CN108956986A - Newcastle disease virus antibody assay kit - Google Patents
Newcastle disease virus antibody assay kit Download PDFInfo
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- CN108956986A CN108956986A CN201810507587.3A CN201810507587A CN108956986A CN 108956986 A CN108956986 A CN 108956986A CN 201810507587 A CN201810507587 A CN 201810507587A CN 108956986 A CN108956986 A CN 108956986A
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- 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
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Abstract
The present invention relates to newcastle disease virus antibody assay kits.Detection kit of the invention includes one or more solid carriers, and the particular polypeptide or polypeptide set that are independently connected on one or more of solid carriers.
Description
Technical field
The invention mainly relates to fowl kit and diagnostic methods.Specifically, the present invention relates to can be used for newcastle disease
The kit and detection method of antibody test.
Background technique
Newcastle disease (ND) is that one kind caused by NDV (Newcastle disease virus, newcastle disease virus) easily passes
The crushing disease of dye, the death rate are often up to 100%, are distributed widely in many countries, OIE is classified as A class epidemic disease.This disease
It is typically characterised by respiratory tract, gastrointestinal mucosal bleeding, people also has neurological susceptibility.This disease is found in India Ni Xi in nineteen twenty-six earliest
Sub- Ba Taweiya, the same year is found in Britain's new city, therefore and gains the name.
NDV is the avian paramyxovirus I type (APMV-1) of paramyxovirus section paramyxovirus category (Avulavirus).Virus tool
Have a cyst membrane structure, genome is the RNA virus of sub-thread minus strand non-segmented negative, be made of 6 kinds of structural proteins, respectively NP, P, M,
F, HN and L albumen.NP albumen is nucleocapsid protein, and conservation of amino acids is higher, and most of NDV virus is in NP albumen 443-460
Position all has a highly conserved B cell epitope；P albumen is the necessary factor of viral RNA synthesis；M, F and HN albumen and disease
The formation of malicious cyst membrane is related；F protein (fusion protein) and HN albumen (hemagglutinin neuraminidase albumen) are mainly exempting from for virus
Epidemic disease immunogenic peptide can induce body to generate neutralizing antibody.F protein mediate retroviral is merged with the film of host cell, and albumen is split
It is related with virus virulence to solve site.HN albumen has the effects that neuraminidase and promotes fusion；In addition, the albumen also with virus
Virulence is related.L albumen is the maximum albumen of virus, it is the most conservative in NDV6 kind structural proteins, has RNA polymerase, turns
The biological activities such as modification, can collectively form viral replication complex with NP and P albumen after record, which participates in viral gene
The transcription and replication of group.
Currently, the laboratory detection technology of newcastle disease specifically includes that conventional separation identification, serological method (hemagglutination test
(HA), hemagglutination-inhibition test (HI), AGP test test (AGP), fluorescent antibody technics (FA), enzyme-linked immunosorbent assay
(ELISA), virus neutralization tests (VNT), latex agglutination test (LAT), neuraminic acid enzyme test (NIT), radioimmunoassay
(RIA), molecular biology method (RNA finger-print, Nucleic Acid Probe Technique, RT-PCR technology) etc..Among these, enzyme linked immunological is surveyed
Therefore the fixed a large amount of samples of specific higher with sensibility and suitable detection are widely used in the inspection of newcastle disease virus antibody
It surveys.
Enzyme-linked immunosorbent assay method for newcastle disease virus antibody test mainly includes competitive enzyme-linked immune measurement (c-
ELISA), enzyme-linked immunosorbent assay (b-ELISA) and indirect enzyme-linked immunosorbent assay are blocked.C-ELISA's and b-ELISA is special
Property and sensibility it is relatively high, be by clinical generally accepted newcastle disease virus antibody detection method.But c-ELISA and b-
ELISA is both needed to using monoclonal antibody, this causes testing cost to greatly improve；Moreover, the operation of c-ELISA and b-ELISA is numerous
It is trivial, detection time is long, criterion is complicated.On the other hand, traditional indirect ELISA use from newcastle disease virus intact proteins or
Recombinant protein detects the antibody in serum as envelope antigen, and cost is lower than c-ELISA and b-ELISA；But due to this
Method uses intact proteins as antigen, is easy to happen the wrong identification and non-specific identification of antibody, therefore, specificity and
Sensibility is all not as good as c-ELISA and b-ELISA.
Therefore, it is necessary to develop the new city that testing cost is low, detection time is short, easy to operate, specific and high sensibility
Epidemic disease virus antibody assay kit.
Summary of the invention
In view of above-mentioned problems of the prior art, low, detection that the purpose of the present invention is to provide a kind of testing costs
Time short, easy to operate, specific and high sensibility newcastle disease virus antibody assay kit, and can be used in preparation should
The polypeptide or polypeptides in combination of kit.
Inventor's discovery: based on the indirect ELISA method of antigen epitope polypeptide due to using antigen epitope polypeptide (20 ammonia
The single polypeptide of base acid or so usually only includes an epitope) it is used as envelope antigen, therefore, specificity is very high；But the party
The sensibility of method is often relatively low, and the sensibility of single polypeptide does not exceed 50% generally.If can find, sensibility is high to be resisted
Former epitope polypeptide, so that it may which the shortcomings that overcoming traditional indirect ELISA, developing specificity and sensibility can match in excellence or beauty c-
ELISA and b-ELISA, even higher newcastle disease virus antibody assay kit.
Inventor has made intensive studies to solve above-mentioned technical problem, as a result, it has been found that: it is more shown in SEQ ID NO:1
Peptide with the sensibility that the polypeptide individually detects newcastle disease virus antibody is 88%, specificity 96%, and being used alone can reach
Standard as detection kit；The detection of the polypeptides in combination shown in the polypeptide shown in SEQ ID NO:1 and SEQ ID NO:2
When newcastle disease virus antibody, sensibility 94%, specificity is 93%, is that the most excellent newcastle disease virus of current comprehensive performance is anti-
One of body detection kit.
Therefore, the present invention includes:
Polypeptide shown in 1.SEQ ID NO:1.
The combination of polypeptide shown in 2.SEQ ID NO:1 and polypeptide shown in SEQ ID NO:2.
3. aforementioned polypeptides or polypeptides in combination are in the biological sample that preparation is used for test object biological source with the presence or absence of anti-
Purposes in the kit of the antibody (IgY) of newcastle disease virus.
4. a kind of newcastle disease virus antibody (IgY) detection kit comprising one or more solid carriers, and it is independent
Ground is connected to following polypeptides in combination 1 on one or more of solid carriers；
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:2.
5. a kind of newcastle disease virus antibody (IgY) detection kit comprising one or more solid carriers, and it is independent
Ground is connected to polypeptide shown in the SEQ ID NO:1 on one or more of solid carriers.
6. newcastle disease virus antibody (IgY) detection kit according to claim 4 or 5 does not include other use
In the probe molecule (such as polypeptide, albumen or nucleic acid) of detection newcastle disease virus antibody (IgY).
7. following polypeptides in combination 1 whether there is anti-newcastle disease in the biological sample that preparation is used for test object biological source
Purposes in the kit of the antibody (IgY) of virus；
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:2.
In the present specification, the object organisms are preferably birds, more preferably chicken.
In the present specification, the biological sample can be whole blood, blood plasma or serum.
Aforementioned polypeptides and kit can be used in the biological sample of test object biological source with the presence or absence of anti-newcastle disease
The antibody (IgY) of virus.In general, the newcastle disease virus antibody in biological sample is since object organisms are by newcastle disease virus infection
Or be immunized and generated by newcastle disease vaccine, therefore, whether aforementioned polypeptides and kit can be used for diagnosing object organisms new
City epidemic disease virus infection or newcastle disease vaccine are immune.
The specific embodiment of invention
Firstly, the present invention provides polypeptide shown in SEQ ID NO:1.Inventors have found that individually detecting new city with the polypeptide
The sensibility of epidemic disease antiviral antibody is 88%, specificity 96%.It is single in the indirect ELISA method based on antigen epitope polypeptide
50% sensibility of being usually no more than of polypeptide is compared, the sensibility of polypeptide shown in SEQ ID NO:1 be it is surprising, individually
It can even be matched in excellence or beauty with traditional ELISA method detection kit using the sensibility of polypeptide detection newcastle disease virus antibody！
Moreover, it also has up to 96% specificity.Therefore, unexpected technical effect is achieved.
In this specification, sensibility refers to: in the positive sample with the confirmation of " goldstandard " method, being measured as by other methods
The ratio of positive sample.Specificity refers to: in the negative sample with the confirmation of " goldstandard " method, being measured as feminine gender by other methods
The ratio of sample.For the detection of newcastle disease virus antibody, " goldstandard " of the art is hemagglutination-inhibition test
(HI)。
Polypeptide shown in the SEQ ID NO:1 can be used as the life that detection probe is used to prepare test object biological source
With the presence or absence of the kit of the antibody of anti-new castle disease virus in object sample.
Inventor also found, when the polypeptide shown in SEQ ID NO:1 and polypeptides in combination shown in SEQ ID NO:2 detect
When newcastle disease virus antibody, sensibility 94%, specificity 93% completely can be with c-ELISA methods and b-ELISA method
It matches in excellence or beauty.Therefore, the present invention also provides following polypeptides in combination 1.
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1, and
Polypeptide shown in SEQ ID NO:2.
Aforementioned polypeptides combination can be used as detection probe be used to prepare in the biological sample of test object biological source whether
There are the kits of the antibody of anti-new castle disease virus.
Therefore, the present invention also provides a kind of newcastle disease virus antibody assay kits comprising one or more solids carry
Body, and the aforementioned polypeptides combination 1 being independently connected on one or more of solid carriers.
In the present specification, solid carrier can be one, be also possible to it is multiple, but preferably one, i.e., whole polypeptides
It is independently connected on same solid carrier.In the present invention, solid carrier is not particularly limited, as long as solid or
The carrier of insoluble material.The connection of polypeptide and solid carrier can be using well known to a person skilled in the art polypeptide and admittedly
The connection method of body carrier carries out.
In the present specification, the object organisms are preferably birds, more preferably chicken.
In the present specification, the biological sample can be whole blood, blood plasma or serum.
It is stated in the biological sample of kit test object biological source in use with the presence or absence of the anti-of anti-new castle disease virus
In the case where body, when any one or more polypeptide in the polypeptides in combination 1 has sound to the biological sample in object organisms source
At once, it is determined as in the biological sample in the object organisms source that there are the antibody of anti-new castle disease virus (i.e. positive)；Conversely, working as institute
It states when there is no any polypeptide to have response to the biological sample in object organisms source in polypeptides in combination 1, is determined as that the object is raw
There is no the antibody of anti-new castle disease virus (i.e. negative) in the biological sample in object source.
In the present specification, " response " refers to: in TMB colour test, the visible bluish violet for being better than negative control point of naked eyes
Signal.
Embodiment
1. the preparation and confirmation of polypeptide
Polypeptide used in embodiment is synthesized by gill biochemistry (Shanghai) Co., Ltd., and has been carried out to it really by mass spectrum
Recognize.Wherein,
SEQ ID NO:1:GDGETQFLDLMRAVANSMRE.
SEQ ID NO:2:IANCKMTTCRCVNPPGIISQ.
2. the preparation of kit (detection chip)
Kit 1
Distinguished on conventional ELISA solid phase carrier using conventional method more shown in the above-mentioned SEQ ID NO:1 of point sample
The solution of peptide, in addition one chicken IgY point of point sample is prepared for examining as positive quality control point and a PB point as negative Quality Control point
Survey chip.
Kit 2
In addition on solid phase carrier distinguish the above-mentioned SEQ ID NO:1 and 2 of point sample shown in polypeptide solution other than, according to it is upper
It states the identical preparation method of kit 1 and is prepared for detection chip, as kit 2.
3. being detected with kit
Checking procedure
1) balance reagent: by all reagents after refrigerator taking-up, balance is placed spare to room temperature.
2) prepare washing lotion: with purified water or distilled water by 1:9 dilution concentration washing lotion, example: washing lotion purified water is concentrated in 50mL
Or distilled water is configured to the washing lotion of 500mL, and mixes well.Unspent washing lotion is put 4 DEG C of refrigerators and is saved, and is finished in 3 months.
3) dilute sample: taking sample to be tested, with serum dilution, dilutes by 1:100, example: adding in 198uL serum dilution
Enter 2uL sample to be tested, and mixes well.Pattern detection need to use the sample after 200uL dilution.
4) wetted chip: detection chip needed for taking out detection.1mL washing lotion is added and infiltrates chip surface, 3min is then abandoned
Go washing lotion.
5) it is loaded: in the implementation case, using single side biological incubation reactor.The sample liquid-transfering gun that dilution is completed
It is added in reactor by liquid feeding venthole, dosage 200uL.After sample-adding, liquid feeding venthole is closed with sealing note.
6) sample incubation: will add excellent reactor to be placed on horizontal shaker, be incubated for 30min at room temperature with 150rpm.
7) it cleans: reaction column being taken out from reaction chamber, discards reaction solution, rinsed in chip surface and reaction chamber with washing lotion
Portion is repeated 3 times (each dosage about 12mL, time 1-2min).
8) ELIAS secondary antibody is incubated for: reaction column and reaction chamber are reconfigured simultaneously clamping.Sealing note is thrown off, is ventilated by liquid feeding
The goat-anti chicken IgY solution 200uL of hole addition horseradish peroxidase-labeled.Liquid feeding venthole is closed again.It is subsequently placed at level
On shaking table, 30min is incubated for 150rpm at room temperature.
9) it cleans again: reaction column being taken out from reaction chamber, discards reaction solution, rinse chip surface and reaction chamber with washing lotion
Inside is repeated 3 times (each dosage about 12mL, time 1-2min).
10) it develops the color: adding TMB developing solution 100uL in each reaction column chip surface respectively, developing solution need to uniformly be laid on core
Piece surface.
Result judgement: observing by the naked eye, and the response condition for counting polypeptide under every part of serum acts on is (visually visible to be better than yin
Property control point royal purple chrominance signal, interpretation be have response).That is,
Newcastle Disease is determined as when detecting that polypeptide shown in SEQ ID NO:1 has response for mentioned reagent box 1
Malicious antibody positive；Conversely, being determined as feminine gender.
For mentioned reagent box 2, when detecting that polypeptide shown in SEQ ID NO:1 and/or 2 has response, it is determined as new
City epidemic disease virus antibody positive；Conversely, being determined as feminine gender.
For 566 parts of chicken serum samples from China regions, the kit prepared in above-mentioned steps 2 is respectively adopted
The hemagglutination-inhibition test (HI) of (the step of by above-mentioned 3) and routine is detected, and testing result is as follows.
Table 1
Sensibility=353/400*100%=88%
Specificity=160/166=96%
Table 2
Sensibility=377/400*100%=94%
Specificity=159/166=93%
As known from the above, mentioned reagent box 1,2 only only used one or two polypeptides and realize as detection probe
85% or more detection sensitivity, and to be 90% or more (and be using HI method validation, be not using control examination specificity
The verifying of agent cassette method), it is sufficient to the kit to match in excellence or beauty using c-ELISA method or b-ELISA method.It should be noted that usually
Think clinically to have the sensibility and specificity of the diagnostic kit of application value all should be 85% or more.
It hereinafter, carrying out more specific description to the present invention by embodiment, but is not the limit to the technology of the present invention range
It is fixed.By the record of this specification, those skilled in the art readily can modify/change to the present invention, these include
Within the technical scope of the present invention.
Sequence table
<110>Suzhou Industrial Park Qiang Dong Pharmaceutical Technology Co., Ltd
<120>newcastle disease virus antibody assay kit
<130> PB00191
<141> 2018-05-24
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Gly Asp Gly Glu Thr Gln Phe Leu Asp Leu Met Arg Ala Val Ala Asn
1 5 10 15
Ser Met Arg Glu
20
<210> 2
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Ile Ala Asn Cys Lys Met Thr Thr Cys Arg Cys Val Asn Pro Pro Gly
1 5 10 15
Ile Ile Ser Gln
20
Claims (8)
- Polypeptide shown in 1.SEQ ID NO:1.
- Polypeptide shown in 2.SEQ ID NO:1 is in the biological sample that preparation is used for test object biological source with the presence or absence of anti-new Purposes in the kit of the antibody (IgY) of city epidemic disease poison.
- 3. a kind of newcastle disease virus antibody (IgY) detection kit comprising one or more solid carriers, and independently connect The following polypeptides in combination 1 being connected on one or more of solid carriers；Polypeptides in combination 1Polypeptide shown in SEQ ID NO:1, andPolypeptide shown in SEQ ID NO:2.
- 4. whether detection kit according to claim 3 is used to deposit in the biological sample of test object biological source In the antibody (IgY) of anti-new castle disease virus.
- 5. following polypeptides in combination 1 whether there is anti-new castle disease virus in the biological sample that preparation is used for test object biological source Antibody (IgY) kit in purposes；Polypeptides in combination 1Polypeptide shown in SEQ ID NO:1, andPolypeptide shown in SEQ ID NO:2.
- 6. purposes according to claim 2 or 5 or detection kit according to claim 4, wherein the object Biology is birds.
- 7. purposes according to claim 2 or 5 or detection kit according to claim 4, wherein the object Biology is chicken.
- 8. purposes according to claim 2 or 5 or detection kit according to claim 4, wherein the biology Sample is whole blood, blood plasma or serum.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810507587.3A CN108956986B (en) | 2018-05-24 | 2018-05-24 | Newcastle disease virus antibody detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810507587.3A CN108956986B (en) | 2018-05-24 | 2018-05-24 | Newcastle disease virus antibody detection kit |
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| CN108956986A true CN108956986A (en) | 2018-12-07 |
| CN108956986B CN108956986B (en) | 2021-07-02 |
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| CN201810507587.3A Active CN108956986B (en) | 2018-05-24 | 2018-05-24 | Newcastle disease virus antibody detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN116519944A (en) * | 2023-02-17 | 2023-08-01 | 中国农业科学院兰州兽医研究所 | Indirect ELISA (enzyme-Linked immuno sorbent assay) detection method for newcastle disease virus antibody and kit thereof |
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| WO2007108568A1 (en) * | 2006-03-23 | 2007-09-27 | Kbnp, Inc. | Hn epitope recognized by avian immune system and antigenic variant newcastle disease viruses carrying changes in the epitope |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN116519944A (en) * | 2023-02-17 | 2023-08-01 | 中国农业科学院兰州兽医研究所 | Indirect ELISA (enzyme-Linked immuno sorbent assay) detection method for newcastle disease virus antibody and kit thereof |
| CN116519944B (en) * | 2023-02-17 | 2024-04-30 | 中国农业科学院兰州兽医研究所 | Indirect ELISA (enzyme-Linked immuno sorbent assay) detection method for newcastle disease virus antibody and kit thereof |
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| CN108956986B (en) | 2021-07-02 |
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