CN108196049A - The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus - Google Patents
The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus Download PDFInfo
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Abstract
The present invention relates to the chemiluminescence antigen immue quantitative detection reagent boxes and detection method of a kind of avian leukosis virus, belong to field of immunodetection, the kit includes Chemiluminescent plate, labelled antibody, peroxide solutions, calibration object, sample diluting liquid and cleaning solution, wherein, Chemiluminescent plate is coated with the monoclonal antibody I of anti-p27 albumen, monoclonal antibody II of the labelled antibody for the anti-p27 albumen of different meter Lu Nuo labels；Monoclonal antibody I and monoclonal antibody II are pairing antibody；Calibration object is prepared by high-purity P27 albumen；Peroxide solutions are the hydrogenperoxide steam generator containing Paracetamol；Sample diluting liquid is leads to and the phosphate buffer of S9 containing horse serum, Qula.The avian leukosis poison chemiluminescence antigen immue quantitative detection reagent box that the present invention is prepared using high-purity P27 albumen and a pair of of pairing monoclonal antibody, its sensibility is high, and specificity is good, and stability is strong, with good reliability, lay a good foundation for preferably purification chicken group avian leukosis.
Description
Technical field
The invention belongs to field of immunodetection, and in particular, to a kind of chemiluminescence antigen of avian leukosis virus quantifies
Detection kit and its detection method.
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 certain 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 countries in the world chicken group, causes chicken death, becomes thin, anaemia, retarded growth, while body is caused to be resisted
Power declines and immunosupress, is to endanger one of principal disease of aviculture.Antigenic difference, virus according to virus envelope glycoprotein
The molecular biological characteristic of interference experiment, host range and genome, chicken infected avian leukosis virus（ALV）Main point of group
For six subgroups of A, B, C, D, E, J.The generation of China's avian leukosis is fairly common, and clinical case is from initial commercial broiler group
Commodity egg, local breeder flock and other poultry are developed to, and along with oncogenicity enhancing and the generation of tumor multiplicity.Research
High infection rate, high incidence is presented in display, the avian leukosis poison in China, and early infection is universal, morbidity age in days significantly shifts to an earlier date, and mixes
Infect it is multiple/take place frequently, host range extension, tumour spectrum expand, pathological change complicate the features such as, caused to the aviculture in China
Huge loss.
At present, which there is no thoroughly feasible control measure, also can be used without effective vaccine.Preventing the main method of this disease is
The new varieties with hereditary resistance are cultivated, positive chicken is eliminated, purifies population, the chicken group of no leukaemia is selected by several generations.
Avian leukosis virus gag gene nucleotides are highly conserved, A, B, C, D subgroup and newly discovered J subgroup homologys
More than 96%, gag gene code group specific antigens（gsa）.P27 albumen is the group specific antigen of gag gene codes
（gsa）The main component of a kind of middle highly conserved albumen and ALV core capsid albumen, content account for viral total protein into
More than 30% divide, there are many viral antigen points, are the preferred antigen for preparing detection antibody.
Chemiluminescence immune assay (Chemiluminescence Immunoassay, CLIA) is by chemiluminescence or life
Object luminescence system is combined with immune response, for detecting a kind of novel markings immunoassay of trace antigen or antibody.
Two parts of immune response system and chemiluminescence system are included in chemiluminescence immune assay.Immune response system, base
The same Enzyme-multiplied immune technique of present principles.The principle of chemiluminescence system is luminous substrate and hydrogen peroxide generation in immune response
Redox reaction simultaneously releases a large amount of energy, generates the intermediate of excitation state.This excitation state intermediate, when it is returned to surely
During fixed ground state, photon can be launched simultaneously.Quantum yield of luminscence, light quantum production are can measure using luminous signal measuring instrument
Volume is directly proportional to the amount of the test substance in sample.Thus standard curve can be established and calculates the content of test substance in sample.
Its with high sensitivity, it is easy to operate, quick the characteristics of, but in practical operation, due to expression albumen and monoclonal antibody
Selection is different or the variability of reaction system, the influence to avian leukosis virus detection sensitivity, specificity or stability compared with
Greatly, unified normalizing operation is not formed, and the batch in the place of production, quantitative detection are difficult to realize.
Invention content
In view of the above-mentioned problems, the chemiluminescence antigen the purpose of the present invention is to provide a kind of avian leukosis virus is quantitatively examined
Test agent box and its detection method.The avian leukosis prepared using high-purity P27 albumen and a pair of of pairing monoclonal antibody is poisoned
The antigen immue quantitative detection reagent box that shines is learned, avian leukosis poison antigen can be detected, sensibility is high, and specificity is good, surely
It is qualitative strong, there is good reliability, directly can carry out batch, quantitative detection in the place of production, for the preferably purification chicken group white blood of fowl
Disease is laid a good foundation.
A kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus, including Chemiluminescent plate, labelled antibody,
Peroxide solutions, calibration object, sample diluting liquid and cleaning solution, the Chemiluminescent plate are coated with anti-avian leukosis virus p27
The monoclonal antibody I of albumen is coated with a concentration of 0.5~0.8ug/ml；
Monoclonal antibody II of the labelled antibody for the anti-avian leukosis virus p27 albumen of different meter Lu Nuo labels；The different luminol
Labeling process be：Different luminol is dissolved in hydrochloric acid solution, sodium nitrite is added in, is protected from light 15min, obtains mixed liquor；With
Borate buffer solution adjusts pH of mixed, adds the monoclonal antibody II of purified anti-avian leukosis virus p27 albumen, low temperature
Under the conditions of be protected from light, dialyse, obtain labelled antibody, -20 DEG C of preservations；
The monoclonal antibody I and monoclonal antibody II belong to IgG2a subclass and to match antibody；
The calibration object is prepared by P27 albumen of the purity more than 97%；
The peroxide solutions are the aqueous hydrogen peroxide solution that volume fraction is 0.06%, wherein the acetyl ammonia containing 0.6g/L
Base phenol；
The phosphate buffer that the pH value that the sample diluting liquid is 10mM is 6.8-7.6, wherein being 5.0%- containing volume fraction
The Qula that 15.0% horse serum, volume fraction are 0.1%-0.5% lead to -100 and mass fraction be 0.05%-0.6% S9.
Further, the potency of the monoclonal antibody I and monoclonal antibody II is all higher than 500,000.
Further, the concentration that the calibration object is prepared for 6 kinds by P27 albumen distinguish 0ng/ml, 1ng/ml, 10ng/ml,
The solution of 100ng/ml, 1000ng/ml and 5000ng/ml.
Further, the P27 albumen that the calibration object is purified by calibration object diluted is made；The calibration object is dilute
Release liquid be pH value be 7.4, a concentration of 10mM phosphate buffers, wherein containing 50ml/L chicken serums.
Further, the coating process of the Chemiluminescent plate is：The monoclonal of anti-avian leukosis virus p27 albumen is resisted
Body I is diluted to coating concentration with coating buffer, is added on Chemiluminescent plate with every hole 100ul, by Chemiluminescent plate be placed in 4 DEG C it is cold
Library is incubated 20h, completes coating, cleaning；Then 150ul confining liquids are added in per hole, Cool Room 4 DEG C is placed in and is incubated 20h, discard closing
Liquid pats dry, and completes closing；The Chemiluminescent plate for completing closing is placed 18h under the conditions of 25 DEG C, humidity are less than 30% does it
It is dry, it is finally fitted into the aluminium foil bag for being placed with drier.
Further, the coating buffer is the carbonate buffer solution that pH is 7.4, wherein containing 0.16g/L sodium carbonate and
0.29g/L sodium bicarbonates；Confining liquid is casein containing 5g/L and the Tris-HCl buffer solutions of 50mM, pH8.0 of 50g/L sucrose.
Further, different luminol label the specific steps are：15mg different luminols are added to 2.5ml, 2M
In HCl, in 0~4 DEG C of stirring and dissolving on magnetic stirring apparatus, the NaNO2 solids of 15mg are then added in, are protected from light 15min, instead
It should finish, obtain mixed liquor；Mixed liquor, in blue, shows potassium iodide starch test paper blue to Congored test paper；Then with 0.5M,
It is 8.5 that the borate buffer solution of pH9.6, which adjusts pH of mixed, adds the Dan Ke of purified anti-avian leukosis virus p27 albumen
Grand antibody II is protected from light for 24 hours under the conditions of 0~4 DEG C, completes label, and dialysis obtains labelled antibody, -20 DEG C of preservations.
Further, the use concentration of labelled antibody is represented with extension rate, is 8000.
Further, labelled antibody is diluted with labelled antibody dilution；The labelled antibody dilution be containing
Fetal calf serum, Proclin300, trehalose 50 mM pH value be 7.6-8.5 Tris-Hcl buffer solutions；Wherein, tire ox blood
Clear volume fraction is 10.0%-50.0%, and the volume fraction of Proclin300 is 0.1%-0.4%, and the mass fraction of trehalose is
1%-10%。
The present invention also provides a kind of method that avian leukosis virus antigen is quantitatively detected using mentioned reagent box, including following
Step：
（1）Chemiluminescent plate from aluminium foil bag is taken out, sample or calibration object 50ul are added in per hole, then adds 50ul per hole
Labelled antibody, 37 DEG C of incubation reactions 30 minutes；
（2）Liquid in hole is got rid of, cleaning solution 250ul is added to be washed per hole, then patted dry, washed repeatedly；
（3）Add peroxide solutions 50ul, Chemiluminescent plate is placed on light-emitting appearance and carries out reading；
（4）Using a concentration of abscissa of calibration object, calibration object luminous value as ordinate, draw standard curve；
（5）The luminous value of sample is substituted into standard curve, calculates the actual concentrations of sample.
Beneficial effects of the present invention：
The present invention is highly conserved between avian leukosis virus difference subgroup using P27 genes, and the high characteristic of content utilizes original
Nuclear expression goes out P27 albumen, and prepares two with confrontation P27 protein monoclonal antibodies with the albumen, then with matching monoclonal antibody
The optimal reaction system of chemical luminescence reagent kit is filtered out, the kit prepared based on this has good sensibility and spy
The opposite sex has a good reactivity with common ALV-A/B/J subgroup virus, and sensitivity can reach 1ng/ml, and with it is common
Other avian virals such as IBV, NDV, AIV, FPV, ILTV, FPV, IBDV, ARV, REV etc. do not react, high specificity.
Compared with the chemiluminescence quantification kit of the present invention has also been done with PCR kit for fluorescence quantitative, 200 parts of anuses after testing
Swab sample is 92% with quantitative fluorescent PCR coincidence rate.Compared with enzyme linked immunological and PCR etc. detect avian leukosis virus method,
Also have the advantage that：
A, the P27 albumen for preparing of the present invention compares by a large amount of ALV genetic fragments, the P27 albumen conservative height of preparation, and common
ALV-A/B/J subgroups have very high homology；
B, the present invention is pairing monoclonal antibody using monoclonal antibody prepared by P27 albumen, and two monoclonal antibodies are for two differences of P27 albumen and farther out
Epitope, reaction is independent of each other, and reacts good, and other common virus do not react with birds with ALV-A/B/J subgroups；
C, luminescent substance different luminol is directly tagged on antibody by the present invention, does not need to enzyme and luminous substrate, and operation is easier,
Sensitivity higher.
D, chemical luminescence reagent kit prepared by the present invention can quantify avian leukosis poison, and susceptibility is high, can
The Infection Status of the clear judgement sample of veterinarian and present infective stage are helped, is very suitable for sample batch detection,
And sterile working is carried out without moving to laboratory, easy to operate, detection time is short, and stability is high, of low cost, is examined for ALV
It surveys and eliminates positive chicken group in time and lay a good foundation so as to preferably realize to the purification of chicken group's avian leukosis.
Description of the drawings
Fig. 1 is the digestion identification collection of illustrative plates of recombinant expression carrier pET32a-p27；Wherein：1：DNA Marker DL2000,2：
BamH I/Sal I endonuclease bamhis, 3：DNA Marker DL15000；
Fig. 2 is the P27 protein SDS-PAGE analysis charts of purifying；
Fig. 3 is canonical plotting.
Specific embodiment
Below by specific embodiment the present invention will be further explained explanation.
The expression and purification of 1 avian leukosis virus of embodiment recombination P27 albumen and the preparation of monoclonal antibody
According to the gene order of ALV-P27 albumen delivered in NCBI GenBank, with reference to both at home and abroad to the research of ALV into
Exhibition filters out the gene order with the homology ＞ 98% of each Reference strains, obtains P27 gene order 726bp, nucleotides sequence
It arranges shown in SEQ. ID. NO.01；The gene order 5,End restriction enzyme site containing NcoI, 3,Hold restriction enzyme site containing XhoI.Above-mentioned base
Because sequence is by doctor's biotechnology of precious day（Beijing）Co., Ltd synthesizes, and synthetic product P27 gene orders are connected to pUC-19 carriers,
Synthetic product is sequenced, and gene order is as shown in SEQ.ID. NO.01.
By pET32a（+）Restriction enzyme NcoI and XhoI are used respectively with the pUC-19 carriers of above-mentioned connection target gene
Under 37 DEG C of water bath conditions carry out double digestion 60min, with after 1% agarose gel electrophoresis according to plastic recovery kit specification
It is required that large fragment and target gene fragment is separately recovered；By the connection, wherein linked system overnight of 22 DEG C of the object of above-mentioned acquisition
For：PET32a (+) carrier 1ul, P27 genetic fragments 1.5ul, ddH2O 20ul, T4DNA ligases 1.5ul, 10 × Green
Buffer 3ul, the common 27ul of total volume obtain connection product pET32a-P27 recombinant plasmids.
Connection product pET32a-P27 recombinant plasmids are conventionally transferred to 0.1%CaCl2The competent bacteria of preparation
In BL21, in coating and the LB solid medium cultures containing ampicillin, 37 DEG C are incubated overnight, next day picking single bacterium colony,
Bacterium is shaken, extraction plasmid sends to sequencing after carrying out double digestion identification, confirms the sequence wherein having as shown in SEQ.ID. NO.01,
It can be seen that successful clone enters in pET32a-P27 recombinant plasmids the segment of the recombination P27 genes 726bp of acquisition.
Positive E. coli BL21 bacterium containing recombinant plasmid are inoculated in containing ampicillin（Amp）The solid training of resistance
It supports in base, after 37 DEG C are incubated overnight, 3~4 single bacterium colonies is taken to be inoculated in the LB fluid nutrient mediums containing Amp, 37 DEG C of concussions
（250rpm）Culture adds in IPTG to OD600=0.8（Isopropyl-beta D-thio galactopyranoside）To final concentration of 1mmol/
L, 37 DEG C are continued culture 4 hours, obtain expression albumen, and avian leukosis P27 expressing quantities are 625mg/L.Culture does not lure simultaneously
The recombinant plasmid transformed BL21 bacterium led are as negative control.The albumen of expression is identified through SDS-PAGE and Western-blot, is tied
Fruit is as shown in Figure 1.
The engineering bacteria bacterium solution of culture is collected, 6000rpm centrifugation 10min abandon supernatant, precipitate and acquisition re-suspension liquid is resuspended with PBS,
Then GuNTA-0Buffer and PMSF is added in re-suspension liquid at low temperature with Ultrasonic Cell Disruptor ultrasonication BL21 bacteriums, 120W,
Work 2s, interval 3s, then ultrasonication bacterium solution is transferred in 50ml centrifuge tubes, trim, 4 DEG C by 100 cycles of ultrasound,
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 collection penetrating component.Chromatography is washed with the GuNTA-60Buffer of 5 times of NTA volumes
De-, flow velocity 12ml/h collects eluent.With the dialysis elution in 24 hours of 0.5mol/LNaCl and 20mmol/L Tris-HCl solution
Liquid, avian leukosis P27 eggs after purification are up to 97% from SDA-PAGE and Western-blot testing goal purity of protein is carried out,
Molecular size range is 36KD.
The preparation of P27 monoclonal antibodies：High-purity P27 albumen will have been purified as immunizing antigen, 5~6 week old are immunized
BALB/c mouse is immunized 10 mouse, without adjuvant emulsion, is directly diluted to 1mg/ml with sterile saline every time, is immunized
Position is axillary gland, every mouse 20ul, and booster immunization after 7 days directly takes lymph node after three days, it is broken after being collected into
Lymphocyte merged with SP2/0 myeloma cell, by after fusion cell 1000r/min centrifuge 5min, abandon supernatant, use
The culture solution containing HAT of 37 degree of water-bath preheatings is resuspended.It is added in 96 well culture plates containing feeder cells, 100 μ l/ holes are put
37 degree, 5% carbon dioxide, saturated humidity culture.Fused cell is cultivated, when cell clone grows to the 1/2 of hole, supernatant is taken to use
The cell in indirect ELISA detection specific antibody positive hole with limiting dilution assay clone three times, makes positive rate up to 100%, in time
Freeze-stored cell.The hybridoma cell strain for screening acquisition is enlarged culture, with 6 × 108Every 0.5ml abdominal cavity of hybridoma
BLAB/C mouse are injected, with syringe collecting ascites after 13 days, 3000rpm centrifugation 10min, supernatant is filtered again with 0.45um after centrifugation
Membrane filtration, then again with caprylic acid-ammonium preliminary purification, coating P27 antigens are to thick pure antibody progress indirect ELISA inspection
It surveys, measures its potency, and which subclass belonged to the monoclonal antibody that IgG subgroup identifications kit measurement screens.To filtering out
5 plants for avian leukosis poison P27 protein specific monoclonal antibodies and secrete the monoclonal antibody hybridoma carry out
It matches two-by-two, detects itself and P27 albumen simultaneous reactions, be independent of each other with reaction and P27 albumen reaction gradient is well mark
Standard, finishing screen select two plants of pairings P27 protein hybridoma cell strains 5H8 and 3D2, and ascites prepared by two strain of hybridoma strains resists
Body potency is both greater than 500,000, belongs to IgG2a subclass.
The preparation of chemical luminescence reagent kit and the use of the kit that 2 avian leukosis antigen of embodiment quantitatively detects
Method
The preparation of labelled antibody：It adds in the HCl of 2.5ml, 2M, 15mg different luminols at 0~4 DEG C on magnetic stirring apparatus
Then stirring and dissolving adds in the NaNO of 15mg2Solid, is protected from light 15min, and reaction makes mixed liquor to Congored test paper when finishing
In blue, blue is shown to potassium iodide starch test paper, then adjusts mixed liquor with the borate buffer solution of 0.5MpH9.6, makes its pH be
8.5 or so, purified 3D2 monoclonal antibodies are added, is protected from light under the conditions of 0~4 DEG C for 24 hours, completes label, finally will
Labelled antibody loading bag filter is put into borate buffer solution and dialyses, and dispensing -20 DEG C after having dialysed freezes.
It is coated with the preparation of plate：The CB buffer solutions of 5H8 monoclonal antibodies pH9.6, a concentration of 0.05M are diluted, 100ul
It is coated with per hole, Chemiluminescent plate is placed in Cool Room 4 DEG C and is incubated 20 hours after coating, is then cleaned 4 times with cleaning solution, then per hole
150ul confining liquids are added in, the luminescent screen after closing is placed in Cool Room 4 DEG C is incubated 20 hours.Confining liquid is discarded later, is patted dry,
Being placed under conditions of temperature is 25 DEG C, humidity is less than 30% makes it fully dry for 18 hours, is finally packed into the aluminium for being placed with drier
Foil bag prepares coating plate.Wherein, the sodium bicarbonate of the sodium carbonate containing 0.16g/L and 0.29g/L in CB buffer solutions；Confining liquid
The Tris-HCl buffer solutions of the preferably 50mMpH8.0 of casein containing 5g/L and 50g/L sucrose.
Optimum antibody coating concentration and labelled antibody concentration are determined with Checkerboard titration method, longitudinal direction 5H8 monoclonal antibodies are pressed
12.8ug/ml, 6.4ug/ml, 3.2ug/ml, 1.6ug/ml, 0.8ug/ml, 0.4ug/ml, 0.2ug/ml, 0.1ug/ml's is
Row dilution is coated with 96 orifice plates, laterally presses 1:500、1:1000、1:2000、1:4000、1:8000、1:16000、1:32000、1:
64000 are diluted labelled antibody, values of chemiluminescence are measured, according to 0 standard items luminous value ﹤, 200,5000 standard items luminous values
In 50000~100000, r2More than 0.99 concentration and labelled antibody optimum dilution degree are coated with for most suitable monoclonal antibody.It is true through overtesting
Determine a concentration of 0.5~0.8ug/ml of coating of 5H8 monoclonal antibodies, labelled antibody optimum dilution degree is 1:8000.
The composition of kit：
Wherein：
It is coated with plate：The coated Chemiluminescent plate of avian leukosis 5H8 monoclonal antibodies prepared as described above；
Calibration object distinguishes 0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml, 1000ng/ for 6 kinds by the concentration that P27 albumen is prepared
The solution of ml and 5000ng/ml.Calibration object dilution used is preferably chicken serum containing 100ml/L and 0.2% in process for preparation
The 10mMpH values of Proclin-300 are 7.4 phosphate buffers；
8000 times of labelled antibody diluted of labelled antibody；Labelled antibody dilution preferably containing fetal calf serum,
Proclin300, trehalose 50 mM pH value for 7.6-8.5Tris-Hcl buffer solutions, the fetal calf serum is in above-mentioned sample
A concentration of 10.0%-50.0% of dilution, the Proclin300 are in a concentration of 0.1%-0.4% of above-mentioned sample diluting liquid, institute
State a concentration of 1%-10% of the trehalose in above-mentioned enzyme combination diluent；
The pH value that sample diluting liquid is preferably the 10mM for leading to -100, S9 and Proclin-300 containing horse serum, Qula is 6.8-
7.6 phosphate buffers, the horse serum above-mentioned sample diluting liquid a concentration of 5.0%-15.0%, the Qula lead to -100
A concentration of 0.1%-0.5% of above-mentioned sample diluting liquid, the S9 are in a concentration of 0.05%-0.6% of above-mentioned sample diluting liquid, institute
State a concentration of the 0.1% of Proclin-300；S9 therein is Tetronic 1307, is a kind of amphoteric surfactant；
Peroxide solutions：2ml, 30% hydrogenperoxide steam generator are drawn, is added in 900ml purified waters, then adds 0.6g pairs
Paracetamol, after dissolving plus water is settled to 1L；
25 × cleaning solution：Weigh NaCl200g, KCl5g, Na2HPO4·12H2O72.5g, KH2PO45g adds water 500ml to dissolve,
Then plus Tween-20 12.5ml, water is added to be settled to 1L.
The application method of chemical luminescence reagent kit that 3 avian leukosis antigen of embodiment quantitatively detects
First, the preparation of solution：
1:The preparation of wash operating solution
25 × cleaning solution is pressed 1 with deionized water:24 volume ratios, which are diluted, prepares wash operating solution, i.e.,：1 part of 25 × cleaning solution
+ 24 parts of deionized waters, for being coated with the washing of plate, wash operating solution can preserve one month in 2 ~ 8 DEG C of environment.
2:The preparation of sample
The cloacal swab of poult is added in the sampling pipe containing 0.5ml sample diluting liquids, then liquid feeding nitrogen is repeatedly
Twice, mixing is for use after finally thawing for freeze thawing.
2nd, operating procedure
1. required reagent is taken out from cold storage environment, it is placed in room temperature（20~25℃）30min is balanced, pays attention to each liquid reagent
It must be shaken up before use.
2. taking out needs the coating plate of quantity, no lath is put into valve bag, is stored in 2 ~ 8 DEG C.
3. wash operating solution also needs back to warm to room temperature before use.
4. number：Sample and calibration object are corresponded to micropore sequentially to number, it is parallel that each sample and standard items do 2 holes, and remembers
Record the position where gauge orifice and sample well.
5. add standard items/sample：It adds in 50 μ L to corresponding micropore of standard items/sample, adds in labelled antibody 50 immediately
μ L/ holes, gently vibrate mixing, react 30min in sealing plate membrane cover plate 37 DEG C of light protected environments of postposition.
6. board-washing：Sealing plate film carefully is opened, liquid in hole is discarded, with 250 μ L/ holes of wash operating solution, fully washs 5
It is secondary, per minor tick 10s, patted dry with blotting paper（The bubble not being eliminated after patting dry can be poked with original pipette tips）.
7. it measures：50 μ L/ holes of peroxide solutions are added in, mixing is gently vibrated, is put into chemiluminescent analyzer reading.
3rd, interpretation of result
Standard curve is drawn first, using calibration object luminous value as ordinate, corresponding calibration object content（ng/ml）For abscissa,
Four parameter curve fits of calibration object are drawn, four parameter modes are Y=(a-d)/[1+ (x/c) b]+d.By the luminous value generation of sample
Enter calibration curve calculating, you can obtain the content of avian leukosis poison P27 albumen in sample.
The specificity analysis of 4 avian leukosis virus antigen immue quantitative detection reagent box of the present invention of embodiment
First, specific detection
Buy 15 parts of poultry diease specific criteria antigen samples of GD companies of Holland：Poultry infectious bursal disease（IBD）, avian infectious branch
Tracheitis（IBV, Beaudette plants）, newcastle disease (NDV), avian encephalomyelitis（AE）, the fowl anaemia factor（CAV）, infectiousness larynx gas
Guan Yan（ILT）, synovia mycoplasma（MS）, bird flu (AIV, 0126 plant）, reovirus（REO）, avian infectious bronchitis
（IBV, Mass plants）, aviadenovirus（FAV, Phelp plants）, aquatic bird/bird pox virus（FPV）, it is viral that fowl subtracts egg（EDS-76）, lose
Blood mycoplasma（MG）, paramyxovirus（APMV）.In addition 3 parts of avian leukosis A subgroups, B subgroups and J subgroup standard antigens are also had purchased
Sample
15 kinds of poultry diease specific criteria antigens and ALV-A/B/J subgroup antigens are all diluted to 1ug/ml, are prepared with the present invention
Avian leukosis chemiluminescence antigen immue quantitative detection reagent box is detected, and operating method is compared with the chemiluminescence antigen in technology
The operating method of immue quantitative detection reagent box.Detection data is as shown in table 1 below.
Table 1：Specific detection data
Conclusion：Avian leukosis virus antigen immue quantitative detection reagent box of the present invention detects 15 parts of poultry diease specific criteria antigen samples,
The testing result of all samples is all negative, shows the present invention and other poultry diease cause of disease no cross reactions, is had good special
Property.Avian leukosis poison A/B/J subgroup virus antigens are detected, luminous signal is very strong, illustrates there is good reactivity, can be used for
Avian leukosis poison A/B/J subgroups are quantified.
2nd, sensitivity Detections
Sample：The avian leukosis positive chicken sample of 200 42 ages in days（Cloacal swab detaches and glimmering by avian leukosis virus
Fluorescent Quantitative PCR method is identified）, from infected area chicken house.
The avian leukosis chemiluminescence antigen immue quantitative detection reagent box prepared with the present invention is detected, operating method co-occurrence
There is the operating method of the chemiluminescence antigen immue quantitative detection reagent box in technology.Detection data is as shown in table 2 below.
Table 2：Sensitivity Detection data
Conclusion：Avian leukosis virus chemiluminescence antigen immue quantitative detection reagent box of the present invention is tested with virus purification and fluorescent quantitation
PCR is compared, and the coincidence rate of sensibility is more than 98%.
3rd, Detection of Stability
Avian leukosis virus chemiluminescence antigen immue quantitative detection reagent box of the present invention is placed in 2-8 DEG C and preserves 0,3,6,9,12 month,
Period grab sample, observes the character of seminal plasma fructose detection kit, while monitors the sensibility and specificity of the kit, assesses reagent
The stability of box.
Avian leukosis virus chemiluminescence antigen immue quantitative detection reagent box is placed in 2-8 DEG C of 0,1,2,3,6,9,12 and of preservation
15 months, the character of seminal plasma fructose detection kit was observed in during which grab sample.
The quality-control product preserved by this laboratory（PC, IBD, IBV, NDV, REO, AIV, SPF）Monitor the sensitivity of the kit
Property and specificity assess the stability of kit.
Method：Operating method is compared with the operating method of the chemiluminescence antigen immue quantitative detection reagent box in technology.As a result
It is as shown in table 3 below.
Table 3：Kit is in the property character result of different time
Table 4：Kit is in the sensibility and specificity testing result of different time
Conclusion：As a result show that the kit preserves 12 months at 2-8 DEG C, the character of all reagents does not become in kit
Change.Its sensibility, specific outcome meet examining report when batch is provided.Show that the kit preserves 12 at 2-8 DEG C
Still there is very stable performance in the moon.
4th, repeatability detection：
The avian leukosis P27 protein samples of known three concentration（50ng/ml, 500ng/ml, 2000ng/ml）.With three batches
Kit, the kit of each batch is repeated 5 times experiment, does experiment every time and at least two laboratory technicians is required for do simultaneously.
The kit experimental result of 3 batches,
It is 5.41% that 50ng/ml P27 albumen, which measures CV percentages,；
It is 6.24% that 500ng/ml P27 albumen, which measures CV percentages,；
It is 8.39% that 2000ng/ml P27 albumen, which measures CV percentages,；
It is separated by certain time with a batch of kit to be detected with same quality-control sample, the CV percentages of sample are
4.28%。
Conclusion：Avian leukosis virus chemiluminescence antigen immue quantitative detection reagent box of the present invention, between batch, batch in it is repeatable steady
Fixed, CV percentages are respectively less than 10%.
In biomaterial used in the present invention：
E. coli bl21 is purchased from China Veterinery Drug Inspection Office, prokaryotic expression carrier pET-32a（+）With cloning vector pMD-
18T is purchased from precious day doctor biotechnology（Beijing）Co., Ltd；
6 week old BALB/c mouse inbred lines are purchased from experimental animal center of henan province；This laboratory of SP2/0 murine myeloma cells is protected
It deposits；
Protein molecular weight Maker, Tricine, Tris, sodium periodate, bovine serum albumin(BSA), PEG 8000 and ammonia benzyl are green
Mycin is purchased from raw work bioengineering（Shanghai）Limited company；
T4DNA ligases, GoTaq enzymes, dNTP, restriction enzyme, IPTG, which are purchased from, cures biotechnology precious day（Beijing）Limited public affairs
Department；
The bag filter of molecular weight 5000kD of shutting off is purchased from Green Bird companies；
0.22 μm, 0.45 μm of water phase pin type filter, 15mL3kD ultrafiltration membrane centrifuge tubes are purchased from Millipore companies；
Plasmid extraction kit, plastic recovery kit etc. are purchased from Tiangeng biochemical technology（Beijing）Co., Ltd；Root
Chemiluminescent plate is purchased from Thermo Fisher companies；
Tryptone, yeast extract are purchased from OXOID companies；
Sodium chloride, potassium chloride, ten sodium dihydrogen phosphate dihydrates, potassium dihydrogen phosphate, that hydrochloric acid is purchased from Chinese medicines group chemical reagent is limited
Company；
RPMI1640 culture mediums, HAT, HT, fetal calf serum are purchased from GibcoBRL；
Affinity chromatography protein purification filler is purchased from GE healthcare；
LB solid mediums are formulated as follows：Tryptone 10g. yeast extracts 5g, NaCl 10g are dissolved in 950ml；Double steamings
In water, it is settled to 1L with NaoH tune pH to 7. 0 and forms；The preparation of LB fluid nutrient mediums：By yeast extract 5g, peptone 10g
NaCl 10g are added in container, then distilled water is added to adjust pH7. 0-7 2, packing sterilizing with NaOH and HCl to 1000ml.
In instrument of the present invention：
All-round table-type high-speed refrigerated centrifuge, specifications and models are Biofuge stratos, purchased from Thermo companies；
Constant temperature/refrigeration landing large capacity shaking table（Forma Orbital Shaker）48l is purchased from Thermo companies；
Gel imaging system is purchased from Beijing Jun Yi companies；
DYY-6C types electrophoresis apparatus, SDS-PAGE electrophoresis tanks, Western-blot film transfer printing devices are purchased from Liuyi Instruments Plant, Beijing；
RNA/DNA quantitative analysis instruments（GeneQuant pro）Purchased from Amersham Biosciences companies；
Chemical illumination immunity analysis instrument is purchased from Zhengzhou Autobio Engineering Co., Ltd.；
Biohazard Safety Equipment, superclean bench are purchased from Sujing Group Co., Ltd., Jiangsu Prov；
Constant temperature CO2Incubator is purchased from Sanyo Electric Co., Ltd；
Ultraviolet specrophotometer（TU-1900）Purchased from Beijing Puxi General Instrument Co., Ltd；
Horizontal steam sterilizer（WDZX-200KC）Purchased from Shenan Medical Appliances Factory, Shanghai；
Cell supersonic wave pulverizer is purchased from NingBo XinZhi Biology Science Co., Ltd.
SEQUENCE LISTING
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture, Luoyang Co., Ltd of modern biotechnology research institute
<120>The chemiluminescence antigen immue quantitative detection reagent box and its detection method of a kind of avian leukosis virus
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 760
<212> DNA
<213>Avian leukosis virus
<400> 1
cctgtagtaa agactggctg atacggtcag gaccaagggc ttacgatccc cgatcactat 60
ggcggaggtg gaagcgctta tgtcctcccc gctgctgccg catgacgtta cgaatctaat 120
gagagttatt ttaagacaga ttaggacctg ccccatatgc cttgtggatg gacgcttggg 180
gtgtccaact acagacggtt gggacccgcc atagcggcag ccactcgcga cccccgacac 240
ccagcgaatg gtcaagggcg gggggaacgg tggacccctc cactaatttg gatcgcttaa 300
agggtttagc tgatgggatg gtgggcaacc cgcagggtca gattgagcca aagccgcatt 360
attaagaccg ggggaattgg ttgctattac ggcgtcggct ctccaggcat ttagactggc 420
tgcgagaggt tgcccggctg gcggaacctg ctggtvvatg ggcggacatt acgcagggac 480
cagctgctgc cgtctgagtc ctttgttgat tttgccaatc ggcttataaa ggcggttgag 540
gggtcagatc tccgaatcta atccgccttc cgcgcgagct ccggtgatca ttgactgctt 600
taggcagaag tcacagccag atgagagtta ttatccagca gcttatacgg gcagcaccct 660
ccacgctgac caccccagga gagataatca aattaggacc tgtatgtgct agacaggcag 720
aagactgccc ctcttacgga tcaaggccat agccgcggcc 760
Claims (10)
1. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus, including Chemiluminescent plate, labelled antibody, mistake
Oxide solution, calibration object, sample diluting liquid and cleaning solution, it is characterised in that：The Chemiluminescent plate is coated with the white blood of anti-fowl
The monoclonal antibody I of sick virus p27 albumen, is coated with a concentration of 0.5~0.8ug/ml；
Monoclonal antibody II of the labelled antibody for the anti-avian leukosis virus p27 albumen of different meter Lu Nuo labels；The different luminol
Labeling process be：Different luminol is dissolved in hydrochloric acid solution, sodium nitrite is added in, is protected from light 15min, obtains mixed liquor；With
Borate buffer solution adjusts pH of mixed, adds the monoclonal antibody II of purified anti-avian leukosis virus p27 albumen, low temperature
Under the conditions of be protected from light, dialyse, obtain labelled antibody, -20 DEG C of preservations；
The monoclonal antibody I and monoclonal antibody II belong to IgG2a subclass and to match antibody；
The calibration object is prepared by P27 albumen of the purity more than 97%；
The peroxide solutions are the aqueous hydrogen peroxide solution that volume fraction is 0.06%, wherein the acetyl ammonia containing 0.6g/L
Base phenol；
The phosphate buffer that the pH value that the sample diluting liquid is 10mM is 6.8-7.6, wherein being 5.0%- containing volume fraction
The Qula that 15.0% horse serum, volume fraction are 0.1%-0.5% lead to -100 and mass fraction be 0.05%-0.6% S9.
2. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as described in claim 1, feature exist
In：The potency of the monoclonal antibody I and monoclonal antibody II is all higher than 500,000.
3. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as described in claim 1, feature exist
In：The concentration that the calibration object is prepared for 6 kinds by P27 albumen distinguish 0ng/ml, 1ng/ml, 10ng/ml, 100ng/ml,
The solution of 1000ng/ml and 5000ng/ml.
4. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as claimed in claim 3, feature exist
In：The P27 albumen that the calibration object is purified by calibration object diluted is made；The calibration object dilution be pH value be 7.4,
The phosphate buffer of a concentration of 10mM, wherein containing 50ml/L chicken serums.
5. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as described in claim 1, feature exist
In：The coating process of the Chemiluminescent plate is：The monoclonal antibody I of anti-avian leukosis virus p27 albumen is dilute with coating buffer
It releases to coating concentration, is added on Chemiluminescent plate with every hole 100ul, Chemiluminescent plate is placed in Cool Room 4 DEG C is incubated 20h, complete
Into coating, cleaning；Then 150ul confining liquids are added in per hole, Cool Room 4 DEG C is placed in and is incubated 20h, discard confining liquid, pat dry, complete envelope
It closes；The Chemiluminescent plate for completing closing is placed 18h under the conditions of 25 DEG C, humidity are less than 30% to make it dry, is finally packed into and is placed with
In the aluminium foil bag of drier.
6. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as claimed in claim 5, feature exist
In：The coating buffer is the carbonate buffer solution that pH is 7.4, wherein containing 0.16g/L sodium carbonate and 0.29g/L sodium bicarbonates；
Confining liquid is casein containing 5g/L and the Tris-HCl buffer solutions of 50mM, pH8.0 of 50g/L sucrose.
7. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as described in claim 1, feature exist
In：Different luminol label the specific steps are：15mg different luminols are added in the HCl of 2.5ml, 2M, at 0~4 DEG C
Then the stirring and dissolving on magnetic stirring apparatus adds in the NaNO2 solids of 15mg, is protected from light 15min, reaction finishes, and must mix
Liquid；Mixed liquor, in blue, shows potassium iodide starch test paper blue to Congored test paper；Then it is buffered with the borax of 0.5M, pH9.6
It is 8.5 that liquid, which adjusts pH of mixed, the monoclonal antibody II of purified anti-avian leukosis virus p27 albumen is added, 0~4
It is protected from light under the conditions of DEG C for 24 hours, completes label, dialysis obtains labelled antibody, -20 DEG C of preservations.
8. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as claimed in claim 7, feature exist
In：The use concentration of labelled antibody is represented with extension rate, is 8000.
9. a kind of chemiluminescence antigen immue quantitative detection reagent box of avian leukosis virus as claimed in claim 8, feature exist
In：Labelled antibody is diluted with labelled antibody dilution；The labelled antibody dilution be containing fetal calf serum,
Proclin300, trehalose 50 mM pH value be 7.6-8.5 Tris-Hcl buffer solutions；Wherein, the volume of fetal calf serum point
Number is 10.0%-50.0%, and the volume fraction of Proclin300 is 0.1%-0.4%, and the mass fraction of trehalose is 1%-10%.
10. using the method such as claim 1-9 kit quantification detection avian leukosis virus antigens as described in any one, spy
Sign is：Include the following steps：
Chemiluminescent plate from aluminium foil bag is taken out, sample or calibration object 50ul are added in per hole, 50ul marks are then added per hole
Remember antibody, 37 DEG C of incubation reactions 30 minutes；
Liquid in hole is got rid of, cleaning solution 250ul is added to be washed per hole, then patted dry, washed repeatedly；
Add peroxide solutions 50ul, Chemiluminescent plate is placed on light-emitting appearance and carries out reading；
Using a concentration of abscissa of calibration object, calibration object luminous value as ordinate, draw standard curve；
The luminous value of sample is substituted into standard curve, calculates the actual concentrations of sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114397449A (en) * | 2022-01-05 | 2022-04-26 | 中科国创(南平)生物科技有限公司 | Sample diluent for ELISA detection of avian leukosis virus antigen in meconium |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053155A (en) * | 2010-06-29 | 2011-05-11 | 华中农业大学 | Homogeneous chemiluminescence immunoassay method for measuring for organophosphorus pesticide Dursban |
| CN102636644A (en) * | 2012-04-18 | 2012-08-15 | 中国农业科学院哈尔滨兽医研究所 | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen |
| CN105198986A (en) * | 2015-09-10 | 2015-12-30 | 中国农业科学院哈尔滨兽医研究所 | Anti-avian leukosis virus p27 protein monoclonal antibody, gold-colloidal strip containing same and application |
| CN105353124A (en) * | 2015-12-04 | 2016-02-24 | 江苏立华牧业股份有限公司 | Method for fast reaction of P27 protein of meconium ALV virus |
| CN105861444A (en) * | 2016-04-08 | 2016-08-17 | 华南农业大学 | Anti-subgroup B avian leukosis virus strain cell line and application thereof |
-
2017
- 2017-12-28 CN CN201711461638.5A patent/CN108196049A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102053155A (en) * | 2010-06-29 | 2011-05-11 | 华中农业大学 | Homogeneous chemiluminescence immunoassay method for measuring for organophosphorus pesticide Dursban |
| CN102636644A (en) * | 2012-04-18 | 2012-08-15 | 中国农业科学院哈尔滨兽医研究所 | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen |
| CN105198986A (en) * | 2015-09-10 | 2015-12-30 | 中国农业科学院哈尔滨兽医研究所 | Anti-avian leukosis virus p27 protein monoclonal antibody, gold-colloidal strip containing same and application |
| CN105353124A (en) * | 2015-12-04 | 2016-02-24 | 江苏立华牧业股份有限公司 | Method for fast reaction of P27 protein of meconium ALV virus |
| CN105861444A (en) * | 2016-04-08 | 2016-08-17 | 华南农业大学 | Anti-subgroup B avian leukosis virus strain cell line and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 中国优秀硕士学位论文全文数据库: "禽白血病病毒p27抗原的单克隆抗体制备及双抗体夹心ELISA检测方法建立", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114397449A (en) * | 2022-01-05 | 2022-04-26 | 中科国创(南平)生物科技有限公司 | Sample diluent for ELISA detection of avian leukosis virus antigen in meconium |
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Application publication date: 20180622 |