CN105759046A - Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof - Google Patents
Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof Download PDFInfo
- Publication number
- CN105759046A CN105759046A CN201610218513.9A CN201610218513A CN105759046A CN 105759046 A CN105759046 A CN 105759046A CN 201610218513 A CN201610218513 A CN 201610218513A CN 105759046 A CN105759046 A CN 105759046A
- Authority
- CN
- China
- Prior art keywords
- encephalitis virus
- antibody
- avian pneumo
- coated
- microsphere
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/125—Newcastle disease virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and a detection method thereof. The detection kit comprises a receptor microballoon coated with a Newcastle disease virus antibody, a biotin labeled Newcastle disease virus antibody and a donor microballoon coated with streptavidin. The detection method comprises the following steps of: successively adding a standard substance or a to-be-detected sample, the receptor microballoon coated with the Newcastle disease virus antibody and the biotin labeled Newcastle disease virus antibody into a porous plate and performing vibration incubation; adding the donor microballoon coated with streptavidin under a light shielding condition and then performing vibration incubation; and detecting a signal value on a AlphaScreen/Lisa detector. The detection kit provided by the invention has high stability and strong specificity. The influence of outside environment on the detection method is small. The detection method has higher sensitivity and specificity, can meet the requirements of high throughput and quick detection and has high popularization and application value.
Description
Technical field
The invention belongs to agricultural biological technical field, relate to veterinary, immunology and biological product, be specifically related to a kind of Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit and detection method thereof.
Background technology
Newcastle (Newcastledisease, ND) is the great epidemic disease of one of harm birds, is classified as A class infectious disease by World Organization for Animal Health.In " national medium-term and long-term animal epidemic control program (2,012 2020) ", one of newcastle great animal epidemic being listed in preferential prevention and control.Some clinical symptoms of this disease are comparatively similar to bird flu, sometimes cause mistaken diagnosis to cause immeasurable economic loss, thus this disease quickly, accurately detected and is particularly important.
At present, diagnosis or the detection of Avian pneumo-encephalitis virus are specifically included that blood clotting suppresses (HI) testing inspection antihemagglutinin (HA) antibody by prior art；Enzyme-linked immunosorbent assay (ELISA) method detects the total specific antibody of Avian pneumo-encephalitis virus or antigen.Although but wherein HI test is relatively sensitive, but being affected relatively big by extraneous factor and weaken its practical value；ELISA detects the positive serum time early, and sensitivity is high, is more suitable for high-volume serosurvey, but because its condition requires height, operation inconvenience, failing to play it in actual applications should have effect compared with testing with HI.And there is false positive (non-specific fluorescence) problem in immunofluorescence technique (IFT).Gene amplification (PCR) method is utilized can directly to detect the gene of virus, newcastle testing laboratory of animal test institute of the Ministry of Agriculture initially sets up One step RT-PCR at home and quickly detects strong poison newcastle, the detection time of newcastle is shortened within 12 hours, but required apparatus expensive, is limited by very large in Clinical detection.CN1827775A (number of patent application the is 200510008997.6) " nucleotide sequences of detection Newcastle virus, test kit and the method for inspection ", this invention selects F gene of NDV strain (synthesis F protein) as target region, it is selected to reflection cracking site characteristic and lacks the multipair primer of conserved regions design and the probe of secondary structure, set up fluorescent PCR detection Avian pneumo-encephalitis virus, more highly sensitive than traditional PCR method 100-1000 times of the method, but need 4 hours from sample treatment to going out result, the enforcement of the method also needs the expensive instruments such as quantitative real time PCR Instrument, should not promote in basic unit.As fully visible, all there is certain defect and weak point in the method for prior art detection Avian pneumo-encephalitis virus, how to study the diagnostic reagent of a kind of quick detection Avian pneumo-encephalitis virus, in order to epidemic situation is carried out on-the-spot monitoring in time, has been urgent problem in current husbandry sector.
Summary of the invention
For prior art above shortcomings, the technical problem to be solved is: how to provide a kind of Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit and detection method thereof, make its by outside environmental elements affect little, do not have a false positive issue, and have easy to operate, detection quickly, specificity is good, highly sensitive, testing cost is low feature.
In order to solve above-mentioned technical problem, the present invention adopts the following technical scheme that and realizes: a kind of Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit, including being coated with the receptor microsphere of Avian pneumo-encephalitis virus antibody, biotin labeled Avian pneumo-encephalitis virus antibody and being coated with the donor microsphere of Streptavidin；Described receptor microsphere is the Nano microsphere being coated with luminophor and lanthanide series, and described donor microsphere is the Nano microsphere being coated with photosensitizer and Streptavidin；Described lanthanide series can be europium.
Adopt the method that above-mentioned Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit carries out detecting, being sequentially added into standard substance or detected sample, the receptor microsphere being coated with Avian pneumo-encephalitis virus antibody and biotin labeled Avian pneumo-encephalitis virus antibody in microwell plate, 15～20min is hatched in 37 DEG C of vibrations；Then adding when lucifuge and be coated with the donor microsphere of Streptavidin, detected signal value on AlphaScreen/Lisa detector is hatched after 15～20min in 37 DEG C of vibrations；Wherein, described standard substance or detected sample, the addition volume ratio that is coated with the receptor microsphere of Avian pneumo-encephalitis virus antibody, biotin labeled Avian pneumo-encephalitis virus antibody and the donor microsphere that is coated with Streptavidin are 1:1:1:7.
Compared to existing technology, there is advantages that
1, Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit of the present invention is prepared for Avian pneumo-encephalitis virus hemagglutinin monoclonal antibody, and with wherein two strains pairing antibody, 1 strain antibody is coated Acceptor beads, another 1 strain antibody is first with biotin labeling, AlphaLISA reaction system is collectively constituted again with the donor microballon of Streptavidin, the positive hybridoma cell good stability that the present invention obtains after testing, successive transfer culture 6 months or still have good titer after frozen 8 months, antibody and Avian pneumo-encephalitis virus display kickback, and be all negative reaction with other viruses, specificity is good, good stability is detected for test kit of the present invention, high specificity, highly sensitive lay a good foundation.
2, Avian pneumo-encephalitis virus has first been carried out purification before preparing monoclonal antibody by the present invention, Avian pneumo-encephalitis virus after verification experimental verification purification has good titer, high without substantially other impurity, purity, and then lay a good foundation for the follow-up monoclonal antibody preparing high specificity, make detection kit of the present invention to have and better detect reliability, do not have false positive issue.
3, detection method is easy and simple to handle, detection is quick, and in the inventive method analysis, the coefficient of variation is 3.48%～5.62% after testing, and between analysis, the coefficient of variation is 5.37%～7.29%, and precision is good；In detection only that Avian pneumo-encephalitis virus detected value is substantially higher, result is judged to the positive, and other pathogen detection value is relatively low, be feminine gender by criterion, and specificity is higher；After detection, room temperature places 7d, and each batch of reagent signal value change is little, and linear relationship is good, and reference standard product signal value has no significantly raised, has good stability；Through mass detection it have been experienced that the sensitivity of detection is 96.5%, specificity 97.4%, result shows that the AlphaLISA detection method set up is affected little by outside environmental elements, there is higher sensitivity and specificity, high flux can be met, the requirement of quickly detection, there is good application value.
4, the inventive method is relative to art methods, detection method is simple, the detection time is short, quick testing goal can be realized, without using expensive detecting instrument, testing cost low, and detection sensitivity is high, high specificity, not havinging false positive test results, detection reliability is higher, has more market prospect.
Accompanying drawing explanation
Fig. 1 is protein quantification standard curve；
Fig. 2 is 10%SDS-PAGE electroresis appraisal result figure；
Fig. 3 is the 14th day cell picture (10 × 10) of cell fusion；
Fig. 4 is test kit dose-response standard curve of the present invention.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention being described in further detail, donor microsphere described in following embodiment is Perkinelmer Products, article No. AL105C；Described receptor microsphere is Perkinelmer Products, article No. 6760002s；Described SUPERDEX200 gel filtration filler is GEHealthcare Products, article No. 17-5175-01；Described 10 × AlphaLISAassayBuffer is purchased from Perkinelmer company, article No. AL000F；Other material if no special instructions, is common commercially available prod.
The purification of embodiment 1 Avian pneumo-encephalitis virus antigen
By Avian pneumo-encephalitis virus AV29 standard strain (NDV-AV29) (purchased from China Veterinery Drug Inspection Office), with the PBS (pH7.4 of sterilizing, 0.01M) 1:50 dilution by volume, allantoic cavity inoculation 9-10 age in days SPF Embryo Gallus domesticus, chick embryo allantoic liquid dead after results 24h.Collecting NDV allantoic fluid 1250mL altogether, prepare 1% (volumetric concentration) chicken red blood cell, the viral allantoic fluid hemagglutination titer recording collection reaches 1:128-1:256.
Successively adopting differential centrifugation, gel filtration to be purified the viral allantoic fluid of results, BCA method measures virus protein concentration, SDS-PAGE electroresis appraisal purity, and hemagglutination test measures viral hemoagglutination titer.
1, differential centrifugation purification NDV
By the NDV allantoic fluid multigelation 3 times of above-mentioned collection, at prior to 4 DEG C, the centrifugal 15min of 5000rpm/min goes precipitation, after at 4 DEG C the centrifugal 15min of 10000rpm/min go precipitation；Collect allantoic fluid supernatant centrifugal 2h under 100000g, most of virion can be made to precipitate, abandoning supernatant, with a small amount of PBS (0.01M, pH7.4) precipitation in centrifuge tube washed out and dissolve, each pipe virus is merged, being sub-packed in little centrifuge tube ,-20 DEG C save backup.
2, coagulation is filtered and is further purified
Select the chromatographic column of SUPERDEX200 gel filtration, process through dress post, sample upper prop and elution process, virus after ultracentrifugation is further purified, remove other foreign proteins wherein contained, selection is purified, collect first eluting peak, adopt PEG6000 embedding to be concentrated into the 1/10 of original volume.
3, the qualification of NDV
By above-mentioned steps 2) NDV of purification BCA method measures virus protein concentration, SDS-PAGE electroresis appraisal purity, and hemagglutination test measures viral hemoagglutination titer.It is 1.26mg/mL that BCA method records NDV protein concentration, and protein quantification normal data is in Table 1, Fig. 1；There is virus band clearly in the sample of SDS-PAGE electrophoresis showed purification, without substantially other impurity bands (in Fig. 2, Fig. 2,1 swimming lane is Mark, and 2 swimming lanes are the NDV purified, and 3 swimming lanes are allantoic fluid stock solution)；The sample virus hemagglutination titer of purification reaches 1:2048.
The A655 absorbance of table 1 variable concentrations standard protein BSA
The preparation of embodiment 2 anti-new castle disease virus hemagglutinin monoclonal antibody
1, the foundation of mouse-anti Avian pneumo-encephalitis virus hemagglutinin monoclonal antibody hybridoma cell strain
By the NDV immunity BALB/c mouse of above-mentioned purification, taking the splenocyte after 4 immunity and carry out cell fusion with SP2/0 myeloma cell with PEG-1500, first time fusion rate is 86.5%, and second time fusion rate is 90.1%.Set up indirect ELISA, purification NDV, blank allantoic fluid coated elisa plate simultaneously is carried out ELISA screening, blank allantoic fluid be antigen selection to positive hole be false positive hole, should get rid of, monoclonal antibody in detection hybridoma supernatant, initial survey positive rate respectively 12.3% and 17.9%.Selecting 42 hole sub-clones that positive value is higher in positive hole purification ndv antigen screened, through 2 sub-clones, continuous repeated screening obtains 5 strains hybridoma cell strain compared with stably excreting antibody, called after 1G7,2A5,2F3,3E12,4E11.The selection result of cell fusion is such as shown in table 2, Fig. 3.Hemagglutination inhibition test confirms that the monoclonal antibody obtained is all for NDV hemagglutinin；The ELISA that is at war with by monoclonal antibody test obtains hybridoma 2A5,4E11 of 2 strain secretion pairing antibody, lumbar injection BALB/C mice respectively, and adopts caprylic acid-ammonium to carry out the purification of antibody, and the antibody of preparation is respectively designated as monoclonal antibody 2A5, monoclonal antibody 4E11.
Table 2 cell fusion and positive rate catalog
2, positive hybridoma cell stability test
2A5 and the 4E11 cell strain successive transfer culture more than 6 months that the present embodiment step 1 purification obtains, its culture supernatant ELISA titer remains at 1:1280-2560, difference frozen 2,4,6, recovering after 8 months, its Mabs titer inducing mouse ascites remains between 1:64000-128000, illustrates to have good stability.
3, the specificity analyses of monoclonal antibody
Select Avian pneumo-encephalitis virus (NDV) respectively, bird flu virus (AIV-H9), infectious bronchitis virus (IBV), bursal disease virus (IBDV) coated elisa plate, adopt indirect elisa method to identify the specificity of monoclonal antibody 2A5 and 4E11.Result is such as shown in table 3, table 4, visible monoclonal antibody 2A5 and 4E11 and the NDV of table 3 and table 4 show kickback, and is all negative reaction with other viruses, illustrates that specificity is good.
The reactivity of table 32A5 cell conditioned medium dilution factor and other virus
The reactivity of table 44E11 cell conditioned medium dilution factor and other virus
4, the hypotype of monoclonal antibody is identified
Using monoclonal antibody hypotype identification kit (Sigma company, article No. ISO2-1KT) that 2A5 and 4E11 antibody is carried out hypotype qualification, result shows that two strain antibodies are IgG1 type (table 5).
Table 5 hypotype qualification result
Embodiment 3 Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit and detection method thereof
1, the preparation of reference standard product
Dilution ndv antigen to concentration respectively 0,0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL, diluent be containing 50mmol/LTris HCl, mass concentration 1.5%BSA, mass concentration 0.9%NaCl, mass concentration 0.05%NaN3, mass concentration 0.01%Tween-20, pH7.8 reaction buffer.
2, the foundation of AlphaELISA system
nullThe mouse-anti NDV hemagglutinin monoclonal antibody 2A5 of preparation is coated receptor microsphere，10 × AlphaLISAassayBuffer ultra-pure water (is diluted to 1 × AlphaLISAassayBuffer with 1xAphaLISA buffer by the receptor microsphere having connected antibody 2A5，Lower same) dilute by 1:200 volume ratio，The mouse-anti NDV hemagglutinin monoclonal antibody 4E11 of preparation is used for biotin labeling，The dilution of 1:400 volume ratio pressed by biotinylated antibody 4E11 and 1xAphaLISA buffer，The donor microsphere of streptavidin is diluted to final concentration of 40 μ g/mL with 1 × AlphaLISAassayBuffer，Receptor microsphere、The donor microsphere of biotinylated antibody and streptavidin collectively constitutes NDV in detectable examination criteria product or detected sample，Detected signal value on AlphaScreen/Lisa detector.Microwell plate is separately added into standard substance or detected sample 25 μ L, connects the receptor microsphere 25 μ L of antibody, biotinylated antibody 25 μ L, 15-20min is hatched in 37 DEG C of vibrations, then adding the donor microsphere l75 μ L of streptavidin when lucifuge, detected signal value on AlphaScreen/Lisa detector is hatched after 15-20min in 37 DEG C of vibrations.
3, dose-response curve
In test kit of the present invention, the logarithm of reference standard product concentration is for abscissa, the logarithm of signal value counting is vertical coordinate, processed by double-log mathematical model Log-Log function, record NDV test kit dose-response curve linearly dependent coefficient r value, being judged dose response linear dependence by r value, dose-response curve is as shown in Figure 4.
4, the range of linearity of sensitivity for analysis and standard curve
Add the signal value of the signal value deduction background of 2 times of standard deviation gained with the average of zero reference standard 20 measured values of product measured value, substitute into standard curve Equation for Calculating.
5, Precision Experiment
Standard substance are prepared low middle high 3 concentration, respectively sets 5 parallel holes, repeatedly measure with in once experiment and in different experiments.Result shows that in detectable analysis of the present invention, the coefficient of variation is 3.48%～5.62%, and between analysis, the coefficient of variation is 5.37%～7.29%, and precision is good.
6, specificity experiments
Select Avian pneumo-encephalitis virus (NDV), bird flu virus (AIV-H9), infectious bronchitis virus (IBV), four kinds of viruses of bursal disease virus (IBDV) carry out AlphaLISA detection, determine the specificity of the method with this.Result is as shown in table 6: only Avian pneumo-encephalitis virus detected value is substantially higher, and result is judged to the positive, and other pathogen detection value is relatively low, is negative by criterion, it was shown that the method specificity is higher.
Table 6 specificity experiments result
Remarks: criterion be the ratio of signal value and negative (NC) of measuring samples more than 2.0, the positive can be judged to.
7, stability experiment
After making AlphaLISA reagent room temperature placement 7d by oneself, observe each batch of reagent signal value change, linear relationship and reference standard product signal value.It is shown that after AlphaLISA reagent room temperature of the present invention placement 7d, each batch of reagent signal value change is little, and linear relationship is good, and reference standard product signal value has no significantly raised, has good stability.
8, the evaluation of AlphaLISA detection method
The present invention learns from else's experience 192 parts of the sample that the virus hemagglutination inhibition test (HI) of standard detects, it has been determined that wherein positive 114 parts, negative sample 78 parts.Detecting through homemade Avian pneumo-encephalitis virus AlphaLISA reagent, Positive rate, negative rate and standard reagent box compare, and the AlphaLISA detection method set up is carried out quality evaluation.Result shows: detect through Avian pneumo-encephalitis virus AlphaLISA reagent of the present invention, find 110 parts of true positives sample in 114 parts of samples, find detection negative sample 76 parts in 78 parts of negative samples, the sensitivity of detection is 96.5%, specificity 97.4%, result shows that the AlphaLISA detection method set up has higher sensitivity and specificity, can meet high flux, and quickly the requirement of detection, has potential application value (table 7).
Table 7AlphaLISA detection method is evaluated
What finally illustrate is, above example is only in order to illustrate technical scheme and unrestricted, although the present invention being described in detail with reference to preferred embodiment, it will be understood by those within the art that, the technical scheme of invention can be modified or equivalent replacement, without deviating from objective and the scope of technical solution of the present invention, it all should be encompassed in the middle of scope of the presently claimed invention.
Claims (10)
1. an Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit, it is characterised in that include being coated with the receptor microsphere of Avian pneumo-encephalitis virus antibody, biotin labeled Avian pneumo-encephalitis virus antibody and being coated with the donor microsphere of Streptavidin；Described receptor microsphere is the Nano microsphere being coated with luminophor and lanthanide series, and described donor microsphere is the Nano microsphere being coated with photosensitizer and Streptavidin.
2. Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit according to claim 1, it is characterised in that on described receptor microsphere, coated Avian pneumo-encephalitis virus antibody and biotin labeled Avian pneumo-encephalitis virus antibody are a pair pairing antibody.
3. Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit according to claim 2, it is characterized in that, described pairing antibody is adopted and is obtained with the following method: by Avian pneumo-encephalitis virus immunity BALB/c mouse, take the mouse boosting cell after immunity and carry out cell fusion with SP2/0 myeloma cell with PEG-1500, cell after merging is carried out indirect ELISA screening, positive colony is selected to carry out 2 sub-clones, after selecting 2 sub-clones, positive hybridoma cell strain carries out hemagglutination inhibition test and competitive ELISA test, obtain hybridoma 2A5 and the 4E11 of secretion pairing antibody, by 2A5 and 4E11 lumbar injection BALB/c mouse respectively, and adopt caprylic acid-ammonium to carry out antibody purification, obtain a pair pairing antibody.
4. Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit according to claim 3, it is characterised in that before described Avian pneumo-encephalitis virus immunity BALB/c mouse, also Avian pneumo-encephalitis virus having been carried out purification, concrete purification step is as follows:
1) Newcastle Disease Virus Vaccine is carried out chick embryo allantoic cavity inoculation, collect chick embryo allantoic liquid；
2) allantoic fluid collected being carried out differential centrifugation, prior under 4 DEG C of 5000rpm/min, centrifugal 15min goes precipitation, after under 4 DEG C of 10000rpm/min centrifugal 15min go precipitation, finally under 100000g, centrifugal 2h abandons supernatant, uses PBS dissolution precipitation, standby；
3) to step 2) to carry out SUPERDEX200 solvent resistant column eluting for solution after differential centrifugation, and collect first eluting peak, obtain the Avian pneumo-encephalitis virus of purification.
5. Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit according to claim 1, it is characterized in that, also include the reference standard liquid of Avian pneumo-encephalitis virus, the solvent of described reference standard liquid is the pH7.8 buffer solution containing 50mmol/LTris HCl, mass concentration 1.5%BSA, mass concentration 0.9%NaCl, mass concentration 0.05%NaN3 and mass concentration 0.01%Tween-20, and the solute of described reference standard liquid is Avian pneumo-encephalitis virus.
6. Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit according to claim 1, it is characterized in that, the described receptor microsphere being coated with Avian pneumo-encephalitis virus antibody is diluted by 1:100 ~ 200 volume ratio with 1xAphaLISA buffer, described in be coated with the donor microsphere 1xAphaLISA buffer of Streptavidin and be diluted to final concentration of 40 μ g/mL.
7. Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit according to claim 1, it is characterised in that described biotin labeled Avian pneumo-encephalitis virus antibody and 1xAphaLISA buffer press 1:200 ~ 400 volume ratio and dilutes.
8. adopt the method that the arbitrary described Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit of claim 1 ~ 7 carries out detecting, it is characterized in that, being sequentially added into standard substance or detected sample, the receptor microsphere being coated with Avian pneumo-encephalitis virus antibody and biotin labeled Avian pneumo-encephalitis virus antibody in microwell plate, 15 ~ 20min is hatched in 37 DEG C of vibrations；Then adding when lucifuge and be coated with the donor microsphere of Streptavidin, detected signal value on AlphaScreen/Lisa detector is hatched after 15 ~ 20min in 37 DEG C of vibrations；Wherein, described standard substance or detected sample, the addition volume ratio that is coated with the receptor microsphere of Avian pneumo-encephalitis virus antibody, biotin labeled Avian pneumo-encephalitis virus antibody and the donor microsphere that is coated with Streptavidin are 1:1:1:7.
9. adopt the method that Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit carries out detecting according to claim 8, it is characterized in that, being sequentially added into standard substance or detected sample 25 μ L in microwell plate, be coated with the receptor microsphere 25 μ L and biotin labeled Avian pneumo-encephalitis virus antibody 25 μ L of Avian pneumo-encephalitis virus antibody, 15-20min is hatched in 37 DEG C of vibrations；Then adding when lucifuge and be coated with the donor microsphere l75 μ L of Streptavidin, detected signal value on AlphaScreen/Lisa detector is hatched after 15-20min in 37 DEG C of vibrations.
10. adopt the method that Avian pneumo-encephalitis virus double-antibody sandwich AlphaLISA detection kit carries out detecting according to claim 8, it is characterized in that, described detected signal value on AlphaScreen/Lisa detector, excitation wavelength is 680nm, and launching light detection wavelength is 615nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610218513.9A CN105759046A (en) | 2016-04-08 | 2016-04-08 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610218513.9A CN105759046A (en) | 2016-04-08 | 2016-04-08 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105759046A true CN105759046A (en) | 2016-07-13 |
Family
ID=56334573
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610218513.9A Pending CN105759046A (en) | 2016-04-08 | 2016-04-08 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105759046A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990248A (en) * | 2017-04-25 | 2017-07-28 | 重庆理工大学 | A kind of preparation method and detection method of the NDV immunosensors based on 81 ° of TFG |
| CN109444414A (en) * | 2018-09-10 | 2019-03-08 | 珠海国际旅行卫生保健中心 | Using the method and corresponding composition, kit of AlphaLISA detection dengue virus |
| CN110488017A (en) * | 2018-05-14 | 2019-11-22 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody assay kit |
| CN110488011A (en) * | 2018-05-14 | 2019-11-22 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody assay kit |
| CN110672844A (en) * | 2019-10-29 | 2020-01-10 | 华中科技大学 | Newcastle disease virus antibody magnetic immuno-chemiluminescence detection kit and application thereof |
| CN112255399A (en) * | 2020-10-20 | 2021-01-22 | 浙江洪晟生物科技股份有限公司 | Hog cholera virus detection kit based on double-antibody sandwich homogeneous phase chemiluminescence method, and preparation method and application thereof |
| CN113341141A (en) * | 2021-06-04 | 2021-09-03 | 重庆理工大学 | AlphaLISA detection kit for avian influenza virus H9 and detection method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196522A (en) * | 2007-12-29 | 2008-06-11 | 华中农业大学 | Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application |
| CN101251540A (en) * | 2008-03-26 | 2008-08-27 | 博阳生物科技（上海）有限公司 | Hepatitis B virus e antigen testing corpuscle, preparation and application thereof |
| WO2009150845A1 (en) * | 2008-06-12 | 2009-12-17 | 株式会社セルフリーサイエンス | Method for selection of most suitable anti-viral agent |
| CN103937751A (en) * | 2014-04-11 | 2014-07-23 | 山东省农业科学院畜牧兽医研究所 | Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody |
| CN104865243A (en) * | 2015-06-12 | 2015-08-26 | 西南大学 | Light-stimulating chemiluminiscence immunological detection method of silkworm mature egg microsporidiosis |
-
2016
- 2016-04-08 CN CN201610218513.9A patent/CN105759046A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101196522A (en) * | 2007-12-29 | 2008-06-11 | 华中农业大学 | Newcastle disease immune body immune colloidal gold fast detecting reagent kit and its application |
| CN101251540A (en) * | 2008-03-26 | 2008-08-27 | 博阳生物科技（上海）有限公司 | Hepatitis B virus e antigen testing corpuscle, preparation and application thereof |
| WO2009150845A1 (en) * | 2008-06-12 | 2009-12-17 | 株式会社セルフリーサイエンス | Method for selection of most suitable anti-viral agent |
| CN103937751A (en) * | 2014-04-11 | 2014-07-23 | 山东省农业科学院畜牧兽医研究所 | Colloidal gold immunochromatograohic assay detection test strip based on NDV (Newcastle Disease Virus) hemagglutinin protein monoclonal antibody |
| CN104865243A (en) * | 2015-06-12 | 2015-08-26 | 西南大学 | Light-stimulating chemiluminiscence immunological detection method of silkworm mature egg microsporidiosis |
Non-Patent Citations (2)
| Title |
|---|
| 王颖 等: "AlphaLISA技术的发展及应用", 《ALPHALISA技术的发展及应用》 * |
| 郭浩 等: "新城疫病毒3种检测方法的研究比较", 《中国生物工程杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106990248A (en) * | 2017-04-25 | 2017-07-28 | 重庆理工大学 | A kind of preparation method and detection method of the NDV immunosensors based on 81 ° of TFG |
| CN110488017A (en) * | 2018-05-14 | 2019-11-22 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody assay kit |
| CN110488011A (en) * | 2018-05-14 | 2019-11-22 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody assay kit |
| CN110488011B (en) * | 2018-05-14 | 2022-11-01 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody detection kit |
| CN110488017B (en) * | 2018-05-14 | 2022-11-04 | 洛阳中科生物芯片技术有限公司 | Newcastle disease virus antibody detection kit |
| CN109444414A (en) * | 2018-09-10 | 2019-03-08 | 珠海国际旅行卫生保健中心 | Using the method and corresponding composition, kit of AlphaLISA detection dengue virus |
| CN110672844A (en) * | 2019-10-29 | 2020-01-10 | 华中科技大学 | Newcastle disease virus antibody magnetic immuno-chemiluminescence detection kit and application thereof |
| CN112255399A (en) * | 2020-10-20 | 2021-01-22 | 浙江洪晟生物科技股份有限公司 | Hog cholera virus detection kit based on double-antibody sandwich homogeneous phase chemiluminescence method, and preparation method and application thereof |
| CN113341141A (en) * | 2021-06-04 | 2021-09-03 | 重庆理工大学 | AlphaLISA detection kit for avian influenza virus H9 and detection method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105759046A (en) | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof | |
| CN102625915A (en) | Method for measurement of glycoprotein, method for detection of hepatic diseases, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein as measure of disease conditions of hepatic diseases | |
| CN104204803A (en) | Signatures and determinants for diagnosing infections and methods of use thereof | |
| CN103323599B (en) | Time resolved immunofluorescence detection kit for rabies virus nucleoprotein, and preparation method thereof | |
| CN104823052A (en) | Biomarkers for diagnosing and/or monitoring tuberculosis | |
| CN104650234B (en) | Anti- AKR1B10 protein monoclonal antibodies and its application | |
| US20200011867A1 (en) | Low Density Microarrays for Vaccine Related Protein Quantification, Potency Determination, and Efficacy Evaluation | |
| CN102507918A (en) | Kit for determining anti-cyclic citrullinated peptide (Anti-CCP) antibody | |
| CN112485438B (en) | Specific protein reaction detection method and device | |
| Shah et al. | Calibration-free concentration analysis of protein biomarkers in human serum using surface plasmon resonance | |
| Atmar et al. | Immunologic Detection and Characterization | |
| CN107045062A (en) | Detect colloidal gold immuno-chromatography test paper strip, kit of human neutrophil genatinase associated lipocalin and preparation method thereof | |
| AU2012271858A1 (en) | Low density microarrays for vaccine related protein quantification, potency determination and efficacy evaluation | |
| CN106119415A (en) | A kind of test kit utilizing one-step method real-time fluorescent quantitative RT qPCR detection hepatitis C virus nucleic acid | |
| CN105467123A (en) | Early diagnosis marker screening system and joint detection kit for pulmonary fibrosis | |
| CN104155450A (en) | Treponema pallidum IgG antibody biotin avidin enzyme-linked immune detection kit and preparation method thereof | |
| CN103235119A (en) | A diagnosis antigen for Toxoplasma gondii infection and a preparation method and applications thereof | |
| CN104459159A (en) | Kit for detecting relevant autoantibody spectrum of autoimmune liver disease | |
| CN102994638A (en) | Sandwich immuno-PCR (polymerase chain reaction) detection method and kit for to-be-detected object in biological samples | |
| Kombe Kombe et al. | Detection of circulating VZV-glycoprotein E-specific antibodies by chemiluminescent immunoassay (CLIA) for varicella–zoster diagnosis | |
| CN102520176B (en) | Kit for quantitatively detecting interleukin 8 | |
| CN201203618Y (en) | Immune colloidal gold test card for new castle disease virus | |
| CN106191286A (en) | Brucellar detection method, test kit and application thereof | |
| CN113999841A (en) | Protein scaffold OVAL100 and application thereof in radioligand method | |
| CN101762703A (en) | Hepatitis C Virus (HCV) antibody detection kit and preparation method and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160713 |
|
| RJ01 | Rejection of invention patent application after publication |