CN105785049A - Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof - Google Patents
Infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof Download PDFInfo
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Abstract
The invention relates to the field of immunological detection and aims at providing an infectious bronchitis virus (IBV) nsp5 (non-structural protein) ELISA (enzyme-linked immunosorbent assay) antibody detection kit and application thereof. The kit comprises a 96-hole ELISA plate coated with recombination expression IBV nsp5, an IgG enzyme-labeled antibody solution diluted with sample diluent, confining liquid, a scrubbing solution, the sample diluent, a positive control sample, a negative control sample, developing liquid and stop liquid. According to the kit, nsp5 is adopted as coating antigen, the kit can have the character of being high in coincidence rate with an import commercialization kit, and cost is greatly reduced. IBV nsp is taken as antigen to assemble the ELISA antibody detection kit for the first time in the industry. The kit has the advantages that using is convenient and quick, sensitivity is high and specificity is strong; a new effective detection means is clinically provided for IBV large-scale antibody monitoring, epidemiological investigation and IB prevention and control.
Description
Technical field
The invention belongs to immunological detection method field, be specifically related to infectious bronchitis virus nsp5 albumen be envelope antigen ELISA antibody assay kit and application.
Background technology
The infectious bronchitis (Infectiousbronchitis, IB) of chicken is that the chicken caused by infectious bronchitis virus (InfectiousbronchitisVirus, IBV) is acute, high degree in contact sexually transmitted disease.Dropping to principal character with dyspnea, nephritis and Egg Quality, this disease is widely current in the whole world, is endanger one of the most serious respiratory infectious disease in poultry husbandry.IBV genome is about as 27.4kb, encodes 4 kinds of structural protein (S, M, N, E), 15 kinds of non-structural proteins (nsp2-nsp16) and 3a, 3b, 5a and 5b4 accessory protein.Transcribe mechanism owing to IBVRNA is distinctive, add that RNase lacks correction system and causes that IBV is easy to morph, occur in that the tissue tropism of complexity, serotype and genotype.Constantly occurring of the variant that IBV is new and being continuously increased of new serotype, bring very big difficulty to the diagnosis of IB and preventing and treating.
Diagnosing IB, antibody horizontal in detection chicken group quickly and accurately, for understanding the popularity of IB in chicken group, adjusting immune programme for children in good time, effective anti-IB processed has vital effect.Elisa (ELISA) owing to it is simple, quickly, sensitive, economical and be suitable to the feature of large-scale use and be widely applied clinically.In IBV antibody test, the commercial kit only having the production of American I DEXX company is more credible, and the sampling observation and the research and development department that are mainly used in IBV antibody horizontal in domestic large-scale chicken farm develop the Comparability test with type new product.But due to import reagent costly, limit this test kit widely using at home.
Homology analysis shows, the gene order of coding IBV non-structural protein has higher stability relative to the gene order of structural protein, and the mutation probability relating to epitope is relatively low.So utilizing IBV non-structural protein as Detection of antigen IBV serum antibody, have different serotypes, or the strain of different tissues preferendum all has the potentiality of versatility.Additionally, due to non-structural protein is not involved in the composition of virion, therefore, also there are the potentiality of differentiation inactivated vaccine immunity and poison infection of living using non-structural protein as detection antigen simultaneously.Non structural protein 5 (nsp5) is a kind of proteolytic enzyme, and it is extremely important in the hydrolysis of polyprotein pp1a and pp1ab, is also designated as major protein enzyme Mpro.The forfeiture of its activity directly affects the duplication of virus, causes virus multiplication failure.In coronavirus genus, the 26S Proteasome Structure and Function of nsp5 is all conservative.Therefore, set up the ELISA antibody detection method based on IBV non-structural protein to have broad application prospects.
At present, not yet there is the non-structural protein that the utilizes IBV report as the Serology test of detection antigen.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, it is provided that a kind of for infectious bronchitis of chicken antibody assay kit, provides economical and practical reliable detection instrument for the diagnosis of clinical IB and the detection of antibody horizontal.
For solving above-mentioned technical problem, the solution of the present invention is:
Thering is provided a kind of avian infectious bronchitis virus nsp5ELISA antibody assay kit, this test kit includes:
(1) by the recombinant expressed coated 96 hole enzyme-linked reaction plates of IBV Non structural protein 5；
(2) with the IgG enzyme labelled antibody solution of sample diluting liquid dilution, described enzyme labelled antibody is the goat-anti chicken igg antibody of horseradish peroxidase (HRP) labelling；
(3) confining liquid: containing the PBS of 10% calf serum；
(4) cleaning mixture: the PBS (i.e. PBST) containing 0.05%Tween-20；
(5) sample diluting liquid: containing the PBST of 5% defatted milk；
(6) positive control sample: anti-IBVnsp5 chicken serum；
(7) negative control sample: negative SPF chicken serum；
(8) nitrite ion: A liquid is substrate solution, containing 0.1mg/ml tetramethyl benzidine (TMB) solution, B liquid is 0.05%H2O2, before using, A liquid and B liquid 1:1 are mixed；
(9) stop buffer: 2MH2SO4；
Described recombinant expressed IBV Non structural protein 5 is prepared by following method:
The gene of IBVnsp5, after RT-PCR method amplification obtains, is cloned in prokaryotic expression carrier pEGX-4T-1, converts escherichia coli；The recombinant bacterium of IBVnsp5 gene is carried in screening, great expression and the GST nsp5 recombiant protein merged under IPTG induces；Utilize GSTrapFF affinity chromatograph column purification, it is thus achieved that the IBVnsp5 recombiant protein after purification, be stored in-20 DEG C standby；
The nucleotide sequence of the gene of described IBVnsp5 is such as shown in SEQIDNO:1；The aminoacid sequence of the gene code of IBVnsp5 is such as shown in SEQIDNO:2.
Invention further provides the using method of aforementioned agents box, comprise the following steps:
(1) preparation of the coated 96 hole enzyme-linked reaction plates of IBVnsp5:
By the IBVnsp5 albumen of restructuring after BCA protein detection kit (BCAproteinAssaykit) (Novagen company of Germany produces) measures concentration, with the 0.01MPBS buffer that pH is 7.4 as being coated liquid, it is diluted to final concentration of 3.64 μ g/ml；Then join in 96 hole enzyme-linked reaction plates by the amount in 100 μ l/ holes, hatch 2h or 4 DEG C of overnight incubation in 37 DEG C, obtain enzyme-linked reaction plate；
(2) close: remove enzyme-linked reaction plate be coated liquid after, add the confining liquid in 200 μ l/ holes, hatch 1 hour, wash plate once with PBST for 37 DEG C；
(3) primary antibodie is hatched: adds 100 μ l/ hole diluents with the 1:200 serum to be checked (primary antibodie) diluted, arranges the positive and negative serum control simultaneously；Hatching 1 hour for 37 DEG C, PBST washes plate 5 times；
(4) two anti-hatch: it is anti-with the sheep anti-chicken IgG two of 1:1000 HRP labelling dilute to add 100 μ l/ hole diluents, hatches 45 minutes for 37 DEG C, and PBST washes plate 5 times；
(5) colour developing: colour developing A liquid and B liquid equal-volume add reacting holes with 100 μ l/ holes after mixing, and develop the color 10 minutes at dark place；
(6) terminate: add 50 μ l/ hole stop buffers；
(7) result judges:
Microplate reader measures OD450nm reading, calculates S/P value by following formula:
S/P value=(sample to be tested OD450nm average-negative control OD450nm average)/(Positive control wells OD450nm average-negative control OD450nm average)；
If the S/P value of blood serum sample is more than 0.12, it is judged to IBV antibody positive, otherwise is then judged to feminine gender.
Compared with prior art, the beneficial effects of the present invention is:
IBVELISA antibody assay kit provided by the invention, using non-structural protein nsp5 as envelope antigen, it is possible to provide have and the characteristic of import test kit height coincidence rate, and cost is substantially reduced.This is the ELISA antibody assay kit assembled as antigen with IBV non-structural protein first in the industry.
The use of this test kit has the advantages such as convenient and swift, sensitivity is high, high specificity, and the prevention and control for the extensive antibody detection of IBV clinically, Epidemiological study and IB provide a kind of new effective detection means.
Accompanying drawing explanation
The purification of Fig. 1 IBVGST-nsp5.
The interval scattergram of the natural logrithm value of the ELISAS/P value of Fig. 2 IFA positive serum (n=595) and negative serum (n=65).
Fig. 3 nsp5-ELISA and IDEXXELISA detects the postvaccinal antibody change curve of IBV.
Detailed description of the invention
The test kit composition utilized in the present invention includes:
(1) recombinant expressed IBV Non structural protein 5 (nsp5), and 96 hole enzyme-linked reaction plates；
(2) with the IgG enzyme labelled antibody solution of sample diluting liquid dilution.Described enzyme labelled antibody is the goat-anti chicken igg antibody of horseradish peroxidase (HRP) labelling；
(3) confining liquid: containing the PBS of 10% calf serum；
(4) cleaning mixture: the PBS containing 0.05%Tween-20, i.e. PBST；
(5) sample diluting liquid: containing the PBST of 5% defatted milk；
(6) positive control sample: anti-IBVnsp5 chicken serum；
(7) negative control sample: negative SPF chicken serum；
(8) nitrite ion: A liquid: i.e. substrate solution, containing 0.1mg/ml tetramethyl benzidine (TMB) solution；B liquid: 0.05%H2O2；Mix with front A liquid and B liquid 1:1.
(9) stop buffer: 2MH2SO4。
The using method of mentioned reagent box, comprises the following steps: successively
(1) preparation of ELISA antigen: utilize IBVnsp5 specific primer (IBVnsp5-F and IBVnsp5-R, primer sequence is such as shown in SEQIDNO:3 and SEQIDNO:4), with the nucleic acid of the chick embryo allantoic liquid extracting of IBVSC021202 virus strain infection for template, the gene of IBVnsp5 is obtained through RT-PCR method amplification, it is cloned in prokaryotic expression carrier pEGX-4T-1, convert escherichia coli, the recombinant bacterium of IBVnsp5 gene is carried in screening, great expression and the GST nsp5 recombiant protein merged under IPTG induces, utilize GSTrapFF affinity chromatograph column purification, obtain highly purified IBVnsp5 recombiant protein be stored in-20 DEG C standby；The nucleotide sequence of the gene of described IBVnsp5 is such as shown in SEQIDNO:1；The aminoacid sequence of the gene code of IBVnsp5 is such as shown in SEQIDNO:2.
(2) preparation of the coated enzyme-linked reaction plate of IBVnsp5:
The recombiant protein GST-nsp15 of preparation measures after concentration through BCA protein detection kit, by the amount being coated liquid and being diluted to 3.64 μ g/ml, join in enzyme-linked reaction plate by 100 μ l/ holes, hatch 2h or 4 DEG C of overnight incubation in 37 DEG C, namely prepare enzyme-linked reaction plate；
(3) closing: the above-mentioned enzyme-linked reaction plate prepared, remove after being coated liquid, PBST washes plate 5 times, adds the confining liquid in 200 μ l/ holes, hatches 1 hour, washes plate once with PBST for 37 DEG C；
(4) primary antibodie is hatched: adds 100 μ l/ hole diluents with the 1:200 serum to be checked (primary antibodie) diluted, arranges the positive and negative serum control simultaneously.Hatching 1 hour for 37 DEG C, PBST washes plate 5 times；
(5) two anti-hatch: it is anti-with the sheep anti-chicken IgG two of 1:1000 HRP labelling dilute to add 100 μ l/ hole diluents, hatches 45 minutes for 37 DEG C, and PBST washes plate 5 times；
(6) colour developing: colour developing A liquid and B liquid equal-volume add reacting holes with 100 μ l/ holes after mixing, and develop the color 10 minutes at dark place；
(7) terminate: add 50 μ l/ hole stop buffers.
(8) result judges: microplate reader measures OD450nm reading.Calculate S/P value.The computing formula of S/P value is: S/P value=(sample to be tested OD450nm average-negative control OD450nm average)/(Positive control wells OD450nm average-negative control OD450nm average).The S/P value of blood serum sample is judged to IBV antibody positive more than 0.12, otherwise is then judged to feminine gender.
It is described further below in conjunction with embodiment and accompanying drawing.Should be appreciated that these embodiments are for illustration purposes only, rather than restriction the scope of the present invention.
The preparation of [embodiment 1] antigen
Activate carrying expressive host bacterium E.coliBL21 (DE3) of pGEX-4T-1-nsp5 carrier through LB fluid medium, transfer mass propgation expanding propagation in the LB fluid medium containing amicillin resistance by 1:100.OD when bacterium solution600nmAfter adding the IPTG abduction delivering 5h of final concentration of 1mM when reaching 0.4-0.6,4 DEG C, 6,000rpm centrifugal 10min collection thalline, use GSTrapFF affinity column, according to product manual purification destination protein.The albumen sampling obtained after eluting carries out SDS-PAGE analysis.As it is shown in figure 1, the recombiant protein GST-nsp5 molecular size range of purification is 60KD, there is significantly high purity.The GST-nsp5 of purification be saved in-20 DEG C standby.
The preparation of [embodiment 2] yin and yang attribute serum and screening
Used by the present invention, yin and yang attribute serum is provided by Zhejiang Tongdian Biological Technology Co., Ltd..Below serum preparation process is illustrated, but the realization being not meant to the present invention must be set about from preparing serum, does not also mean that method prepared by serum is confined to described content.
(1) preparation of IBVnsp5 yin and yang attribute serum
The positive serum that this test kit uses can pass through restructuring GST-nsp5 albumen embodiment 1 being purified to, by after 1mg/ dose immunization SPF chicken only three times, collect and separating immune chicken serum, indirect immunofluorescence assay (IFA) is utilized to verify in serum containing after the anti-nsp5 antibody of high-titer, it is sub-packed in-70 DEG C of preservations, as IBVnsp5 positive serum.Not immunity SPF chicken serum, after being verified as IBVnsp5 negative antibody with IFA, subpackage-70 DEG C preservation, it is IBVnsp5 negative serum.
(2) screening of clinical serum
Positive and the negative serum sample with the clinical IBV of IFA method screening.Specific practice: the DF-1 cell inoculation IBVSC021202 strain of 80-90% monolayer will be covered with in 96 porocyte plates, and set and do not inoculate IBV hole as comparison.37 DEG C, 5%CO2 cultivate after 24-36h, discard culture fluid, add fixative (methanol: acetone=1:1) with the amount in 100 μ l/ holes, places after-20 DEG C of refrigerators fix 20min, get rid of fixative and dry standby in fume hood.In 96 orifice plates fixed, add confining liquid (PBS solution containing 0.05%Tween-20 and 5% defatted milk) with the amount in 200 μ l/ holes, 37 DEG C of calorstats are closed 1h.With confining liquid, 660 parts of clinical serum samples are pressed 1:20 and 1:100 dilution respectively, join (repetition of two, each sample) in 96 orifice plates closed, 37 DEG C of calorstats hatch 1h.Wash the sheep anti-chicken IgG (1:400) adding FITC labelling after 5 times with PBST, 37 DEG C of calorstats act on the positive and negative result observing and recording each part serum after 45min, PBST wash 5 times under inverted fluorescence microscope.The person that has specificity fluorescent when diluting with 1:100 is for the positive；Person is feminine gender not to have specificity fluorescent during 1:20 dilution；And there is no specificity fluorescent during 1:100 dilution, person is the weak positive to have specificity fluorescent during 1:20 dilution.As a result in 660 parts of clinical serum 595 parts be that IBV is positive, 65 parts be IBV feminine gender.
The optimization of [embodiment 3] ELISA testing conditions
The base program of 1.ELISA reaction
(1) be coated: with the Tris-HCl buffer of pH8.0 by antigen diluent to working concentration, by 100 μ l/ hole coated elisa plates, 37 DEG C be coated 2h or 4 DEG C be coated overnight, PBST washes plate 5 times；
(2) close: add confining liquid with the amount in 200 μ l/ holes, close 2h, PBST for 37 DEG C and wash plate 5 times；
(3) the hatching of primary antibodie: add the serum to be checked of dilution with the amount in 100 μ l/ holes, 37 DEG C of calorstats are hatched 1h, PBST and are washed plate 5 times；
(4) two anti-hatching: adding the sheep anti-chicken IgG of the HRP labelling diluted with the amount in 100 μ l/ holes, hatch 1h (time before optimization) in 37 DEG C of calorstats, PBST washes plate 5 times；
(5) colour developing: add tmb substrate with the amount in 100 μ l/ holes, lucifuge colour developing 10min in 37 DEG C of calorstats；
(6) terminate: add 2MH2SO4 stop buffer with the amount in 50 μ l/ holes and terminate reaction；
(7) reading: use microplate reader to measure each hole at OD450nm place light absorption value.
(8) judged result.Calculating S/P value, formula is S/P value=(sample to be tested OD450nm average-negative control OD450nm average)/(Positive control wells OD450nm average-negative control OD450nm average).By S/P value and the threshold comparison finally determined, it is determined that result.
The optimization of 2.ELISA reaction condition
Based on above-mentioned base program, the reaction condition of nsp5ELISA is optimized.
(1) selection of antigen coated concentration and serum diluting multiple
Adopting square formation method to optimize on 96 hole enzyme-linked reaction plates, horizontally-arranged 1:500,1:1000,1:2000, the 1:4000 of being made by antigen respectively dilutes；The positive and negative serum are done 1:25,1:50,1:100,1:200,1:400,1:800,1:1600 dilution, each dilution factor two repetition by tandem respectively.Measure OD450nm value after ELISA reaction, choose P/N value (positive serum OD450nm meansigma methods/negative serum OD450nm meansigma methods) and be the final condition of optimization to the maximum.By analysis, when antigen coated concentration is 3.64 μ g/ml, when serum diluting multiple to be checked is 1:200, P/N value is maximum.
(2) selection of enzyme labelled antibody working concentration
In the antigen coated concentration of the best determined and serum diluting multiple basis, ELIAS secondary antibody being done 1:1000,1:2000,1:4000,1:8000 dilution respectively, carries out ELISA detection, the enzyme labelled antibody extension rate selecting P/N value maximum is best effort concentration.When enzyme labelled antibody extension rate is that 1:1000, P/N value is maximum.
(3) selection of liquid it is coated
Respectively singly to steam water (H2O, pH5.0), the normal saline (NS of 0.9%NaCl, pH7.0), 0.01M phosphate buffer (PBS, pH7.4), 0.05MTris-HCl buffer (TB, pH8.5), 0.05M carbonate buffer solution (CB, pH9.6) and 0.1MNaOH solution (NaOH, pH13) are as being coated liquid, carrying out ELISA detection, that selectes the maximum correspondence of P/N value is coated liquid.When the PBS of 0.01MpH7.4 is for being coated liquid, P/N value is maximum.
(4) selection of confining liquid
Make confining liquid with 2% defatted milk, 5% defatted milk, 0.1%BSA, 0.5%BSA, 1%BSA, 5% calf serum and 10% calf serum respectively, carry out ELISA detection, the confining liquid of the selected maximum correspondence of P/N value.When 10% new-born calf serum is confining liquid, P/N value is maximum.
(5) selection of primary antibodie diluent
Respectively with PBS, the PBS (PBST1) of 0.05%Tween-20, the PBS (PBST2) of 0.1%Tween-20, the PBS of 0.1%BSA, the PBS (BSAT) of 0.1%BSA, 0.05%Tween-20, PBS and 5% defatted milk (SM) of 5% defatted milk, 0.05%Tween-20 PBS (SMT) make primary antibodie diluent, carry out ELISA detection, the primary antibodie diluent of the selected maximum correspondence of P/N value.When 5% defatted milk containing 0.05% tween 20 is sample diluting liquid, P/N value is maximum.
(6) selection in primary antibodie response time
After adding the primary antibodie of dilution, hatch 8min, 15min, 30min, 45min, 60min, 90min at 37 DEG C respectively, carry out ELISA detection, the primary antibodie response time of the selected maximum correspondence of P/N value.When the seroreaction time to be checked is 60min, P/N value is maximum.
(7) selection in ELIAS secondary antibody response time
After adding the enzyme labelled antibody of dilution, hatch 8min, 15min, 30min, 45min, 60min, 90min at 37 DEG C respectively, carry out ELISA detection, the enzyme labelled antibody response time of the selected maximum correspondence of P/N value.When the ELIAS secondary antibody response time is 45min (45min is the time after optimizing), P/N value is maximum.
(8) selection in tmb substrate response time
Hatching 3min, 5min, 8min, 10min, 15min, 20min, 25min at 37 DEG C respectively after A and the B nitrite ion of the volume ratio mixing of addition 1:1, the 2MH2SO4 adding 50 μ l/ holes afterwards terminates, the tmb substrate response time of the selected maximum correspondence of P/N value.As substrate reactions time 10min, P/N value is maximum.
Therefore, the optimum reaction condition finally determined is: the optimization of reaction condition, it is determined that optimum reaction condition: antigen coated concentration is 3.64 μ g/ml, and serum diluting multiple to be checked is 1:200, and enzyme labelled antibody extension rate is 1:1000；The PBS of 0.01MpH7.4 is for being coated liquid；10% new-born calf serum is confining liquid；5% defatted milk containing 0.05% tween 20 is sample diluting liquid；Serum to be checked and ELIAS secondary antibody response time respectively 60min and 45min, substrate reactions time 10min.
[embodiment 4] ELISA detects the determination of marginal value
By the ELISA reaction system optimized, the 660 parts of clinical samples detected through IFA are detected, calculate the S/P value of each part serum respectively, and utilize software GraphPadPrism2.0 that result is analyzed, find the critical point (S/P value) that the positive and negative testing result of ELISA testing result and IFA at utmost meets, in this, as the Critical Standard that ELISA method positive and negative judge.
The ELISA testing result of 660 parts of blood serum samples and IFA testing result are as shown in Figure 2.IFA detection is that the nsp5ELISAS/P value of IBV positive serum (595 parts) is between 0.0696 2.731；IFA detection is that the nsp5ELISAS/P value of IBV negative serum (65 parts) is between-0.386 0.131.When take S/P value equal to 0.10 as the marginal value of positive and negative serum time, now ELISA method is relative to the positive coincidence rate of IFA, negative match-rate and total coincidence rate respectively 96.6%, 76.9% and 94.7%.When take S/P value equal to 0.11 as the marginal value of positive and negative serum time, now ELISA method is relative to the positive coincidence rate of IFA, negative match-rate and total coincidence rate respectively 94.8%, 84.6% and 93.8%.When take S/P value equal to 0.12 as the marginal value of positive and negative serum time, now ELISA method is relative to the positive coincidence rate of IFA, negative match-rate and total coincidence rate respectively 93.1%, 95.4% and 93.3% (table 1).Consider both goodness of fit, take 0.12 the most desirable as the marginal value of positive and negative serum.Namely when a certain blood serum sample is judged to IBV antibody positive through the IBVnsp5ELISA S/P value detected more than 0.12, otherwise feminine gender then it is judged to.
Under the S/P value that table 1 is different, nsp5-ELISA and IFA detects coincidence rate and compares
Note: the total number of samples of detection 660 parts, wherein positive 595 parts of IFA, negative 65 parts of IFA.
[embodiment 5] replica test
(1) replica test in criticizing
10 parts of positive serums and 5 parts of negative serums are detected respectively by the coated ELISA Plate of GST-nsp5 albumen of same Batch purification, repeated trials 3 times, calculate the meansigma methods (X) of the S/P value of each sample, standard deviation (SD) and the coefficient of variation (CV) respectively, it is determined that nsp5ELISA is with the coefficient of variation scope of batch antigen.S/P value coefficient of variation circle (table 2) between 3%-16% with the 10 parts of serum and 5 parts of negative serums of criticizing Detection of antigen, it was shown that batch interior repeatability of antigen is better.
Table 2 is with a collection of antigen coated microplate variation within batch coefficient
(2) criticize between replica test
The GST-nsp5 albumen coated elisa plate purified with different batches, above-mentioned 10 parts of positive serums and 5 parts of negative serums are carried out 3 repeated trials, calculate the meansigma methods (X) of the S/P value of each sample, standard deviation (SD) and the coefficient of variation (CV) respectively, it is determined that the coefficient of variation scope of nsp5ELISA different batches antigen.10 parts of serum of different batches Detection of antigen and S/P value coefficient of variation circle of 5 parts of negative serums (table 3) between 7%-19%, it was shown that antigen batch between reproducible.
The different batch antigen coated microplate interassay coefficient of variation of table 3
The specificity of [embodiment 6] nsp5ELISA method
Respectively by newcastle disease virus (NDV) positive serum, bird flu virus (AIV) positive serum and infectious bursa of Fabricius virus (IBDV) positive serum do the best dilution, carry out ELISA detection, set up IBV positive and negative serum control simultaneously, determine the specificity of IBVELISA with this.Testing result shows that the S/P value of above-mentioned serum is respectively less than 0.12, and result is feminine gender, it was shown that recombinant antigen and above-mentioned serum are absent from cross reaction, have good specificity.
[embodiment 7] nsp5ELISA test kit validation verification
(1) the detection checking of clinical serum
Take 126 parts of clinical serum samples, respectively with nsp5ELISA method and import IBVELISA test kit (production of American I DEXX company) Parallel testing, compare nsp5ELISA and the sensitivity of import ELISA kit, specificity and coincidence rate.Import IBVELISA kit test method carries out with reference to its description.It is shown that the positive rate of nsp5ELISA is 83.33% (105/126), negative recall rate 16.67% (21/126)；And the positive rate of commercialization import ELISA kit (IDEXX company) is 84.13% (106/126), negative recall rate is 15.87% (20/126).Relative to IDEXXELISA test kit, the sensitivity of nsp5ELISA is 98.11% (104/106), and specificity is 95% (19/20), and total coincidence rate is 97.62% [(104+19)/126] (table 4).Data above shows, the nsp5ELISA that the present invention sets up has good specificity and sensitivity.And relative to the virion of the purification IDEXX test kit as envelope antigen, the nsp5ELISA that the present invention the sets up albumen that to be used in escherichia coli recombinant expressed is more economical and practical as envelope antigen.
Table 4nsp5-ELISA and the importer comparison of IBV test kit
(2) detection of artificial infection chicken's Antibody dynamics change
In order to assess the effectiveness that the nsp5ELISA method set up changes for antibody positive after detecting IBV inoculation, carry out the experiment of artificial vaccination SPF chicken: IBVH52 Attenuate vaccine recommended doses to specifications is inoculated 1 monthly age SPF chicken, and separated in time gathers serum.The nsp5-ELISA method that the serum gathered is set up detects antibody, makes Parallel testing with import IDEXX company test kit simultaneously.And draw the antibody variation curve of immunity chicken.Testing result is as it is shown on figure 3, H52 Attenuate vaccine group inoculation SPF chickling starts to occur IBVnsp5 antibody for 15 days, and blood serum sample S/P meansigma methods exceedes marginal value 0.12, rising of hereafter fluctuating gradually.After inoculating about 19 weeks, nsp5 antibody is turned out cloudy；The IBV antibody variation tendency that IDEXX company ELISA kit detects consistent (Fig. 3).Show that IBVnsp5ELISA detection method can embody real antibody change after IBV inoculates.The nsp5ELISA detection method that the present invention sets up can be used in the Serologic detection of IBV.
Claims (2)
1. avian infectious bronchitis virus nsp5ELISA antibody assay kit, it is characterised in that this test kit includes:
(1) by the recombinant expressed coated 96 hole enzyme-linked reaction plates of IBV Non structural protein 5；
(2) with the IgG enzyme labelled antibody solution of sample diluting liquid dilution, described enzyme labelled antibody is the goat-anti chicken igg antibody of horseradish peroxidase-labeled；
(3) confining liquid: containing the PBS of 10% calf serum；
(4) cleaning mixture: the PBS containing 0.05%Tween-20；
(5) sample diluting liquid: containing the PBST of 5% defatted milk；
(6) positive control sample: anti-IBVnsp5 chicken serum；
(7) negative control sample: negative SPF chicken serum；
(8) nitrite ion: A liquid is substrate solution, containing 0.1mg/ml tetramethyl biphenyl amine aqueous solution, B liquid is 0.05%H2O2, before using, A liquid and B liquid 1:1 are mixed；
(9) stop buffer: 2MH2SO4；
Described recombinant expressed IBV Non structural protein 5 is prepared by following method:
The gene of IBVnsp5, after RT-PCR method amplification obtains, is cloned in prokaryotic expression carrier pEGX-4T-1, converts escherichia coli；The recombinant bacterium of IBVnsp5 gene is carried in screening, great expression and the GST nsp5 recombiant protein merged under IPTG induces；Utilize GSTrapFF affinity chromatograph column purification, it is thus achieved that the IBVnsp5 recombiant protein after purification, be stored in-20 DEG C standby；
The nucleotide sequence of the gene of described IBVnsp5 is such as shown in SEQIDNO:1；The aminoacid sequence of the gene code of IBVnsp5 is such as shown in SEQIDNO:2.
2. the using method of test kit described in claim 1, it is characterised in that comprise the following steps:
(1) preparation of the coated 96 hole enzyme-linked reaction plates of IBVnsp5:
The IBVnsp5 albumen of restructuring is measured after concentration through BCA protein detection kit, with the 0.01MPBS buffer that pH is 7.4 as being coated liquid, is diluted to final concentration of 3.64 μ g/ml；Then join in 96 hole enzyme-linked reaction plates by the amount in 100 μ l/ holes, hatch 2h or 4 DEG C of overnight incubation in 37 DEG C, obtain enzyme-linked reaction plate；
(2) close: remove enzyme-linked reaction plate be coated liquid after, add the confining liquid in 200 μ l/ holes, hatch 1 hour, wash plate once with PBST for 37 DEG C；
(3) primary antibodie is hatched: adds 100 μ l/ hole diluents with the 1:200 serum to be checked diluted, arranges the positive and negative serum control simultaneously；Hatching 1 hour for 37 DEG C, PBST washes plate 5 times；
(4) two anti-hatch: it is anti-with the sheep anti-chicken IgG two of 1:1000 HRP labelling dilute to add 100 μ l/ hole diluents, hatches 45 minutes for 37 DEG C, and PBST washes plate 5 times；
(5) colour developing: colour developing A liquid and B liquid equal-volume add reacting holes with 100 μ l/ holes after mixing, and develop the color 10 minutes at dark place；
(6) terminate: add 50 μ l/ hole stop buffers；
(7) result judges:
Microplate reader measures OD450nm reading, calculates S/P value by following formula:
S/P value=(sample to be tested OD450nm average-negative control OD450nm average)/(Positive control wells OD450nm average-negative control OD450nm average)；
If the S/P value of blood serum sample is more than 0.12, it is judged to IBV antibody positive, otherwise is then judged to feminine gender.
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| CN108680741A (en) * | 2018-03-28 | 2018-10-19 | 华南农业大学 | Avian infectious bronchitis virus 5b ELISA antibody assay kits and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107727854A (en) * | 2017-09-30 | 2018-02-23 | 南京农业大学 | A kind of avian infectious bronchitis virus protein chip antibody assay kit and its application |
| CN108680741A (en) * | 2018-03-28 | 2018-10-19 | 华南农业大学 | Avian infectious bronchitis virus 5b ELISA antibody assay kits and its application |
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