CN108776221A - A kind of kit of avian leukosis virus antibody and the detection simultaneously of S. pullonum antibody - Google Patents
A kind of kit of avian leukosis virus antibody and the detection simultaneously of S. pullonum antibody Download PDFInfo
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Abstract
The present invention relates to biotechnology, more particularly to the kit of a kind of avian leukosis virus antibody and the detection simultaneously of S. pullonum antibody.The antigen combination of the kit includes p27 albumen, gp85 albumen and GroEL- Δ 8-1 albumen.The prokaryotic expression protein of the truncate GroEL- Δs 8-1 of avian leukosis virus capsid protein p27, the prokaryotic expression protein of envelope protein gp85 and S. pullonum dominant antigen GroEL is used to develop the ELISA kit that can detect avian leukosis virus and S. pullonum antibody simultaneously as envelope antigen.Compared with the kit of currently used independent detection avian leukosis or white diarrhea, kit of the invention achieves while detecting the effect of avian leukosis and white diarrhea, can greatly mitigate the detection purification work of avian leukosis and white diarrhea.
Description
Technical field
The present invention relates to biotechnology, more particularly to a kind of avian leukosis virus antibody and S. pullonum are anti-
The kit that body detects simultaneously.
Background technology
Avian leukosis (Avian Leukosis) be by avian leukosis virus (Avian Leukosis Virus, ALV) and
The system of birds kinds of tumors disease caused by virus in viral (Avian Sarcoma Virus, the ASV) group of fowl sarcosis
Claim.The disease can cause many kinds to have communicable benign and malignant tumour chicken.
Since Roloff in 1868 reports chicken lymphosarcoma, avian leukosis has been distributed in all over the world, and China also has certainly
Right case occurs.The disease can cause chicken weightening slowly, sexal maturity delay, egg is small, eggshell is thin, egg production decline, rate of fertilization and
Hatching rate is low, trunk discards rate increase, the death rate increases, and causes direct economic loss.Also body can be caused non-specific simultaneously
Property resistance decline and immunosupress, cause indirect economic loss.Due to disease energy vertical transmission, very to aviculture harm
Greatly, it is to endanger one of principal disease of poultry husbandry.
White diarrhea (Pullorum Disease) is drawn by S. pullonum (Salmonella pullorum, SP)
It rises, using chick as main infection object, using the white excrement of row and egg drop reduction as main feature, antenatal each age in days chicken can be caused to lose
The birds acute infectious disease of courageous and upright typhoid fever.
The circulation way of S. pullonum is in diversity, can not only pass through the progress such as feed, air, drinking-water, muroid
Horizontal transmission, the ovum that can also be carried disease germs by production carry out vertical transmission, and a large amount of pathogens carried in the young villus of the disease hatched can be dirty
Drinking-water, brooder, feed etc. are contaminated, by the infection such as alimentary canal, respiratory tract with the young bird of henhouse, resistance to cross after chicken grows up arranges band again
Complicated communication network is consequently formed in bacterium ovum.Infection rate is high at home for S. pullonum, and distribution is wide, is that kind of fowl cultivation is net
The disease of emphasis and commodity egg the cultivation keypoint control of change.
Avian leukosis and white diarrhea are all that can cause serious harm to poultry husbandry with the infectious disease of vertical transmission.At present
Control avian leukosis and the most effective measure of white diarrhea are to implement the purification of infectious disease, mainly by periodically being examined to breeder flock
Epidemic disease eliminates positive chicken, by the chicken group for constantly purifying to select no avian leukosis and white diarrhea.Various countries are in numerous detections
In method, ELISA detection method is sensitive, special, easy to operate with it and is widely used in clinical detection.The method is suitable for base
The diagnosis and Serologic detection of layer Veterinary office and chicken house to the disease.Currently, the commercialization avian leukosis for having external production is anti-
Former or antibody assay kit, detection white diarrhea are total to mainly by plate agglutination test without avian leukosis and white diarrhea
The kit or method of detection.Therefore, avian leukosis and white diarrhea antibody with China's independent intellectual property right is developed to detect altogether
Kit, and by its industrialization production, have great importance for the purification of China's avian leukosis and white diarrhea.
The p27 genes of avian leukosis virus are one section of highly conserved genes between avian leukosis virus difference subgroup.
Its p27 albumen encoded is the main component of virus core shell, and content is high, accounts for 30% or more of viral total protein component,
Body can be induced to generate specific antibody.The gp85 genes of avian leukosis virus are a cyst membrane eggs for avian leukosis virus
In vain, body can be induced to generate specific antibody.Heat-shock protein 60 (GroEL) are S. pullonums
Important proteantigen can induce when S. pullonum invades body and generate lot of antibodies.Therefore, can use p27,
Gp85 and GroEL establishes the indirect ELISA testing kit of detection avian leukosis and white diarrhea antibody as envelope antigen.
1 avian leukosis of table and white diarrhea detection kit statistics
Mainly detect the avian leukosis of chicken cloacal swab with external ELISA kit to the purification of avian leukosis at present
The avian leukosis virus antibody of viral antigen p27 and chicken serum mainly detect chicken to the purification of white diarrhea with plate agglutination test
The S. pullonum antibody of serum.These kits or method all just for single infectious disease detection, Bu Nengtong
When detection avian leukosis and white diarrhea.
Avian leukosis and white diarrhea are all that chicken farm needs the infectious disease purified, are mainly to the purification of avian leukosis at present
The avian leukosis antibody of the avian leukosis virus antigen p27 and chicken serum of chicken cloacal swab are detected with external ELISA kit,
The S. pullonum antibody of chicken serum, these kits or side are mainly detected with plate agglutination test to the purification of white diarrhea
Method both for single infectious disease detection, there are no a kind of Single agents box can detect avian leukosis and white diarrhea both
Disease.The method that individually kit of detection avian leukosis and plate agglutination test individually detect white diarrhea, makes clinical detection purify
Avian leukosis and white diarrhea work expend a large amount of human and material resources and financial resources.Therefore there is an urgent need to develop to facilitate handy fowl
Leukaemia and white diarrhea detection kit mitigate the purification work of avian leukosis and white diarrhea.
Invention content
In view of this, the present invention provides a kind of avian leukosis virus antibody and S. pullonum antibody to detect simultaneously
Kit.The kit can detect avian leukosis and white diarrhea simultaneously.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of antigen combination, including p27 albumen, gp85 albumen and GroEL- Δ 8-1 albumen.
In some specific embodiments of the present invention, p27 albumen, gp85 albumen and GroEL- in the antigen combination
The total concentration of Δ 8-1 albumen is (1~8) μ g/mL and mass ratio is (0.5~1):(0.5~1):1.
In some specific embodiments of the present invention, p27 albumen, gp85 albumen and GroEL- in the antigen combination
The total concentration of Δ 8-1 albumen is (1~4) μ g/mL and mass ratio is 1:1:(0.25~4).
On this basis, the present invention also provides the antigen combinations to prepare detection avian leukosis and/or white diarrhea
Kit in application.
The present invention also provides kit, it is coated with the antigen combination.
In some specific embodiments of the present invention, the kit further includes serum, primary antibody, ELIAS secondary antibody, coating
Dilution, developing solution and the terminate liquid of buffer solution, washing buffer, confining liquid, sample or secondary antibody.
The present invention also provides the application method of the kit, the dilution of the serum is 1:(100~400),
Preferably, the dilution of the serum is 1:200；The ELIAS secondary antibody dilution is 1：(4000~16000), as excellent
Choosing, the ELIAS secondary antibody dilution are 1：8000.
In some specific embodiments of the present invention, the sealing condition of the confining liquid is 5% skimmed milk, 37 DEG C of closings
2h。
In some specific embodiments of the present invention, the incubation conditions of the primary antibody are 37 DEG C of incubation (45~90) min,
Preferably, the incubation conditions of the primary antibody are 37 DEG C of incubation 60min；The incubation conditions of the ELIAS secondary antibody are 37 DEG C of incubations
(45~90) min, preferably, the incubation conditions of the ELIAS secondary antibody are 37 DEG C of incubation 45min.
In some specific embodiments of the present invention, the developing time of the developing solution is (5~15) min, as excellent
The developing time of choosing, the developing solution is 10min.
The present situation that present invention combination avian leukosis and white diarrhea will purify simultaneously, it is white with fowl using the basic principle of ELISA
Blood disease viral capsid proteins p27, the prokaryotic expression protein of envelope protein gp85 and S. pullonum dominant antigen GroEL
The prokaryotic expression protein of truncate GroEL- Δs 8-1 is developed as envelope antigen can detect avian leukosis and white diarrhea simultaneously
ELISA kit.It is coated with condition by p27, gp85 and GroEL- Δ 8-1 for groping best, optimizes the reaction condition of ELISA
Establish the indirect ELISA method of detection avian leukosis and white diarrhea.With currently used independent detection avian leukosis or white diarrhea
Kit compare, kit of the invention can detect avian leukosis and white diarrhea simultaneously, as long as the chicken of test positive is
It eliminates, achievees the purpose that while purifying, greatly mitigate the work of clinical detection purification.
Specific implementation mode
The invention discloses the examinations that a kind of avian leukosis virus antibody and S. pullonum antibody detect simultaneously
Agent box, those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that institute
There are similar replacement and change apparent to those skilled in the art, they are considered as being included in the present invention.
The method of the present invention and application are described by preferred embodiment, and related personnel can obviously not depart from the present invention
Hold, method described herein and application are modified or are suitably changed and combined in spirit and scope, to realize and apply this
Inventive technique.
1. research method：
The present situation of purification avian leukosis and white diarrhea is needed according to chicken house, and there is presently no can detect the white blood of fowl simultaneously
The kit or method of disease and white diarrhea.Using the basic principle of ELISA, with avian leukosis virus capsid protein p27, cyst membrane egg
White gp85 and S. pullonum dominant antigen GroEL- Δs 8-1 be common envelope antigen establish detection avian leukosis virus and
The indirect ELISA new method of S. pullonum antibody.This method concrete operations are as follows：With the p27 albumen of purifying, gp85 eggs
White and GroEL- Δ 8-1 albumen is envelope antigen, by grope best coating condition, sealing condition, serum dilution ratio and
Action time, secondary antibody dilution and action time and developing time establish detection avian leukosis virus antibody and white diarrhea sramana
The method of Salmonella antibody, and verification is detected to the method established.
2. the optimal conditions operating method of the indirect ELISA detection method determined
(1) antigen coat：The coating buffer of pH9.6 dilutes antigen, and coating total concentration is 4 μ g/mL and GroEL- Δs 8-1:p27
+ gp85 is 1:2, i.e. GroEL- Δs 8-1:p27:Gp85 is 1:1:1, it is added in 96 hole elisa Plates, 100 holes μ L/, 4 DEG C were coated with
Night.
(2) it washs：PBST board-washings, 300 holes μ L/, pat dry, and washing is three times.
(3) it closes：5% skimmed milk, 200 holes μ L/, 37 DEG C of closing 2h are added in ELISA Plate.
(4) it washs：Repeat step (2).
(5) antigen-antibody reaction：With 0.5% BSA dilute serums to 1:200, it is added in ELISA Plate, 100 holes μ L/, 37
DEG C be incubated 60min.
(6) it washs：Repeat step (2).
(7) enzyme labelled antibody reacts：With 0.5% BSA dilution HRP labels rabbit anti-chicken IgG mother liquor to 1：8000, enzyme is added
In target, 100 holes μ L/, 37 DEG C of incubation 45min.
(8) it washs：Repeat step (2).
(9) it develops the color：TMB developing solutions are added in ELISA Plate, 100 holes μ L/.
(10) it terminates：50 μ L terminate liquids are added per hole and terminate reaction by color development at room temperature 10min.
(11) it reads：Microplate reader preheating is opened within 1 minute in advance, microplate reader parameter (reference wavelength is set：620nm measures wave
It is long：450nm shakes 3s, interval 2s), read OD450nm values.
(12) judge：When detection serum OD450nm values be more than yin and yang attribute critical value (mean value+3 of standard female sample ×
The standard deviation of standard female sample), then it detects in serum containing in avian leukosis virus antibody or S. pullonum antibody
One or two.
3.p27+gp85 the best coating condition with GroEL- Δs 8-1：
Packet is amounted in p27+gp85 the and GroEL- Δ 8-1 antigen coat groups of different coating total concentrations and different coating ratios
By ELISA Plate, each antigen coat combination use respectively doubling dilution avian leukosis positive serum and white diarrhea positive serum as
Primary antibody, remaining operation are carried out by ELISA routine operations, detect serum titer.Calculate each white blood of antigen coat combine detection fowl
The valence value of sick positive serum and white diarrhea positive serum takes the detection all higher antigen coat group cooperation of two kinds of serum titer values
For best antigen coat condition.As table 2 and table 3 are chosen as a result, it has been found that there are two antigen coat combination is preferable in conjunction with valence value
Antigen coat total concentration is 4 μ g/mL and GroEL- Δs 8-1:P27+gp85 is 1:2, i.e. GroEL- Δs 8-1:p27:Gp85 is 1:
1:1 is used as best antigen coat condition.
4. people is mixing white diarrhea positive serum and the detection of avian leukosis positive serum samples：
White diarrhea positive serum and avian leukosis positive serum are mixed according to different proportion respectively, then use has optimized
S. pullonum antibody ELISA detection method, avian leukosis virus antibody ELISA detection method and S. pullonum
Antibody detects the potency of different pooled serums from avian leukosis virus antibody ELISA co-detection method.As a result with expected consistent,
Such as table 14, the ELISA method singly detected detects the potency of serum as the ratio of corresponding positive serum increases and constantly increases, and
The serum titer of the ELISA method detection detected altogether shows that the S. pullonum antibody established and fowl are white without significant changes
Blood disease antiviral antibody ELISA co-detection methods can detect this two kinds of diseases well.
5. infection experiment chicken serum sample detection：
Respectively (GroEL- Δ 8-1 packets are used with the self-built S. pullonum antibody ELISA detection method optimized
Quilt), self-built avian leukosis virus antibody ELISA detection method (being coated with p27+gp85), self-built S. pullonum
Antibody and avian leukosis virus antibody ELISA co-detection method (GroEL- Δs 8-1 and p27+gp85 are coated with altogether), IDEXX companies
The ALV A/B subgroups and J subgroups antibody assay kit of production, white diarrhea plate agglutination test detect 104 parts of infection experiments
Chicken serum sample.Such as table 16 and table 17, as a result the ALV A/B subgroups of IDEXX and J subgroups antibody assay kit and self-built fowl
For leukemia virus antibody ELISA detection method coincidence rate up to 90.4%, white diarrhea plate agglutination test is husky with self-built white diarrhea
The coincidence rate of door Salmonella antibody ELISA detection method illustrates individually to be coated with g85 and individually be wrapped with GroEL- Δs 8-1 up to 89.4%
The detection method coincidence rate of the ELISA method and commercialization that are established is all higher.Such as table 18 and table 19, as a result g85 and GroEL-
Δ 8-1 is individually coated with coating together and is more conform with ALV A/B subgroup and J subgroup antibody test of the rate up to 93.3%, IDEXX
Kit/white diarrhea plate agglutination test is total with self-built S. pullonum antibody and avian leukosis virus antibody ELISA
The coincidence rate of detection method shows the indirect ELISA method for being coated with foundation altogether with p27+g85 and GroEL- Δs 8-1 up to 87.5%
The detection method that can achieve the effect that and be commercialized, and avian leukosis virus and S. pullonum can be detected simultaneously
Antibody can detect the infection such as table 15 of avian leukosis virus and S. pullonum.
Table 19 the result shows that, be aggregated with the ALV A/B subgroups of IDEXX and J subgroups antibody assay kit/white diarrhea tablet
Experiment is reference, and self-built S. pullonum antibody is accorded with the positive of avian leukosis virus antibody ELISA co-detection method
Conjunction rate is 85.4%, negative match-rate 88.9%, illustrates that the ELISA method sensibility and specificity established is good.
Avian leukosis virus capsid protein p27, the prokaryotic expression protein of envelope protein gp85 genes and chicken are used in this research
The prokaryotic expression protein of the truncate GroEL- Δs 8-1 of Salmonella pullorum dominant antigen GroEL is that envelope antigen establishes detection
The ELISA kit of avian leukosis and white diarrhea.Other methods that can detect avian leukosis and white diarrhea simultaneously can use it
His S. pullonum dominant antigen and avian leukosis antigen establishes ELISA kit as envelope antigen altogether, but uses
The validity of other antigens is also uncertain.The detection white blood of fowl is established using p27, gp85 and GroEL- Δ 8-1 as common antigen
The colloidal gold kit of disease and white diarrhea, but this method is not suitable for the detection of a large amount of samples, and sensitivity is too late
ELISA.If with S. pullonum dominant antigen GroEL genes and avian leukosis virus p27, gp85 gene with merging
The method of PCR links together three genes, and by Fusion gene construction to expression vector, three Gene Fusions of expression and purification
Albumen, the albumen that GroEL is merged with p27, gp85 establishes detection white diarrhea and avian leukosis as ELISA envelope antigens
Method, but the possible false folding of albumen that GroEL is merged with p27, gp85, influence antigenicity.
For the present invention according to the gene constructed p27 recombinant prokaryotic expression vectors of p27 of avian leukosis virus, gp85 is gene constructed
Gp85 recombinant prokaryotic expression vectors, with IPTG induced expressions fusion p27 albumen and gp85 albumen, ni-sepharose purification p27 albumen with
Gp85 albumen.It is best by groping using the p27 albumen of purifying and gp85 albumen and GroEL- Δ 8-1 albumen as envelope antigen
Coating condition, sealing condition, serum dilution ratio and action time, secondary antibody dilution and action time and developing time are established
The method for detecting avian leukosis virus antibody and S. pullonum antibody, and the validity of the method to being established is tested
Card.
The present situation that present invention combination avian leukosis and white diarrhea will purify simultaneously, it is white with fowl using the basic principle of ELISA
The truncate of the prokaryotic expression protein and white diarrhea dominant antigen GroEL of blood disease viral capsid proteins p27, envelope protein gp85
The prokaryotic expression protein of GroEL- Δs 8-1 is developed for envelope antigen can detect avian leukosis and the ELISA of white diarrhea simultaneously
Kit.Compared with the kit of currently used independent detection avian leukosis or white diarrhea, kit of the invention can be same
When detection avian leukosis and white diarrhea achieve the purpose that while purifying, greatly mitigate as long as the chicken of test positive is eliminated
The work of clinical detection purification.
ELISA immune detection related reagents：
1. reagent configures：
It is coated with buffer solution (pH9.6 0.05M carbonate buffer solutions):
Washing buffer (pH7.4PBST)：
| Reagent | Dosage |
| KH2PO4 | 0.2g |
| Na2HPO4·12H2O | 2.9g |
| NaCl | 8.0g |
| KCl | 0.2g |
| Tween-20 | 0.5mL |
| Distilled water | 1000mL |
Confining liquid:
| Reagent | Dosage |
| Skimmed milk | 5g |
| Washing buffer | 100mL |
Sample and secondary antibody diluent (0.5% BSA)：
| Reagent | Dosage |
| Bovine serum albumin(BSA) (BSA) | 0.5g |
| Washing buffer | 100mL |
Terminate liquid (2M H2SO4) configuration method
| Reagent | Dosage |
| The concentrated sulfuric acid | 22mL |
| Water | 178mL |
Conventional ELISA experimental methods：
(1) antigen coat：The coating buffer of pH9.6 dilutes coating protein to required concentration, is added in 96 hole elisa Plates, 100 μ
The holes L/, 4 DEG C of coatings are overnight.
(2) it washs：PBST board-washings, 300 holes μ L/, pat dry, and washing is three times.
(3) it closes：5% skimmed milk, 200 holes μ L/, 37 DEG C of closing 2h are added in ELISA Plate.
(4) it washs：Repeat step (2).
(5) antigen-antibody reaction：With 0.5% BSA dilute serums 1:200, it is added in ELISA Plate, 100 holes μ L/, 37 DEG C
It is incubated 1h.
(6) it washs：Repeat step (2).
(7) enzyme labelled antibody reacts：With 0.5% BSA dilution HRP labels rabbit anti-chicken IgG mother liquor to 1：8000, enzyme is added
In target, 100 holes μ L/, 37 DEG C of incubation 1h.
(8) it washs：Repeat step (2).
(9) it develops the color：TMB developing solutions are added in ELISA Plate, 100 holes μ L/.
(10) it terminates：50 μ L terminate liquids are added per hole and terminate reaction by color development at room temperature 10min.
(11) it reads：Microplate reader preheating is opened within 1 minute in advance, microplate reader parameter (reference wavelength is set：620nm measures wave
It is long：450nm shakes 3s, interval 2s), read OD450nm values.
The kit that a kind of avian leukosis virus antibody and S. pullonum antibody provided by the invention detect simultaneously
In raw materials used and reagent be available on the market.
With reference to embodiment, the present invention is further explained：
The preparation of embodiment 1p27 albumen, gp85 albumen and GroEL- Δ 8-1 albumen
(1) avian leukosis virus p27 Prokaryotic expression vector constructions and protein expression and purification
It is set according to the sequence of p27 genes in the avian leukosis virus gene complete sequence (M37980) that oneself logs in Genbank
Count the specific primer with restriction enzyme site：Sense primer (as shown in SEQ ID No.1)
CGGGATCCATGCCTGTAGTGATTAAGAC, downstream primer (as shown in SEQ ID No.2)
CGCTCGAGCTAGGGCTGGATAGCAGACG.P27 genes are inserted by PCR amplification p27 genetic fragments with the method for digestion enzyme even
PET-30a prokaryotic expression carriers.Correct pET-30a-p27 will be sequenced and be transformed into Transetta (DE3).By pET-30a-
37 DEG C of shaken cultivations of p27/Transetta (DE3) are to OD600It is 0.6 to 0.8, IPTG, which is added, makes its final concentration of 1mmol/L, and 37
DEG C, 200rpm induced expression 6h, thalline were collected by centrifugation.Thalline through ultrasound cracking (ultrasonic power than 35%, ultrasonic 2s, pause 2s,
Ultrasonic time (including ultrasound and time out) totally 1800 seconds), rear 12 000r/min centrifuges 20min, collects supernatant.With with
The affinity column of Ni ions carries out affinitive layer purification, the p27 albumen purified to supernatant.
(2) avian leukosis virus gp85 Prokaryotic expression vector constructions and protein expression and purification
According to gp85 full length genes in the J subgroup avian leucosis gene complete sequences (Z46390) that oneself logs in Genbank
Specific primer of the sequence design with restriction enzyme site：Sense primer (as shown in SEQ ID No.3)
CGGGATCCGGAGTTCATCTATTGCAACA, downstream primer (as shown in SEQ ID No.4)
CGCTCGAGTTAGCGCCTGCTACGGTGGTGAC.PCR amplification gp85 genetic fragments, with the method for digestion enzyme even by gp85 bases
Because being inserted into pET-28a prokaryotic expression carriers.Correct pET-28a-gp85 will be sequenced and be transformed into Transetta (DE3).By pET-
37 DEG C of shaken cultivations of 28a-gp85/Transetta (DE3) are to OD600It is 0.6 to 0.8, IPTG, which is added, keeps its final concentration of
1mmol/L, 37 DEG C, 200rpm induced expression 6h, thalline were collected by centrifugation.(ultrasonic power is ultrasonic than 35% through ultrasound cracking for thalline
2s suspends 2s, ultrasonic time (including ultrasound and time out) totally 1800 seconds), rear 12 000r/min centrifuges 20min, collects
Clearly.Affinitive layer purification is carried out to supernatant with the affinity column with Ni ions, the gp85 albumen purified.
(3) structure of S. pullonum GroEL- Δs 8-1 prokaryotic expression carriers and protein expression and purification according to
S. pullonum GroEL gene orders (the gene ID that Genbank is retrieved:CP003047.1 upstream and downstream) is designed
Restriction enzyme site is respectively the specific primer of BamH I and Xho I：Sense primer (as shown in SEQ ID No.5)
CGCGGATCCCTGATCATCGCTGAAGAT；Downstream primer (as shown in SEQ ID No.6)
CCGCTCGAGTTCCATTGCACGCAGCGC.PCR amplification GroEL- Δ 8-1 genetic fragments, will with the method for digestion enzyme even
GroEL- Δ 8-1 genes are inserted into pGEX-6p-1 prokaryotic expression carriers.Correct pGEX-6p-1-GroEL- Δs 8-1 will be sequenced to turn
Transetta (DE3) is dissolved into, 37 DEG C of shaken cultivations to OD600 are 0.6 to 0.8, and IPTG, which is added, makes its final concentration of 1mmol/L,
30 DEG C, 200rpm induced expression 10h, thalline were collected by centrifugation.(ultrasonic power is 2 seconds ultrasonic than 35%, temporarily through ultrasound cracking for thalline
Stop 2s, ultrasonic time (including ultrasound and time out) totally 1800 seconds), rear 12 000r/min centrifuges 20min, collects supernatant.With
Glutathion SepharoseTM4B affinity columns carry out affinitive layer purification to supernatant, and purifying obtains GroEL- Δs 8-1
Albumen.
The foundation of 2 indirect ELISA detection method of embodiment and the determination of condition
The determination of 2.1 best antigen coat conditions
Determine that the ratio of p27+gp85 is p27 first:Gp85 is 1:1, then press p27+gp85 and GroEL- Δ 8-1 albumen
The μ g/mL of final concentration of 0.25,0.5,1,2,4 and 8, and GroEL- Δs 8-1：P27+gp85 protein ratios are 1:0.125,1:
0.25,1:0.5,1:1,1:2,1:4 and 1:8 different antigen coat conditional combinations, each antigen coat combination is respectively with coating
Liquid dilution mixing p27+gp85 and GroEL- Δ 8-1 antigen coat ELISA Plates, 100 holes μ L/.Each antigen coat combination is examined respectively
The potency for surveying white diarrhea positive serum and avian leukosis positive serum dilutes white diarrhea positive serum and the white blood of fowl with 0.5%BSA
Sick positive serum, dilution are respectively 1:200,1:400,1:800,1:1600,1：3200,1：6400,1：12800,1：
25600, each 2 ELISA Plates of combination repeat, and 100 holes μ L/ use sample diluting liquid as negative control.ELISA is pressed in remaining operation
Routine operation carries out, and calculates the serum titer value of each antigen coat combination, takes the white diarrhea for measuring positive and avian leukosis
The all higher antigen coat combination of positive serum valence value is used as best antigen coat condition.
The valence value of the different antigen coat condition detection mono- positive serums of ALV and SP of table 2
Note：" * " represents detection white diarrhea positive serum highest valence value, and " △ " represents detection avian leukosis positive serum most
Efficiently value.
The ratio between the potency and highest potency of the detection mono- positive serums of ALV and SP under the conditions of the different antigen coats of table 3
Note：" * " represents all higher antigen coat combination of valence value of the detection mono- positive serums of ALV and SP
As it is always dense to choose antigen coat as a result, it has been found that there are two antigen coat combination is preferable in conjunction with valence value for table 2 and table 3
Degree is 4 μ g/mL and GroEL- Δs 8-1:P27+gp85 is 1:2, i.e. GroEL- Δs 8-1:p27:Gp85 is 1:1:1 as best anti-
Primordial covering condition.
The determination of 2.2 best confining liquids and off-period
With best antigen coat condition coated elisa plate, respectively with 5% skimmed milk, 1%BSA, 5% standard tire ox blood
Clearly, 0.7% gelatin is in 4 DEG C of closings overnight, and 37 DEG C of off-periods are set to 0.5h, 1h, 1.5h, 2h, 2.5h and 3h, according to chess
Disk method is laid out, remaining operation is carried out by ELISA routine operations.Calculate separately detection white diarrhea positive serum and avian leukosis
The P/N values of positive serum, determine best confining liquid and off-period.
Table 4 detects ALV antibody sealing conditions and gropes
Table 5 detects SP antibody sealing conditions and gropes
As a result all it is with 37 DEG C of 5% skimmed milk to the P/N values of detection white diarrhea positive serum and avian leukosis positive serum
2h highests are closed, therefore sealing condition is 5% skimmed milk, 37 DEG C of closing 2h.
The determination of 2.3 best serum dilutions and ELIAS secondary antibody dilution
It carries out, is laid out in a manner of chessboard method, serum dilution is set to 1 according to the condition optimized：50,1：100,
1：200,1：400 and 1：800, ELIAS secondary antibody dilution is set to 1：2000,1：4000,1：8000 and 1：16000.It counts respectively
The P/N values for calculating detection white diarrhea positive serum and avian leukosis positive serum, determine that best serum dilution and ELIAS secondary antibody are dilute
Degree of releasing.
Table 6 detects ALV antibody serums dilution and secondary antibody dilution is groped
Table 7 detects SP antibody serums dilution and secondary antibody dilution is groped
As a result all it is 1 in serum dilution to the P/N values of detection white diarrhea positive serum and avian leukosis positive serum：
200 and ELIAS secondary antibody dilution be 1：8000 highests, it is thus determined that serum dilution is 1：200 and ELIAS secondary antibody dilution be 1：
8000。
The determination of the best incubation time of 2.4 primary antibodies
Under the reaction condition optimized, antigen-antibody action time is set to 30min, 45min, 60min,
75min, 90min, remaining operation are carried out by ELISA routine operations.Calculate separately detection white diarrhea positive serum and avian leukosis
The P/N values of positive serum, determine the Best Times of antigen-antibody reaction.
Table 8 detects ALV antibody primary antibody incubation times and gropes
Table 9 detects SP antibody primary antibody incubation times and gropes
As a result P/N values are all when being incubated 60min at 37 DEG C to detection white diarrhea positive serum and avian leukosis positive serum
It is all higher, it is final to determine that 37 DEG C are incubated 60min as primary antibody reaction condition.
The determination of the best incubation time of 2.5 ELIAS secondary antibodies
The rabbit anti-chicken IgG of HRP labels react at 37 DEG C, the reaction time is respectively set as 15min, 30min, 45min,
60min, 75min, 90min, remaining operation are carried out by ELISA routine operations.Compare detection white diarrhea positive serum and the white blood of fowl
The P/N values of sick positive serum.
Table 10 detects the ALV antibody secondary antibody reaction time and gropes
Table 11 detects the SP antibody secondary antibody reaction time and gropes
As a result P/N values are all when being incubated 45min at 37 DEG C to detection white diarrhea positive serum and avian leukosis positive serum
Highest, it is final to determine that 37 DEG C are incubated 45min as secondary antibody reaction condition.
The determination of 2.6 developing times
It is tested according to the condition optimized, developing time is set to 5min, 8min, 10min, 15min, 20min,
Remaining operation is carried out by ELISA routine operations.Compare the P/N values of detection white diarrhea positive serum and avian leukosis positive serum.
Table 12 detects ALV antibody developing times and gropes
Table 13 detects SP antibody developing times and gropes
As a result when developing time is 10min to the P/N values of detection white diarrhea positive serum and avian leukosis positive serum
All highests, it is thus determined that developing time is 10min.
The optimal conditions operating method of 2.7 indirect ELISA detection methods finally determined
(1) antigen coat：The coating buffer of pH9.6 dilutes antigen, and coating total concentration is 4 μ g/mL and GroEL- Δs 8-1:p27
+ gp85 is 1:2, i.e. GroEL- Δs 8-1:p27:Gp85 is 1:1:1, it is added in 96 hole elisa Plates, 100 holes μ L/, 4 DEG C were coated with
Night.
(2) it washs：PBST board-washings, 300 holes μ L/, pat dry, and washing is three times.
(3) it closes：5% skimmed milk, 200 holes μ L/, 37 DEG C of closing 2h are added in ELISA Plate.
(4) it washs：Repeat step (2).
(5) antigen-antibody reaction：With 0.5% BSA dilute serums to 1:200, it is added in ELISA Plate, 100 holes μ L/, 37
DEG C be incubated 60min.
(6) it washs：Repeat step (2).
(7) enzyme labelled antibody reacts：With 0.5% BSA dilution HRP labels rabbit anti-chicken IgG mother liquor to 1：8000, enzyme is added
In target, 100 holes μ L/, 37 DEG C of incubation 45min.
(8) it washs：Repeat step (2).
(9) it develops the color：TMB developing solutions are added in ELISA Plate, 100 holes μ L/.
(10) it terminates：50 μ L terminate liquids are added per hole and terminate reaction by color development at room temperature 10min.
(11) it reads：Microplate reader preheating is opened within 1 minute in advance, microplate reader parameter (reference wavelength is set：620nm measures wave
It is long：450nm shakes 3s, interval 2s), read OD450nm values.
(12) judge：When detection serum OD450nm values be more than yin and yang attribute critical value (mean value+3 of standard female sample ×
The standard deviation of standard female sample), then it detects in serum containing in avian leukosis virus antibody or S. pullonum antibody
One or two.
3 white diarrhea positive serum of embodiment and avian leukosis positive serum mix sample detection
By white diarrhea positive serum and avian leukosis positive serum respectively according to 1:0,100:1,50：1,20:1,10:1,1:
1,1:10,1:20,1:50,1:100 and 0:1 different proportion mixing, respectively with the self-built S. pullonum optimized
Antibody ELISA detection method, self-built avian leukosis virus antibody ELISA detection method, self-built S. pullonum are anti-
Body detects the potency of different pooled serums from avian leukosis virus antibody ELISA co-detection method.
Table 14 detects the potency result of SP positive serums and ALV positive serum different mixing proportions with different envelope antigens
As a result with expected consistent, such as table 14, the potency of the ELISA method detection serum singly detected is with corresponding positive blood
Clear ratio increases and constantly increases, and the serum titer of the ELISA method detection detected altogether shows foundation without significant changes
S. pullonum antibody can detect this two kinds of diseases well with avian leukosis virus antibody ELISA co-detection method.
4 infection experiment chicken serum sample detection of embodiment
Infection experiment chicken serum：Avian myeloblastosis virus (AMV) infected group, S. pullonum (SP) infection
The acquisition in 1,4,7,15 and 21 day of group, AMV and S. pullonum coinfection group and blank control group after Chickens Infected
Chicken serum.Respectively (GroEL- Δ 8-1 packets are used with the self-built S. pullonum antibody ELISA detection method optimized
Quilt), self-built avian leukosis virus antibody ELISA detection method (being coated with p27+gp85), self-built S. pullonum
Antibody and avian leukosis virus antibody ELISA co-detection method (GroEL- Δs 8-1 and p27+gp85 are coated with altogether), IDEXX companies
The ALV A/B subgroups and J subgroups antibody assay kit of production, white diarrhea plate agglutination test detect 104 parts of infection experiments
Chicken serum sample.
The self-built S. pullonum antibody of table 15 is infected with the detection of avian leukosis virus antibody ELISA co-detection method
Test sera positive rate
The ALV A/B subgroups and J subgroups antibody assay kit of 16 IDEXX of table and self-built avian leukosis virus antibody
The coincidence rate of ELISA detection method
The coincidence rate of 17 white diarrhea plate agglutination test of table and self-built S. pullonum antibody ELISA detection method
The self-built S. pullonum antibody ELISA detection method of table 18 and self-built avian leukosis virus antibody
Symbol of the ELISA detection method with self-built S. pullonum antibody and avian leukosis virus antibody ELISA co-detection method
Conjunction rate
The ALV A/B subgroups and J subgroups antibody assay kit and white diarrhea plate agglutination test of 19 IDEXX of table is with certainly
The coincidence rate of the S. pullonum antibody and avian leukosis virus antibody ELISA co-detection method built
Such as table 16 and table 17, as a result the ALV A/B subgroups of IDEXX and J subgroups antibody assay kit and self-built fowl are white
Blood disease antiviral antibody ELISA detection method coincidence rate is up to 90.4%, white diarrhea plate agglutination test and self-built white diarrhea sramana
The coincidence rate of Salmonella antibody ELISA detection method illustrates up to 89.4% with the coated single detection methods of p27+g85 and GroEL- Δs
The ELISA method that 8-1 is individually coated with foundation is all higher compared to coincidence rate with the detection method of commercialization.Such as table 18 and table 19, knot
Coated co-detection method is more conform with rate and reaches coated single detection method fruit p27+g85 and GroEL- Δ 8-1 together respectively
The ALV A/B subgroups and J subgroups antibody assay kit/white diarrhea plate agglutination test of 93.3%, IDEXX are white with self-built chicken
The coincidence rate of dysentery antibodies toward salmonella and avian leukosis virus antibody ELISA co-detection method shows to use p27+g85 up to 87.5%
It is coated with the detection method that the indirect ELISA method of foundation can achieve the effect that and be commercialized, and energy altogether with GroEL- Δs 8-1
Enough while avian leukosis virus and S. pullonum antibody are detected, avian leukosis virus and white diarrhea Salmonella can be detected
The infection of bacterium such as table 5.
Table 19 the result shows that, be aggregated with the ALV A/B subgroups of IDEXX and J subgroups antibody assay kit/white diarrhea tablet
Experiment is reference, and self-built S. pullonum antibody is accorded with the positive of avian leukosis virus antibody ELISA co-detection method
Conjunction rate is 85.4%, negative match-rate 88.9%, illustrates that the ELISA method sensibility and specificity established reaches commercialization examination
The detection result of agent box achieves while detecting the effect of both antibody of avian leukosis virus and S. pullonum.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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Claims (10)
1. a kind of antigen combination, which is characterized in that including p27 albumen, gp85 albumen and GroEL- Δ 8-1 albumen.
2. antigen combination as described in claim 1, which is characterized in that p27 albumen, gp85 albumen and GroEL- Δ 8-1 albumen
Total concentration be (1~8) μ g/mL and mass ratio is (0.5~1):(0.5~1):1.
3. antigen combination as described in claim 1, which is characterized in that p27 albumen, gp85 albumen and GroEL- Δ 8-1 albumen
Total concentration be (1~4) μ g/mL and mass ratio is 1:1:(0.25~4).
4. antigen combination as described in any one of claims 1 to 3 is in the kit for preparing detection avian leukosis and/or white diarrhea
In application.
5. kit, which is characterized in that be coated with antigen combination as described in any one of claims 1 to 3.
6. kit as claimed in claim 5, which is characterized in that further include serum, primary antibody, ELIAS secondary antibody, coating buffer solution,
Dilution, developing solution and the terminate liquid of washing buffer, confining liquid, sample or secondary antibody.
7. the application method of kit as claimed in claim 6, which is characterized in that the dilution of the serum is 1:(100~
400)；The ELIAS secondary antibody dilution is 1：(4000~16000).
8. application method as claimed in claim 7, which is characterized in that the sealing condition of the confining liquid is 5% skimmed milk 37
DEG C closing 2h.
9. application method as claimed in claim 7 or 8, which is characterized in that the incubation conditions of the primary antibody are 37 DEG C and are incubated (45
~90) min；The incubation conditions of the ELIAS secondary antibody are 37 DEG C of incubation (45~90) min.
10. such as claim 7 to 9 any one of them application method, which is characterized in that the developing time of the developing solution is (5
~15) min.
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| CN201810575209.9A CN108776221B (en) | 2018-06-06 | 2018-06-06 | Kit for simultaneously detecting avian leukosis virus antibody and salmonella pullorum disease antibody |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113341140A (en) * | 2021-06-02 | 2021-09-03 | 贵州大学 | Indirect ELISA (enzyme-linked immunosorbent assay) method for detecting avian leukosis P27 |
| CN113519455A (en) * | 2021-08-02 | 2021-10-22 | 北京市华都峪口禽业有限责任公司 | Chicken white diarrhea field detection method |
| CN114532295A (en) * | 2022-03-19 | 2022-05-27 | 广西参皇养殖集团有限公司 | Method for jointly purifying leukemia and pullorum disease of high-quality chicken breeding core group |
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| CN114532295A (en) * | 2022-03-19 | 2022-05-27 | 广西参皇养殖集团有限公司 | Method for jointly purifying leukemia and pullorum disease of high-quality chicken breeding core group |
| CN114532295B (en) * | 2022-03-19 | 2024-04-16 | 广西参皇养殖集团有限公司 | Combined purification method for leukemia and pullorum disease of high-quality chicken breeding core group |
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