阅读说明：本技术 新城疫病毒抗体检测试剂盒 (Newcastle disease virus antibody assay kit ) 是由 马宏伟 杨兰 李静芝 于 2018-05-24 设计创作，主要内容包括：本发明涉及新城疫病毒抗体检测试剂盒。本发明的检测试剂盒包括一个或多个固体载体,以及独立地连接于所述一个或多个固体载体上的特定多肽或多肽组合。(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.)

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,

Polypeptide shown in SEQ ID NO:5, and

Polypeptide shown in SEQ ID NO:6.

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,

Polypeptide shown in SEQ ID NO:5, and

Polypeptide shown in SEQ ID NO:6.

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.

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.

The specificity and sensibility of c-ELISA and b-ELISA is relatively high, is by clinical generally accepted newcastle disease virus Antibody detection method.But c-ELISA and b-ELISA are both needed to using monoclonal antibody, this causes testing cost to greatly improve； Moreover, the cumbersome of c-ELISA and b-ELISA, detection time are long, criterion is complicated.On the other hand, traditional indirect ELISA Intact proteins or recombinant protein from newcastle disease virus are used to detect the antibody in serum as envelope antigen, it is at low cost In c-ELISA and b-ELISA；But since this method uses intact proteins as antigen, it is easy to happen the wrong identification of antibody It is identified with non-specificity, therefore, specificity and sensibility are 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:1~6 institute When the polypeptides in combination detection newcastle disease virus antibody shown, sensibility 90%, specificity 95% 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,

Polypeptide shown in SEQ ID NO:5, and

Polypeptide shown in SEQ ID NO:6.

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,

Polypeptide shown in SEQ ID NO:5, and

Polypeptide shown in SEQ ID NO:6.

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:1~6 SEQ ID detects newcastle disease virus antibody Perception is 90%, specificity 95%, 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,

Polypeptide shown in SEQ ID NO:5, and

Polypeptide shown in SEQ ID NO:6.

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