(250 mL): solutions of
1mol/L urea;
4. solutions of
(250 mL): solutions of
2mol/L urea;
5. inclusion body lysis solution (250 mL): solutions of
8mol/L urea.
The purified His-cENA protein and His-gp85 protein were used to coat ELISA plates, respectively, and the biological activity of the protein was measured by enzyme-linked immunosorbent assay (ELISA). Chicken serum which is positive by detecting the avian leucosis virus A/B/J antibody by using a His tag antibody and an IDEXX detection kit is used as a positive control, healthy SFP chicken serum is used as a negative control, and an HRP-labeled goat anti-mouse IgG and goat anti-chicken IgG enzyme-labeled secondary antibody are used according to the instruction. The result shows that the ratio of the OD value of the positive control to the OD value of the negative control is larger than 2.1, which indicates that the protein has good biological activity and antigenicity.
Wherein, the ELISA comprises the following specific steps:
1. coating: diluting purified recombinant protein with 0.05MpH9.6 carbonate buffer solution to optimal concentration, coating reaction plate, 100 ul/well, and standing at 4 deg.C overnight;
2. washing: throwing off coating liquid in the ELISA plate, filling each hole with PBST (PBS, 0.05% Tween-20), standing at room temperature for 3min, discarding PBST, washing for 3 times, 3 min/time, and patting to dry;
3. and (3) sealing: adding blocking solution (2% BSA) into each well at a rate of 200 ul/well, standing at 4 ℃ overnight, blocking, and patting dry;
4. sample adding: diluting the serum to be detected according to gradient, adding the diluted serum into a coating plate at 100 ul/hole, and simultaneously establishing a negative and positive serum control. Incubating in an incubator at 37 ℃ for 1h, washing for 3 times, 3 min/time, and patting to dry;
5. diluting HRP-labeled goat anti-mouse IgG and goat anti-chicken IgG enzyme-labeled secondary antibody with PBST at a ratio of 1:2000, incubating at 37 ℃ for 1h, washing for 3 times, 3 min/time, and patting to dry; 6. Color development: adding TMB single-component color developing solution (product of TIANGEN) into the coated plate at a concentration of 100 ul/well, developing at 37 deg.C in dark for 15min, and adding stop solution (2M H)2SO4) Terminating the color development; 7. detecting the OD value of each hole at the wavelength of 450nm by using an enzyme-linked immunosorbent detector, wherein the OD450 value of the negative control is N, and the OD450 value of the positive control is P. If P/N>2.1 the test piece was determined to be positive.
Experimental example 2 preparation of acceptor microspheres and donor microspheres
Taking 0.2mL of AlphaPlex 645 receptor microspheres (20 mg/mL) and placing the AlphaPlex 645 receptor microspheres into a 1.5mL EP centrifuge tube, adding 1mL of 0.1mol/L phosphate buffer solution with the pH value of 8.0, centrifugally washing and suspending for 3 times, centrifuging 16000g/min for 15 minutes each time, sucking the supernatant, adding 0.2mL of MES buffer solution, then adding 0.3-0.6 mg of purified high-activity recombinant protein avian leukosis virus A/B subgroup His-cNAV with the concentration of 1-3 mg/mL, adding 8-12 uL of cyano sodium borohydride solution with the concentration of 400-500 mmol/L and 12.5uL of 1% Tween-20, carrying out rotary reaction at the temperature of 2-8 ℃ in a dark place for 40-48 hours, adding 10uL of CMO, stirring and reacting at the temperature of 37 ℃ in the dark place for 1 hour to terminate the reaction. Centrifuging the marked receptor microspheres for 30min at 4 ℃ at 13000g/min, removing supernatant by using a pipettor, adding 0.5mL of HEPES buffer solution with the concentration of 50mmol/L and the pH value of 7.4 for resuspension, and oscillating the receptor microspheres at the bottom of a suspension tube; the above steps are repeated for 2 times of centrifugation, and 50mmol/L of preservation solution with pH7.4 is added for standby.
Putting 0.2mL of AlphaPlex 545 receptor microspheres (20 mg/mL) into a 1.5mL EP centrifuge tube, adding 1mL of 0.1mol/L, pH 8.0.0 phosphate buffer solution, centrifuging, washing and suspending for 3 times, centrifuging 16000g/min for 15 minutes each time, adding 0.2mL of MES buffer solution into the sucked supernatant, then adding 0.2 mg-0.5 mg of purified high-activity recombinant protein avian leukosis virus J subgroup His-gp85 protein with the concentration of 1-2 mg/mL, adding 8-12 uL of sodium cyanoborohydride solution with the concentration of 400-500 mmol/L and 12.5uL of 1% Tween-20, carrying out rotary reaction at the temperature of 2-8 ℃ in a dark place for 40-48 hours, adding 10uL of CMO in the dark place, and stirring and reacting at the temperature of 37 ℃ for 1 hour to terminate the reaction. Centrifuging the marked receptor microspheres for 30min at 4 ℃ at 13000g/min, removing supernatant by using a pipettor, adding 0.5mL of HEPES buffer solution with the concentration of 50mmol/L and the pH value of 7.4 for resuspension, and oscillating the receptor microspheres at the bottom of a suspension tube; the above steps are repeated for 2 times of centrifugation, and 0.2mL of 50mmol/L preservation solution with pH of 7.4 is added for standby.
Putting 0.2mL of aldehyde alphaScreen donor microspheres (20 mg/mL) into a 1.5mL EP centrifuge tube, adding 1mL of 0.02mol/L phosphate buffer solution with pH7.6, centrifugally washing and suspending for 3 times, centrifuging 16000g/min for 15 minutes each time, adding 0.2mL of MES buffer solution into the sucked supernatant, then adding 0.4-0.6 mg of purified high-activity rabbit chicken-resistant secondary antibody with the concentration of 1-2 mg/mL, adding 8-12 uL of cyano sodium borohydride solution with the concentration of 400-500 mmol/L and 12.5uL of 1% Tween-20, carrying out rotary reaction at the temperature of 2-8 ℃ in a dark place for 40-48 hours, adding 10uL of CMO, stirring and reacting at the temperature of 37 ℃ in a dark place for 1 hour, and stopping the reaction. Centrifuging the marked receptor microspheres for 30min at 4 ℃ at 13000g/min, removing supernatant by a liquid-moving machine, adding 0.5mL of HEPES buffer solution with the concentration of 50mmol/L and the pH value of 7.4 for resuspension, and oscillating the receptor microspheres at the bottom of a suspension tube; the above steps are repeated for 2 times of centrifugation, and 50mmol/L of preservation solution with pH7.4 is added for standby.
Preparation of the HEPES buffer: weighing 5.96g of HEPES and 5g of Bovine Serum Albumin (BSA), adding 800mL of water to dissolve, adding 5mL of Tween-20, and adding water to a constant volume of 1L.
The preservation solution is 50m mol/L Tris-HCl buffer solution with pH7.4: weighing 6.05g of Tris, adding 800mL of water for dissolution, and adjusting the pH value to 7.4; adding 10g of trehalose, 10g of glucose, 30g of mannitol, 20g of BSA, 5g of proline and Proclin-3001mL, dissolving, and adding water to a constant volume of 1L.
Preparation of the 0.1mol/L, pH 8.0.0 phosphate buffer: weighing Na2HPO4·12H2O33.9 g and NaH2PO40.731g, adding 800mL of water to dissolve, adding 0.5mL of Tween-20, adding water to a constant volume of 1L.
The preparation of the 0.02mol/L phosphate buffer solution with the pH value of 7.6 comprises the following steps: weighing Na2HPO4·12H2O 5.8g、NaH2PO40.496g and NaCl 8g, adding 800mL of water to dissolve, adding 0.5mL of Tween-20, adding water to fix the volume to 1L.
Preparing the MES buffer: weighing 21.3g of MES, adding 800mL of distilled water for dissolving, adjusting the pH value to 6.0 by using 10mol/L of NaOH, and fixing the volume to 1L.
Test example 3 Assembly of homogeneous chemiluminescent assay kit for avian leukosis subgroup A/B/J specific antibody
The homogeneous chemiluminescence detection kit for avian leukosis A/B/J subgroup specific antibody comprises a luminescence microporous plate, a sealing film, acceptor microspheres, donor microspheres, a calibrator and an analysis buffer solution; the receptor microspheres comprise Alphaplex-645 receptor microspheres coupled with avian leukosis virus subgroup A/B His-cENV proteins and Alphaplex-545 receptor microspheres coupled with avian leukosis virus subgroup J His-gp85 proteins; the donor microspheres are AlphaScreen Donor microspheres which mark rabbit anti-chicken secondary antibody; the Tris-HCl buffer solution with the analysis buffer solution of 50mmol/L and the pH value of 7.4 contains 0.8 percent of NaCl, 1 percent of bovine serum albumin, 0.1 percent of casein, 0.1 percent of surfactant S9, 0.1 percent of triton-100 and 0.05 percent of Proclin-300; the luminescent microplate is a 96-well luminescent microplate (Perkin Elmer company, USA); the sealing film was TopSeal-A sealing film (PerkinElmer, USA).
The calibration product is prepared by the following method: mixing 50 parts of detected high-value serum to prepare positive standard serum, centrifuging at 10000rpm/min for 30min, purifying by affinity chromatography, collecting pure antibody, subpackaging the pure antibody with an ampoule and freeze-drying to form freeze-dried powder, and preparing the freeze-dried powder into 6 calibrator concentrations by using an analysis buffer solution: 0ng/mL, 10ng/mL, 40ng/mL, 160ng/mL, 640ng/mL, and 2560 ng/mL.
After the preparation, the kit is stored at-20 ℃ for later use.
Experimental example 4 optimization of conditions for detecting avian leukosis subgroup A/B/J specific antibody by using homogeneous chemiluminescence detection kit disclosed by the invention
(1) Optimum dilution ratio of acceptor microspheres and donor microspheres
A chessboard titration method is adopted, the Alphaplex 645 receptor microspheres of the avian leukosis virus A/B subgroup His-cNEV and the Alphaplex 545 receptor microspheres of the avian leukosis virus J subgroup His-gp85 are respectively provided with 6 concentrations of 0.005 mg/mL, 0.010mg/mL, 0.015mg/mL, 0.020mg/mL, 0.025mg/mL and 0.030mg/mL, the AlphaScreen donor microspheres marking rabbit anti-chicken secondary antibodies are provided with 4 concentrations of 0.010mg/mL, 0.020mg/mL, 0.030mg/mL and 0.040mg/mL, and the measurement results are fitted with a Signal/blank ratio (Signal/Background, S/B) and a four-parameter curve to determine the optimal dilution ratio. Finally, the optimal concentration of the AlphaPlex 645 receptor microspheres of the A/B subgroup His-cENV of the tagged protein avian leukosis virus is determined to be 0.015mg/ml, the optimal concentration of the AlphaPlex 545 receptor microspheres of the J subgroup His-gp85 of the tagged protein avian leukosis virus is determined to be 0.020mg/ml, and the optimal concentration of the AlphaScreen donor microspheres of the tagged rabbit anti-chicken secondary antibody is determined to be 0.030 mg/ml.
(2) Reaction temperature and time
Setting the reaction time to 10min, 20min, 30min, 40min, 50min and 60min, setting the reaction temperature to 25 ℃ and 37 ℃, selecting the temperature and the time with the highest S/B value and the shortest S/B value according to the S/B value, adding 100 mu L of prepared acceptor microspheres and donor microspheres in a luminescent microplate respectively, then adding 20 mu L of sample serum, mixing uniformly, carrying out reaction according to the preset temperature and time, and then reading the signal value in a light-excited chemiluminescence apparatus. As a result, incubation at 37 ℃ was selected for 20min for optimum temperature and incubation time.
(3) Selection of optimal assay buffer
The following 4 buffers were selected: HEPES buffer, PBS buffer, MES buffer, Tris buffer, with S/B as optimization criteria. Finally, Tris-HCl buffer solution with the highest S/B value, 50mmol/L and pH7.4 is selected, wherein the Tris-HCl buffer solution contains 0.8% of NaCl by mass fraction, 1% of bovine serum albumin by mass fraction, 0.1% of casein by mass fraction, 0.1% of S9, 0.1% of triton-100 by volume fraction and 0.05% of Proclin-300 by volume fraction
Test example 5 method for detecting avian leukosis subgroup A/B/J specific antibody using the kit of the present invention
The method for detecting the specific antibody of the avian leukosis subgroup A/B/J by using the kit comprises the following steps:
step one, collecting and storing clinical specimens: collecting a chicken vein whole blood sample by using a vacuum blood collection tube without an anticoagulant, standing for 2 hours at 37 ℃, standing overnight at 2-8 ℃, sucking out precipitated serum, centrifuging for 5 minutes at 3000rpm/min, taking supernatant, and subpackaging in an EP tube, and marking a blood collection date and an identification number to obtain sample serum; if the sample serum is used on the same day, directly placing the sample serum in a refrigerator at the temperature of 2-8 ℃; if used every other day, the product is stored at-20 deg.C.
And step two, respectively adding 100 mu L of prepared acceptor microspheres and donor microspheres into a luminescent microplate, then adding 20 mu L of sample serum, uniformly mixing, incubating for 20min at 37 ℃, and then reading a luminescent value in a light-excited chemiluminescence apparatus.
And step three, according to the operation flow of the homogeneous phase chemiluminescence kit detection in the step one and the step two, measuring the prepared calibrator with six concentrations, and fitting four parameters of the obtained result to perform regression calculation to obtain a regression equation and a calibrator dose response curve. And calculating the dosage value of the specific antibody in the sample serum according to the luminous value and the reaction curve of the sample serum measured in the step two.
Test example 6 Main index of method for detecting avian leukosis virus subgroup A/B/J specific antibody by kit of the present invention
(1) Sensitivity of the composition
3 parts of strong positive serum samples of the avian leukemia subgroup A, subgroup B and subgroup J are subjected to gradient dilution 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024, and the detection method is used for detection, and the results are shown in the following table 1. 30 parts of positive specimen serum is selected, and a virus separation experiment, the detection method and an imported reagent kit (IDEXX) are used for simultaneous determination, so that the results are known, and the comparison coincidence rate of the results of the three detection methods for detecting 30 known positive serum samples is 100%.
Table 1: kit sensitivity detection
As can be seen from the above table 1, the detection method of the present invention can detect strongly positive sera of avian leukemia subgroup A and subgroup B diluted 256 times, the lowest detectable dosage value of the specific antibody of subgroup A is 22ng/mL, and the lowest detectable dosage value of the specific antibody of subgroup B is 26 ng/mL; the detection method can detect 512-time diluted avian leukosis J subgroup strong positive serum, and the lowest dosage value of the detected specific antibody of the J subgroup is 12 ng/mL.
(2) Specificity of
Purchase of 15 avian disease-specific standard positive serum samples from the netherlands GD: avian Infectious Bursal Disease (IBD), avian infectious bronchitis (IBV, Beaudette strain), Newcastle Disease (NDV), Avian Encephalomyelitis (AE), avian anemia factor (CAV), Infectious Laryngotracheitis (ILT), Mycoplasma Synoviae (MS), avian influenza (AIV, strain 0126), Reovirus (REO), avian infectious bronchitis (IBV, strain Mass), avian adenoviruses (FAV, strain Phelp), waterfowl/Fowl Pox Virus (FPV), avian egg drop virus (EDS-76), Mycoplasma septicum (MG) and paramyxovirus (APMV). In addition, 3 standard positive serum samples of avian leukosis subgroup A, subgroup B and subgroup J were purchased
The 15 avian disease specific standard positive sera and ALV-A/B/J subgroup standard positive sera are diluted 1:8, and are detected by the method for quantitatively detecting the antibody by using the homogeneous chemiluminescence kit, and the specific detection data are shown in the following table 2.
Table 2: kit specificity detection
As shown in the table 2, the detection results of all 15 poultry disease specific standard serum samples detected by the detection method of the kit are negative, which indicates that the kit has no cross reaction with other poultry disease sera and has good specificity. The standard positive serum for detecting the avian leukosis virus A/B/J subgroup has strong luminescent signal, which indicates that the kit has good reactivity and can be used for quantitatively detecting the avian leukosis virus A/B/J subgroup antibody.
(3) Precision degree
1 part of avian leukosis subgroup A strong positive serum specimen, 1 part of subgroup B strong positive serum specimen and 1 part of subgroup J strong positive serum specimen are taken. The three serum samples were repeated 20 times per person by two experimenters (a and B), and the measured Coefficient of Variation (CV) was calculated, showing that: a, the average percent CV of the results measured by 20 experimenters is 6.28 percent; the average percent of CV of the results measured by the experimenter B for 20 times is 7.44 percent, the percent of CV is less than 10 percent, and the results show good repeatability and stable and reliable results.
(4) Stability of
The method comprises the steps of placing the AlphaPlex 645 receptor microspheres for marking avian leukosis virus A/B subgroup His-cENV, the AlphaPlex 545 receptor microspheres for marking avian leukosis virus J subgroup His-gp85, the AlphaScreen donor microspheres for marking rabbit anti-chicken secondary antibody and analysis buffer solution in a temperature range of 2-8 ℃ for 0, 3, 6, 9 and 12 months, detecting quality control products (PC, IBD, IBV, NDV, REO, AIV and SPF) stored in a laboratory by using the homogeneous chemiluminescence method established by the invention, observing the sensitivity and specificity change trend of the homogeneous chemiluminescence method, and evaluating the stability of the method, wherein the results are shown in the following table 3.
Table 3: kit stability detection
As shown in the above Table 3, the main reagents used in the kit of the present invention are stored at 2-8 ℃ for 12 months, and the sensitivity and specificity results are almost unchanged, indicating that the kit has strong stability within 12 months.
SEQUENCE LISTING
<110> centers for prevention and control of animal epidemic diseases in Heilongjiang province, Luoyang research institute of modern biotechnology, Inc
<120> kit for detecting avian leukosis virus A/B/J subgroup specific antibody and detection method thereof
<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
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<213> Escherichia coli BL21
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Met Gly Thr Pro Leu Leu Pro Thr Arg Val Asn Tyr Ile Leu Ile Ile
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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
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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
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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
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Gln Ala Gly Gly Ser Thr Thr Ser Leu Leu Gly Asp Leu Leu Asp Asp
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Val Thr Ser Ile Arg His Ala Val Leu Gln Asn Arg Gly Gly Ser Ile
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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
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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
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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
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1 5 10 15
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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
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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
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Ser Ile Leu Ala Asn Ser Gly Gly Ser His Lys Arg Ser Val Thr His
225 230 235 240
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Gly Ser Ala Arg Ile Phe Ala Ser Ile Leu Ala Pro Gly Val Ala Ala
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Ala Leu Gln Ala Leu Arg Gly Gly Ser Arg Leu Ala Cys Trp Ser Val
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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
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Ile Asp Phe Leu Leu Leu Ala His Gly His Gly Cys Glu Asp Ile Ala
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Gly Met Cys Cys Phe Asn Leu Ser Gly Gly Ser Glu Trp Ala Val His
340 345350
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Val Cys Leu Pro Cys Phe Leu Gln Phe Val Ser Ser Ser Leu Glu
370 375 380
Claims (5)
1. A homogeneous phase chemiluminescence kit for detecting specific antibodies of avian leukosis virus subgroup A/B/J is characterized in that: the kit comprises a luminous microporous plate, a sealing film, an acceptor microsphere I, an acceptor microsphere II, a donor microsphere, a calibrator and an analysis buffer solution;
the receptor microsphere I is marked with avian leukosis virus subgroup A/B His-cenV protein, and the receptor microsphere II is marked with avian leukosis virus subgroup J His-gp85 protein; the donor microsphere marks a rabbit anti-chicken secondary antibody;
the marking process of the acceptor microsphere I, the acceptor microsphere II or the donor microsphere is realized by the following method: placing a to-be-marked object in a centrifuge tube, adding a phosphate buffer solution, centrifuging, washing and suspending, sucking up a supernatant, adding an MES buffer solution, then sequentially adding the purified marked object, a sodium cyanoborohydride solution and Tween-20, carrying out a light-shielding rotary reaction at 2-8 ℃ for 40-48 h, adding CMO, and stirring at 37 ℃ for 1h to terminate the reaction; centrifuging to remove supernatant, adding HEPES buffer solution for heavy suspension, centrifuging to remove supernatant, and adding preservation solution to obtain receptor microsphere I, receptor microsphere II or donor microsphere of the coupled marker;
the preservation solution is 50mmol/L Tris-HCl buffer solution with pH7.4, which comprises 6.05g/L Tris, 10g/L trehalose, 10g/L glucose, 30g/L mannitol, 20g/L bovine serum albumin, 5g/L proline and 0.1% Proclin-300 by volume fraction;
the calibrator is prepared by pure antibodies extracted from positive standard serum mixed by multiple high-value sera;
the analysis buffer solution is 50mmol/L Tris-HCl buffer solution with pH7.4, wherein the analysis buffer solution comprises 0.8% of NaCl by mass fraction, 1% of bovine serum albumin by mass fraction, 0.1% of casein by mass fraction, 0.1% of surfactant S9 by mass fraction, 0.1% of triton-100 by volume fraction and 0.05% of Proclin-300 by volume fraction.
2. The kit of claim 1, wherein: the donor microspheres are AlphaScreen ^ donor microspheres which mark rabbit anti-chicken secondary antibodies.
3. The kit of claim 1, wherein: the receptor microsphere I is an AlphaPlex region 645 microsphere marked with His-cENV protein; the receptor microsphere II is an AlphaPlex 545 microsphere marked with His-gp 85.
4. The kit of claim 1, wherein: the marking process of the acceptor microsphere I, the acceptor microsphere II or the donor microsphere is realized by the following method: placing 20mg/mL of a substance to be marked in a centrifuge tube, adding 1mL of 0.1mol/L, pH 8.0.0 phosphate buffer solution into every 0.2mL of the substance to be marked, repeatedly centrifuging, washing and suspending for 3-5 times, sucking out supernatant, adding 0.2mL of MES buffer solution, then adding 0.2-0.6 mg of purified marker with the concentration of 1-3 mg/mL, adding 8-12 uL of 400-500 mmol/L sodium cyanoborohydride solution and 12.5uL of 1% Tween-20 in volume fraction, carrying out lucifugal rotation reaction at the temperature of 2-8 ℃ for 40-48 h, adding 10uL of CMO, and stirring for 1h at the temperature of 37 ℃ to terminate the reaction; centrifuging to remove the supernatant, adding 0.5mL of 50mmol/L HEPES buffer solution with pH7.4 for resuspension, centrifuging to remove the supernatant, and adding a preservation solution to prepare the receptor microsphere I, the receptor microsphere II or the donor microsphere of the coupled marker.
5. The kit of claim 1, wherein: the calibration product is prepared by the following method: mixing 50 parts of detected high-value serum to prepare positive standard serum, centrifuging at 10000rpm for 30min, purifying by affinity chromatography, collecting pure antibody, subpackaging the pure antibody with an ampoule and freeze-drying to form freeze-dried powder, and preparing the freeze-dried powder into 6 calibrator concentrations by using an analysis buffer solution: 0ng/mL, 10ng/mL, 40ng/mL, 160ng/mL, 640ng/mL, and 2560 ng/mL.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1646913A (en) * | 2002-04-10 | 2005-07-27 | 反应生物医学公司 | Sensitive immunochromatographic assay |
| CN103472235A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting protein solution stabilizing agent |
| CN103837675A (en) * | 2014-03-07 | 2014-06-04 | 常艳敏 | Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method |
| CN102735833B (en) * | 2012-07-09 | 2015-01-21 | 沃克(天津)生物科技有限公司 | Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof |
| CN104548087A (en) * | 2014-12-26 | 2015-04-29 | 山东农业大学 | Epitope vaccine for resisting A/B subgroup avian leucosis virus infection and preparation method and application of epitope vaccine |
| CN106442982A (en) * | 2016-08-31 | 2017-02-22 | 扬州大学 | Fowl leucovirus subgroup J specific antigenic polypeptide and application thereof |
| CN106932583A (en) * | 2015-12-29 | 2017-07-07 | 北京大成生物工程有限公司 | Human epidermal growth factor acceptor Her-2/neu immue quantitative detection reagent boxes and preparation method and application |
-
2017
- 2017-08-23 CN CN201710730963.0A patent/CN107543923B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1646913A (en) * | 2002-04-10 | 2005-07-27 | 反应生物医学公司 | Sensitive immunochromatographic assay |
| CN102735833B (en) * | 2012-07-09 | 2015-01-21 | 沃克(天津)生物科技有限公司 | Thyroperoxidase antibody homogeneous-phase luminescent immunoassay kit and detection method thereof |
| CN103472235A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting protein solution stabilizing agent |
| CN103837675A (en) * | 2014-03-07 | 2014-06-04 | 常艳敏 | Homogeneous luminescence immunoassay method for quantitatively analyzing multiple components simultaneously and kit used for method |
| CN104548087A (en) * | 2014-12-26 | 2015-04-29 | 山东农业大学 | Epitope vaccine for resisting A/B subgroup avian leucosis virus infection and preparation method and application of epitope vaccine |
| CN106932583A (en) * | 2015-12-29 | 2017-07-07 | 北京大成生物工程有限公司 | Human epidermal growth factor acceptor Her-2/neu immue quantitative detection reagent boxes and preparation method and application |
| CN106442982A (en) * | 2016-08-31 | 2017-02-22 | 扬州大学 | Fowl leucovirus subgroup J specific antigenic polypeptide and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| AB亚群禽白血病病毒gp85蛋白的克隆表达及其单克隆抗体的制备;吴艳，等;《中国畜牧兽医学会禽病学分会第十五次学术研讨会论文集》;20101001;第115页左栏第2行-右栏第18行 * |
| J亚群禽白血病病毒gp85基因的原核表达及间接ELISA方法建立;刘丽娜，等;《中国预防兽医学报》;20140131;第36卷(第1期);摘要，第39页左栏-第41页左栏 * |
| 检测J亚群禽白血病病毒抗体的间接ELISA方法建立和针对gp85蛋白的单克隆抗体研制;朱陈娟;《中国优秀硕士学位论文全文数据库 农业科技辑》;20130615(第6期);摘要，第5-25页 * |
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