CN110488010A - Newcastle disease virus antibody assay kit - Google Patents
Newcastle disease virus antibody assay kit Download PDFInfo
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- CN110488010A CN110488010A CN201810455110.5A CN201810455110A CN110488010A CN 110488010 A CN110488010 A CN 110488010A CN 201810455110 A CN201810455110 A CN 201810455110A CN 110488010 A CN110488010 A CN 110488010A
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- 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
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- 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
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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 polypeptides in combination 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 has made intensive studies to solve above-mentioned technical problem, as a result, it has been found that, when with SEQ ID NO:1~5 institute
When the polypeptides in combination detection newcastle disease virus antibody shown, sensibility 94%, specificity 99% completely can be with c-ELISA
Method and b-ELISA method match in excellence or beauty.
Therefore, the present invention includes:
1. 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,
Polypeptide shown in SEQ ID NO:2,
Polypeptide shown in SEQ ID NO:3,
Polypeptide shown in SEQ ID NO:4, and
Polypeptide shown in SEQ ID NO:5.
2. being used in the biological sample of test object biological source whether there is according to detection kit described in item 1
The antibody (IgY) of anti-new castle disease virus.
3. according to detection kit described in item 2, wherein the object organisms are birds.
4. according to detection kit described in item 2, wherein the object organisms are chickens.
5. according to detection kit described in item 2, wherein the biological sample is whole blood, blood plasma or serum.
6. 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,
Polypeptide shown in SEQ ID NO:2,
Polypeptide shown in SEQ ID NO:3,
Polypeptide shown in SEQ ID NO:4, and
Polypeptide shown in SEQ ID NO:5.
7. according to purposes described in item 6, wherein the object organisms are birds.
8. according to purposes described in item 6, wherein the object organisms are chickens.
9. according to purposes described in item 6, wherein the biological sample is 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
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)。
Inventor also found, quick when the polypeptides in combination shown in NO:1~5 SEQ ID detects newcastle disease virus antibody
Perception is 94%, specificity 99%, can be matched in excellence or beauty completely with c-ELISA method and b-ELISA method.Therefore, the present invention is gone back
Following polypeptides in combination 1 are provided.
Polypeptides in combination 1
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:2,
Polypeptide shown in SEQ ID NO:3,
Polypeptide shown in SEQ ID NO:4, and
Polypeptide shown in SEQ ID NO:5.
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: signal-to-noise ratio (SNR) is greater than or equal to 2, wherein signal-to-noise ratio=(polypeptide point letter
Number value-negative control point signal value)/negative control point signal value.
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:DYIGGIGKELIVDDASDVTS.
SEQ ID NO:2:IANCKMTTCRCVNPPGIISQ.
SEQ ID NO:3:GSIPCQASARCPNSCVTGVY.
SEQ ID NO:4:CSKVTETEEEDYNSAVPTRM.
SEQ ID NO:5:KVTFDKLEKKIRSLDLSVGL.
2. the preparation of kit (detection chip)
Kit 1
Distinguished shown in NO:1~5 point sample above-mentioned SEQ ID on conventional ELISA solid phase carrier using conventional method
The solution of polypeptide, in addition one chicken IgY point of point sample is prepared for as positive quality control point and a PB point as negative Quality Control point
Detection chip.
3. being detected with kit
Polypeptide chip is placed in room temperature 20min, visually observes chip surface, there will be the polypeptide chip of defect to eliminate, simultaneously
The correct direction for determining polypeptide chip, is sandwiched in tongue, and the number of chip is carried out according to the quantity of serum, then in qualified polypeptide
TBST (0.4M Tris-HCl, 2.74M NaCl, 2%Tween20, pH7.2 ± 0.2) is filled in chip, is placed at room temperature for 2-
4min checks for that in the dead of night, the polypeptide chip of leakage being discarded.The polypeptide chip of vacancy is filled up, operation is same as above.It is qualified to examine
Polypeptide chip be sandwiched on tongue, every 8 chips are one group, and with TBST rinse 2 times, watering can elution a, hole Kong Jinyi goes out, and are formed
Flowing, is finally gently patted on gauze, another one takes lid to dry, but it is noted that holding polypeptide chip surface wettability, standby
With.
Serum is diluted 50 times (5ul serum sample+245ul serum dilutions) in super-clean bench to number respectively, is shaken
It mixes, chip number is made to number consistent with each other, the loading on the chip of rinse with serum, the hole 200ul/ avoids generating gas
Bubble, is added serum dilution 200ul, room temperature, and shaking table 150rpm is incubated for 30min.
Liquid is abandoned, 4 times (method is same as above) is eluted, drying is added secondary antibody (goat-anti chicken IgY), room temperature, shaking table 150rpm, is incubated for
30min
Liquid is abandoned, lid is abandoned, is eluted 4 times, drying, every 8 chips are one group, add luminescent solution (Thermo, the Prod# in the hole 20ul/
37074) 2min, is exposed.Data are acquired using GenePix Pro6.0.
For every a serum, whether each polypeptide counted in the kit respectively has response (that is, signal-to-noise ratio
(SNR) it is more than or equal to 2), and determined.
For mentioned reagent box 1, when detect any one or two in polypeptide shown in NO:1~5 SEQ ID with
On when having response, be determined as the peste des petits ruminants positive；Conversely, being determined as feminine gender.
Wherein, signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point
Signal value refers to the chemiluminescence intensity value for the polypeptide point that software is read, and negative control point signal value refers to the feminine gender that software is read
The chemiluminescence intensity value of control point.
For 420 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=302/320*100%=94%
Specificity=99/100=99%
As known from the above, mentioned reagent box 1 realizes 94% or more detection sensitivity, and specificity up to 99% (and
And be using HI method validation, it is not to be verified using contrast agents cassette method), it is sufficient to it matches in excellence or beauty using c-ELISA method or b-
The kit of ELISA method.
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>Luoyang Zhong Ke Bioarray Solutions Ltd.
<120>newcastle disease virus antibody assay kit
<130> NDV31
<141> 2018-04-26
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Asp Tyr Ile Gly Gly Ile Gly Lys Glu Leu Ile Val Asp Asp Ala Ser
1 5 10 15
Asp Val Thr Ser
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
<210> 3
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Gly Ser Ile Pro Cys Gln Ala Ser Ala Arg Cys Pro Asn Ser Cys Val
1 5 10 15
Thr Gly Val Tyr
20
<210> 4
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Cys Ser Lys Val Thr Glu Thr Glu Glu Glu Asp Tyr Asn Ser Ala Val
1 5 10 15
Pro Thr Arg Met
20
<210> 5
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Lys Val Thr Phe Asp Lys Leu Glu Lys Lys Ile Arg Ser Leu Asp Leu
1 5 10 15
Ser Val Gly Leu
20
Claims (9)
1. 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 1
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:2,
Polypeptide shown in SEQ ID NO:3,
Polypeptide shown in SEQ ID NO:4, and
Polypeptide shown in SEQ ID NO:5.
2. whether detection kit according to claim 1 is used to deposit in the biological sample of test object biological source
In the antibody (IgY) of anti-new castle disease virus.
3. detection kit according to claim 2, wherein the object organisms are birds.
4. detection kit according to claim 2, wherein the object organisms are chickens.
5. detection kit according to claim 2, wherein the biological sample is whole blood, blood plasma or serum.
6. 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 1
Polypeptide shown in SEQ ID NO:1,
Polypeptide shown in SEQ ID NO:2,
Polypeptide shown in SEQ ID NO:3,
Polypeptide shown in SEQ ID NO:4, and
Polypeptide shown in SEQ ID NO:5.
7. purposes according to claim 6, wherein the object organisms are birds.
8. purposes according to claim 6, wherein the object organisms are chickens.
9. purposes according to claim 6, wherein the biological sample is whole blood, blood plasma or serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810455110.5A CN110488010B (en) | 2018-05-14 | 2018-05-14 | Newcastle disease virus antibody detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810455110.5A CN110488010B (en) | 2018-05-14 | 2018-05-14 | Newcastle disease virus antibody detection kit |
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| Publication Number | Publication Date |
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| CN110488010A true CN110488010A (en) | 2019-11-22 |
| CN110488010B CN110488010B (en) | 2022-11-01 |
Family
ID=68543795
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|---|---|---|---|
| CN201810455110.5A Active CN110488010B (en) | 2018-05-14 | 2018-05-14 | Newcastle disease virus antibody detection kit |
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Citations (7)
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|---|---|---|---|---|
| JPH06100591A (en) * | 1991-04-23 | 1994-04-12 | Ishihara Sangyo Kaisha Ltd | Peptide, antibody derived therefrom and anti-newcastle disease agent containing the same antibody |
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|---|---|---|---|---|
| JPH06100591A (en) * | 1991-04-23 | 1994-04-12 | Ishihara Sangyo Kaisha Ltd | Peptide, antibody derived therefrom and anti-newcastle disease agent containing the same antibody |
| US20100261206A1 (en) * | 2007-11-30 | 2010-10-14 | Kang Seuk Choi | Peptide fragments reacting specifically with antibodies against highly pathogenic newcastle disease virus and uses thereof |
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| Publication number | Publication date |
|---|---|
| CN110488010B (en) | 2022-11-01 |
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