CN108956986B - Newcastle disease virus antibody detection kit - Google Patents
Newcastle disease virus antibody detection kit Download PDFInfo
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- CN108956986B CN108956986B CN201810507587.3A CN201810507587A CN108956986B CN 108956986 B CN108956986 B CN 108956986B CN 201810507587 A CN201810507587 A CN 201810507587A CN 108956986 B CN108956986 B CN 108956986B
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- newcastle disease
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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
Abstract
The invention relates to a Newcastle disease virus antibody detection kit. The detection kit comprises one or more solid supports and a specific polypeptide or a polypeptide set independently connected to the one or more solid supports.
Description
Technical Field
The invention mainly relates to a kit for poultry and a diagnostic method. In particular, the invention relates to a kit and a detection method for detecting antibodies against newcastle disease.
Background
Newcastle Disease (ND) is a very contagious and devastating disease caused by NDV (Newcastle disease virus), the mortality rate of which is often as high as 100%, and is widely distributed in many countries, and OIE classifies it as a type a epidemic disease. The disease is characterized by mucosal hemorrhage of respiratory tract and digestive tract, and is also susceptible to human beings. This disease was first discovered in 1926 in balava, indonesia, and in the same year in new british city, hence its name.
NDV is avian paramyxovirus type I (APMV-1) of Paramyxovirus (Avulavirus) of Paramyxoviridae. The virus has envelope structure, and its genome is RNA virus with single negative strand non-segmental, and is composed of 6 structural proteins, NP, P, M, F, HN and L proteins. The NP protein is nucleocapsid protein, the amino acid conservation is higher, most NDV viruses have a highly conserved B cell epitope at the position of the NP protein 443-; the P protein is an essential factor for the synthesis of viral RNA; m, F and HN proteins are involved in the formation of viral envelope; the F protein (fusion protein) and HN protein (hemagglutinin neuraminidase protein) are the main immunogenic proteins of the virus and can induce the body to produce neutralizing antibodies. The F protein mediates the membrane fusion of the virus with the host cell, and the protein cleavage site is related to the virulence of the virus. HN protein has effects of neuraminidase and fusion promotion; in addition, the protein is also associated with viral virulence. The L protein is the largest protein of the virus, is most conserved in NDV6 structural proteins, has biological activities such as RNA polymerase and post-transcriptional modification, and can form a virus replication complex together with NP and P proteins, and the complex is involved in transcription and replication of virus genomes.
At present, the laboratory detection technology of newcastle disease mainly comprises: conventional separation and identification, serological methods (hemagglutination assay (HA), hemagglutination inhibition assay (HI), agar diffusion Assay (AGP), fluorescent antibody technology (FA), enzyme-linked immunosorbent assay (ELISA), virus neutralization assay (VNT), latex agglutination assay (LAT), neuraminidase assay (NIT), Radioimmunoassay (RIA), molecular biological methods (RNA fingerprint, nucleic acid probe technology, RT-PCR technology) and the like, wherein the specificity and sensitivity of the enzyme-linked immunosorbent assay are high, and the enzyme-linked immunosorbent assay is suitable for detecting a large number of samples, and is widely applied to the detection of Newcastle disease virus antibodies.
The enzyme-linked immunoassay method for detecting the newcastle disease virus antibody mainly comprises competitive enzyme-linked immunoassay (c-ELISA), blocking enzyme-linked immunoassay (b-ELISA) and indirect blocking enzyme-linked immunoassay. The specificity and the sensitivity of the c-ELISA and the b-ELISA are higher, and the method is a clinically generally accepted newcastle disease virus antibody detection method. However, both c-ELISA and b-ELISA need monoclonal antibody, which results in greatly increased detection cost; moreover, c-ELISA and b-ELISA are complicated in operation, long in detection time and complex in criterion. On the other hand, the conventional indirect ELISA uses complete protein or recombinant protein derived from Newcastle disease virus as a coating antigen to detect antibodies in serum, which is less costly than c-ELISA and b-ELISA; however, since this method uses the intact protein as an antigen, erroneous recognition and non-specific recognition of the antibody easily occur, and thus, its specificity and sensitivity are inferior to those of c-ELISA and b-ELISA.
Therefore, there is a need to develop a newcastle disease virus antibody detection kit with low detection cost, short detection time, simple operation, and high specificity and sensitivity.
Disclosure of Invention
In view of the problems in the prior art, the present invention aims to provide a newcastle disease virus antibody detection kit which has low detection cost, short detection time, simple operation, and high specificity and sensitivity, and a polypeptide or a polypeptide combination which can be used for preparing the kit.
The inventor finds that: the indirect ELISA method based on epitope polypeptides has high specificity because epitope polypeptides (a single polypeptide of about 20 amino acids usually only contains one epitope) are used as coating antigens; however, the sensitivity of the method is often low, and the sensitivity of a single polypeptide generally does not exceed 50%. If the epitope polypeptide with high sensitivity can be found, the defects of the traditional indirect ELISA can be overcome, and a newcastle disease virus antibody detection kit with the specificity and the sensitivity comparable to those of c-ELISA and b-ELISA, or even higher than those of the traditional indirect ELISA is developed.
The inventors have conducted intensive studies to solve the above-mentioned technical problems and found, as a result, that: SEQ ID NO: 1, the sensitivity and the specificity of the polypeptide for independently detecting the newcastle disease virus antibody are respectively 88% and 96%, and the polypeptide can reach the standard of a detection kit when being independently used; when using SEQ ID NO: 1 and SEQ ID NO: 2, the sensitivity is 94% and the specificity is 93%, and the kit is one of the newcastle disease virus antibody detection kits with the most excellent comprehensive performance at present.
Accordingly, the present invention comprises:
1, SEQ ID NO: 1.
SEQ ID NO: 1 and the polypeptide shown in SEQ ID NO: 2.
3. Use of the above polypeptide or combination of polypeptides in the preparation of a kit for detecting the presence of antibodies against newcastle disease virus (IgY) in a biological sample of biological origin of a subject.
4. A newcastle disease virus antibody (IgY) detection kit, comprising one or more solid supports, and the following polypeptide combination 1 independently linked to the one or more solid supports;
polypeptide combination 1
SEQ ID NO: 1, and
SEQ ID NO: 2.
5. A newcastle disease virus antibody (IgY) detection kit comprising one or more solid supports and SEQ ID NO: 1.
6. The newcastle disease virus antibody (IgY) detection kit according to claim 4 or 5, which does not comprise other probe molecules (e.g. polypeptides, proteins or nucleic acids) for detecting newcastle disease virus antibodies (IgY).
7. The use of the polypeptide combination 1 in the preparation of a kit for detecting the presence of antibodies against newcastle disease virus (IgY) in a biological sample of biological origin of a subject;
polypeptide combination 1
SEQ ID NO: 1, and
SEQ ID NO: 2.
In the present specification, the subject organism is preferably an avian, more preferably a chicken.
In the present specification, the biological sample may be whole blood, plasma or serum.
The polypeptide and the kit can be used for detecting whether an anti-Newcastle disease virus antibody (IgY) exists in a biological sample of a biological source of a subject. Generally, antibodies against newcastle disease virus in a biological sample are generated by a subject organism infected with newcastle disease virus or immunized with newcastle disease vaccine, and thus the polypeptide and the kit can be used to diagnose whether a subject organism is infected with newcastle disease virus or immunized with newcastle disease vaccine.
Detailed description of the invention
First, the present invention provides SEQ ID NO: 1. The inventor finds that the sensitivity and the specificity of the polypeptide for detecting the Newcastle disease virus antibody are respectively 88% and 96%. Compared to the sensitivity of typically not more than 50% for a single polypeptide in an epitope-polypeptide-based indirect ELISA method, the amino acid sequence of SEQ ID NO: 1 is surprising, and the sensitivity of the polypeptide alone for detecting antibodies to newcastle disease virus is comparable to that of the conventional ELISA detection kit! Moreover, it also has a specificity of up to 96%. Therefore, it achieves unexpected technical effects.
In the present specification, sensitivity means: among positive samples confirmed by the "gold standard" method, the proportion of positive samples determined by other methods was determined. The specificity refers to: among negative samples confirmed by the "gold standard" method, the proportion of negative samples was determined by other methods. For the detection of newcastle disease virus antibodies, the "gold standard" in the art is the hemagglutination inhibition assay (HI).
The nucleotide sequence of SEQ ID NO: 1 can be used as a detection probe for preparing a kit for detecting whether an antibody against Newcastle disease virus exists in a biological sample of a biological source of a subject.
The inventors have also found that when using SEQ ID NO: 1 and the polypeptide shown in SEQ ID NO: 2, the sensitivity is 94 percent, the specificity is 93 percent, and the polypeptide combination can completely compare favorably with a c-ELISA method and a b-ELISA method. Accordingly, the present invention also provides the following polypeptide combination 1.
Polypeptide combination 1
SEQ ID NO: 1, and
SEQ ID NO: 2.
The polypeptide combination can be used as a detection probe for preparing a kit for detecting whether an antibody for resisting the Newcastle disease virus exists in a biological sample of a biological source of a detected object.
Therefore, the invention also provides a newcastle disease virus antibody detection kit, which comprises one or more solid carriers and the polypeptide combination 1 independently connected to the one or more solid carriers.
In the present specification, the solid support may be one or a plurality of solid supports, but preferably one, that is, all the polypeptides are independently attached to the same solid support. In the present invention, the solid carrier is not particularly limited as long as it is a carrier which is a solid or an insoluble material. The polypeptide can be linked to the solid support by methods known to those skilled in the art.
In the present specification, the subject organism is preferably an avian, more preferably a chicken.
In the present specification, the biological sample may be whole blood, plasma or serum.
In the case of detecting whether an antibody against newcastle disease virus is present in a biological sample of biological origin of a subject using the above-mentioned kit, when any one or more of the polypeptides in the polypeptide combination 1 responds to the biological sample of biological origin of the subject, it is determined that the antibody against newcastle disease virus is present in the biological sample of biological origin of the subject (i.e., positive); on the other hand, when none of the polypeptides in the polypeptide combination 1 responds to the biological sample of biological origin of the subject, it is determined that the biological sample of biological origin of the subject does not contain an antibody against newcastle disease virus (i.e., is negative).
In the present specification, "response" means: in the TMB color test, a bluish-purple signal stronger than that of the negative control point was observed with the naked eye.
Examples
1. Preparation and validation of Polypeptides
The polypeptides used in the examples were synthesized by gill biochemical (shanghai) ltd and confirmed by mass spectrometry. Wherein the content of the first and second substances,
SEQ ID NO：1：GDGETQFLDLMRAVANSMRE。
SEQ ID NO：2：IANCKMTTCRCVNPPGIISQ。
2. preparation of kit (detection chip)
Kit 1
The above-mentioned SEQ ID NOs: 1, and additionally spotting one chicken IgY spot as a positive quality control spot and one PB spot as a negative quality control spot to prepare the detection chip.
Kit 2
Except that the above-mentioned SEQ ID NOs: 1 and 2, preparing a detection chip according to the same preparation method as the kit 1, namely the kit 2.
3. Detection with a kit
Inspection step
1) Balancing reagent: all reagents were taken out of the refrigerator and allowed to equilibrate to room temperature for use.
2) Preparing a washing liquid: the concentrated washings were diluted 1:9 with purified or distilled water, examples: 50mL of the concentrated washing solution was prepared as 500mL of washing solution with purified water or distilled water, and mixed well. The unused lotion is stored in a refrigerator at 4 ℃ and is used up within 3 months.
3) Diluting the sample: taking a sample to be tested, diluting the sample with serum diluent according to a ratio of 1:100, for example: 198uL serum diluent is added with 2uL sample to be tested and fully mixed. For one sample detection, 200uL of diluted sample is used.
4) Chip wetting: and taking out the detection chip required by detection. 1mL of the wash solution was added to soak the chip surface for 3min, and then the wash solution was discarded.
5) Sample adding: in this embodiment, a single-sided biological incubation reactor is used. The diluted sample was added to the reactor through the liquid addition vent using a pipette gun in an amount of 200 uL. After the sample is added, the liquid adding vent hole is sealed by the sealing patch.
6) Sample incubation: the well-loaded reactor was placed on a horizontal shaker and incubated at 150rpm for 30min at room temperature.
7) Cleaning: the reaction column was taken out of the reaction chamber, the reaction solution was discarded, and the surface of the chip and the inside of the reaction chamber were washed with the washing solution and repeated 3 times (each time, about 12mL, for 1-2 min).
8) Incubation with enzyme-labeled secondary antibody: the reaction column is recombined with the reaction chamber and clamped. And (4) removing the sealing label, and adding 200uL of horseradish peroxidase-labeled goat anti-chicken IgY solution through the liquid-adding vent hole. The liquid feeding vent hole is closed again. Then placed on a horizontal shaker and incubated at 150rpm for 30min at room temperature.
9) And (3) cleaning again: the reaction column was taken out of the reaction chamber, the reaction solution was discarded, and the surface of the chip and the inside of the reaction chamber were washed with the washing solution and repeated 3 times (each time, about 12mL, for 1-2 min).
10) Color development: 100uL of TMB color development liquid is added on the surface of each reaction column chip respectively, and the color development liquid needs to be uniformly paved on the surface of the chip.
And (4) judging a result: the response of the polypeptide under the action of each serum is counted by visual observation (a bluish purple signal stronger than that of a negative control point can be seen by the naked eyes, and the response is judged). That is to say that the first and second electrodes,
for the above kit 1, when SEQ ID NO: 1, judging the polypeptide to be positive by the Newcastle disease virus antibody when the polypeptide responds; otherwise, the result is judged to be negative.
For the above kit 2, when SEQ ID NO: 1 and/or 2, and determining that the newcastle disease virus antibody is positive; otherwise, the result is judged to be negative.
566 chicken serum samples from each region of China were tested by the kit prepared in the above step 2 (according to the above step 3) and a conventional hemagglutination inhibition assay (HI), and the test results are shown below.
TABLE 1
Sensitivity 353/400 x 100% 88%
Specificity 160/166 96%
TABLE 2
Sensitivity 377/400 × 100%
Specificity 159/166 93%
As can be seen from the above, the above-mentioned kits 1 and 2 achieve a detection sensitivity of 85% or more by using only one or two polypeptides as a detection probe, and the specificity is 90% or more (and the specificity is verified by the HI method, not by the control kit method), which is sufficient to compare with the kits using the c-ELISA method or the b-ELISA method. It should be noted that the sensitivity and specificity of diagnostic kits generally considered to be clinically useful should be over 85%.
The present invention will be described more specifically with reference to the following examples, but the technical scope of the present invention is not limited thereto. The present invention can be easily modified/changed by those skilled in the art according to the description of the present specification, and these are included in the technical scope of the present invention.
Sequence listing
<110> Jiandong medicine science and technology Co., Ltd, Industrial park, Suzhou province
<120> newcastle disease virus antibody detection 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 (3)
1. The use of the polypeptide combination 1 in the preparation of a kit for detecting whether an anti-newcastle disease virus antibody IgY exists in a biological sample of biological origin of a subject;
polypeptide combination 1
SEQ ID NO: 1, and
SEQ ID NO: 2.
2. The use according to claim 1, wherein the subject organism is a chicken.
3. The use of claim 1, wherein the biological sample is whole blood, plasma or serum.
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| CN201810507587.3A CN108956986B (en) | 2018-05-24 | 2018-05-24 | Newcastle disease virus antibody detection kit |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| CA2029586A1 (en) * | 1990-11-08 | 1992-05-09 | Roberta Elaine K. Fulton | Amplified fluorogenic enzyme immunoassay |
| WO2002036617A2 (en) * | 2000-11-02 | 2002-05-10 | Akzo Nobel N.V. | A recombinant newcastle disease virus nucleoprotein mutant as a marker vaccine |
| 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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2018
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|---|---|---|---|---|
| CA2029586A1 (en) * | 1990-11-08 | 1992-05-09 | Roberta Elaine K. Fulton | Amplified fluorogenic enzyme immunoassay |
| WO2002036617A2 (en) * | 2000-11-02 | 2002-05-10 | Akzo Nobel N.V. | A recombinant newcastle disease virus nucleoprotein mutant as a marker vaccine |
| 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 |
| CN104820095A (en) * | 2015-04-22 | 2015-08-05 | 河南省康星药业股份有限公司 | Newcastle disease virus virulent strain colloidal gold test strip and preparation method thereof |
| CN206725579U (en) * | 2017-01-10 | 2017-12-08 | 深圳芬德生物技术有限公司 | A kind of more titres of newcastle epidemic disease antibody quantitatively detect box |
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| Title |
|---|
| La Sota与V4疫苗F蛋白的B细胞表位活性检测与比较;孙建宏 等;《动物医学进展》;20051031;第26卷(第10期);第55-60页 * |
| Localization of the antigenic sites of newcastle disease virus nucleocapsid using a panel of monoclonal antibodies;Raha Ahmad-Raus等;《Research in Veterinary Science》;20090228;第86卷(第1期);第174-182页， * |
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