CN110240635A - Detect the polypeptide and its ELISA detection kit of newcastle disease virus antibody - Google Patents
Detect the polypeptide and its ELISA detection kit of newcastle disease virus antibody Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 40
- 238000002965 ELISA Methods 0.000 title claims abstract description 30
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- 208000010359 Newcastle Disease Diseases 0.000 claims abstract description 9
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
- C12N2760/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a kind of polypeptide and its ELISA detection kit for detecting newcastle disease virus antibody, the polypeptide coated elisa plate that the kit passes through the newcastle disease virus HN albumen of screening, pass through the binding ability of serum antibody in test sample and polypeptide, by means of the antibody of the goat-anti chicken Ig γ of horseradish peroxidase-labeled, to detect newcastle disease virus antibody level in serum.It is feminine gender that the kit, which detects avian influenza virus H5, H7, H9 subclass antibodies, chicken infectivity bursa of Fabricius virus antibody, avian infectious bronchitis virus antibody,.The kit can detect blood clotting and (HI) potency inhibited to be greater than or equal to 4log2Serum, meet the requirement of clinical serum detection sensitivity.When detecting clinical serum simultaneously with blood clotting suppressing method, coincidence rate is up to 98.7%.And it is easy to operate, no special material requirement, result judgement objectivity is good, is not influenced by operator's subjective determination.It is suitable as the tool of newcastle disease vaccine immune rear antibody level detection and monitoring.
Description
Technical field
The present invention relates to agricultural biological technical fields, and in particular to it is a kind of detect newcastle disease virus antibody polypeptide and its
ELISA detection kit.
Background technique
Newcastle disease (Newcastle disease, ND) is the acute height of the chicken as caused by newcastle disease virus and a variety of birds
Contagious disease is spent, which propagates rapidly, and it is to endanger one of great epidemic disease of aviculture that case fatality rate is high.The main spy of this disease
Sign is expiratory dyspnea, diarrhea, neurological disorders, mucous membrane and serous coat bleeding.The disease can result in significant economic losses, and be provided by OIE
For the epidemic disease that must be reported, therefore countries in the world popular are paid much attention to the occurrence of this disease and.
Newcastle disease virus (Newcastle disease virus, NDV) belongs to paramyxovirus section, fowl Rubulavirus,
Complete virus particle subcircular, diameter are 100~500nm.The virus have cyst membrane structure, viral nucleic acid type be sub-thread regardless of
The RNA of segment is arranged in sequence with NP, P, M, F, HN and L gene on NDV genome, is separately encoded nucleocapsid protein (NP), phosphorus
Albumen (P), stromatin (M), fusion protein (F), hemagglutinin-neuraminidase albumen (HN) and high molecular weight protein (L).NP egg
White is a kind of and the closely related albumen of virus multiplication, and most important to the formation of virus nucleocapsid, it can wrap up genome
RNA forms the nucleocapsid of a spiral, has protective effect to viral RNA.P albumen is located in nucleocapsid, is the RNA that RNA is relied on
One of Liang Ge subunit of polymerase, main function are to participate in virus genomic duplication and transcription.In addition, P albumen also passes through
Its N-terminal inhibits self assembly of N protein, to prevent the nucleocapsid without viral RNA in conjunction with the N protein of newcastle disease virus
Formation.M albumen is a kind of non-glycosylated protein for being present in virus envelope internal layer, to viral capsule during Virus assemble
Identification between the formation of film, nucleocapsid and cyst membrane maintains Vims particle structures integrality to have important role.F protein is
The main structural proteins of NDV are located on virus envelope, are responsible for virus and merge with permissive cell, to keep cell entry thin
Born of the same parents simultaneously generate haemolysis, and protein cleavage site is related with virus virulence.HN albumen is a kind of existing hemagglutinin (HA) activity
There is neuraminidase (NA) active glycoprotein again, is NDV important virulence protein and protective antigens, determines jointly with F gene
The virulence for determining NDV is strong and weak.L albumen is the maximum albumen of NDV, it is the main composition portion of the RNA polymerase of RNA dependence
Point, viral replication complex can be collectively formed with NP albumen and P albumen, which participates in virus genomic transcription and answer
System.
Currently, China takes the prevention and control of newcastle disease the comprehensive prevention and control measure based on vaccine immunity.For a long time with
Carry out the method that the assessment to immune effect of vaccine mainly uses hemagglutination-inhibition test, it is believed that blood clotting inhibits potency to be greater than or equal to
4log2It is determined as the antibody level positive, immuno-competent；Blood clotting inhibits potency to be less than 4log2Then it is determined as antibody level feminine gender；It is logical
Often think that blood clotting inhibits potency to reach 1:8log2Or more when, then have High antibody level.But this method needs freshly prepared
Chicken red blood cell obtains to be related to complicated biomaterial, and the quality of chicken red blood cell is affected to the evaluation of result；Behaviour
As when need doubling dilution serum and step is more, more complicated when this method being caused to operate, especially conventional detection or monitoring is anti-
It is usually directed to very more samples when body level, needs great workload so as to cause this method, cause very big difficulty；Separately
Outside, it is affected when the result judgement of this method by Quality of Erythrocyte, artificial subjective determination factor etc., causes result judgement usual
Having a certain difference property.
For a long time, researcher always strive to develop it is a kind of it is easy, be easy to standardize, a large amount of blood serum samples be suitble to detect
Method.Elisa technique is common one kind, standardization, technology that is more sensitive and being suitble to a large amount of blood serum samples detections.But
For a long time, itself or sensitivity of the ELISA detection kit of the detection newcastle disease virus antibody level of serum of early development is not
Clinical requirement can be reached, and (blood clotting inhibits potency 4log2) or specificity it is bad (have with the antibody of other chicken cause of diseases intersects instead
It answers).
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of polypeptides for detecting newcastle disease virus antibody
And its ELISA detection kit, the polypeptide and kit can high sensitivity and newcastle disease virus in detection serum with high specificity
Antibody level；Be suitable as newcastle disease vaccine it is immune after antibody level detection and monitoring tool, be of great significance and valence
Value, and clinical urgent need.
In order to realize the first purpose, the present invention provides a kind of polypeptide for detecting newcastle disease virus antibody, the polypeptide is
Any one in NDV-F, NDV-H1, NDV-H2, NDV-H3 and NDV-H4, wherein
The amino acid sequence of NDV-F is LNKLEESNRKLDKVN, as shown in SEQ ID NO.1；
The amino acid sequence of NDV-H1 is
SAVFDSTSRSRITRVSSSSTKAAYTTSTCFK, as shown in SEQ ID NO.2；
The amino acid sequence of NDV-H2 is PDEQDYQIR, as shown in SEQ ID NO.3；
The amino acid sequence of NDV-H3 is
PDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, as shown in SEQ ID NO.4；
The amino acid sequence of NDV-H4 is
RYNDTCPDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, as shown in SEQ ID NO.5.
Preferably, the polypeptide is NDV-H4, amino acid sequence RYNDTCPDEQDYQIRMAKSSYKPG
RFGGKRIQQAILS, as shown in SEQ ID NO.5.
The polypeptide of above-mentioned five detections newcastle disease virus antibody is in analysis newcastle disease virus HN albumen and F protein sequence
On the basis of, it is analyzed according to sequence conservation, sequence hydrophily, B cell Antigen Epitope Prediction etc., has screened 5 kinds of polypeptides.Wherein,
NDV-H4 reactivity is best.
In order to realize second purpose, the present invention also provides a kind of ELISA for detecting newcastle disease virus antibody to detect examination
Agent box, it is characterised in that: the ELISA detection kit includes the ELISA Plate with the polypeptide of bovine serum albumin(BSA) BSA coupling.
Preferably, the ELISA detection kit further includes the goat-anti chicken of horseradish peroxidase (HRP) label
Ig γ, serum dilution, cleaning solution, standard positive control, negative control, substrate solution A, substrate solution B and terminate liquid.
Preferably, contain KH in every liter of the cleaning solution2PO40.2g, Na2HPO4·12H2O 2.9g, NaCl
8.0g, KCl 0.2g, Tween-20 0.5mL；
The confining liquid: skimmed milk 5g adds washing buffer to 100mL；
The serum dilution: bovine serum albumin(BSA) (BSA) 2g, skimmed milk 5g adds washing buffer to 100mL；
The enzyme labelled antibody: horseradish peroxidase (HRP) goat-anti chicken Ig γ marked is diluted using serum dilution
8000 times of preparations；
The substrate solution A:0.2M Na2HPO425.7mL, 0.1M citric acid 24.3mL add distilled water to 50mL, before use
3%H is added2O2150μL；
The substrate solution B:0.2M Na2HPO425.7mL, 0.1M citric acid 24.3mL add distilled water to 50mL, before use
The TMB ethanol solution 2.5mL of 2mg/mL is added；
The substrate solution: substrate solution A and substrate solution B equal proportion mix, ready-to-use；
The terminate liquid: 2.5mL hydrofluoric acid (HF) is added in 900mL water for injection, and is settled to 1000mL；
The standard positive control: the serum of SPF chicken preparation is immunized in Newcastle Disease Virus Vaccine, and blood clotting inhibition potency is
10log2；
The negative control: the serum of monthly age SPF chicken acquisition.
The method of newcastle epidemic disease antibody in the ELISA detection kit detection chicken serum of above-mentioned detection newcastle disease virus antibody,
The following steps are included:
1) preparation of sample to be tested: taking animal's whole blood, and after blood sufficiently solidifies, 4000r/min is centrifuged 10min, collects
Supernatant；Obtain sample to be tested
2) dilution of each sample:
A. the dilution of measuring samples: taking sample to be tested, is diluted with sample diluting liquid by 1:40, and mix well；
B. the dilution of negative serum: taking 1 part of negative serum, is diluted with sample diluting liquid by 1:40, and mix well；
C. the dilution of standard positive serum: 1 part of NDV standard positive serum of known HI potency is taken, is pressed with sample diluting liquid
1:40~1:2560 doubling dilution is sequentially added after mixing well into ELISA Plate hole；
3) ELISA Plate contained with the BSA polypeptide being coupled for taking -20 DEG C of preservations, first washs antigen coat with 1 × cleaning solution
Plate 1 time, the measuring samples for taking 100 μ L to dilute add in antigen coated microplate, 37 DEG C of incubation 45min；
4) liquid in hole is discarded, 1 × cleaning solution, 200 μ L is added in every hole, and it washes repeatedly 3 times, stands 3min every time, last
It is secondary to pat dry；
5) goat-anti chicken Ig γ secondary antibody 100 μ L, 37 DEG C of incubation 30min is added in every hole；
6) liquid in hole is discarded, is repeated board-washing 5 times with 1 × cleaning solution, is stood 3min every time, pat dry for the last time；
7) 100 μ L substrate developing solutions are added in every hole, and 37 DEG C are protected from light colour developing 15min；
8) every hole is added 50 μ L terminate liquids and terminates reaction after the completion of developing the color, and OD is surveyed in microplate reader in 10min630nmValue；
9) result judgement: the OD value as obtained by standard positive serum doubling dilution draw standard curve, and carry out OD value with
The conversion of HI potency (is faced by comparing the criterion whether the conversion HI potency for detecting serum reaches clinical HI test yin and yang attribute
With HI potency >=4log in bed2Antibody be determined as NDV positive antibody, otherwise be feminine gender), so that it is determined that detection serum yin
It is positive:
As HI potency >=4log that the serum testing result to be checked is conversion2When, it is judged to existing in serum to be checked anti-
The antibody (i.e. positive) of newcastle disease virus；
Conversely, as HI potency < 4log that the serum testing result to be checked is conversion2When, it is determined as in serum to be checked not
There are the antibody of anti-new castle disease virus (i.e. negative).
Beneficial effects of the present invention:
A kind of ELISA detection kit using polypeptide detection newcastle disease virus antibody of the present invention is that one kind is easy to standard
Change, technology that is more sensitive and being suitble to a large amount of blood serum sample detections.Using the ELISA kit to 449 portions of chickens of clinical source
Serum sample is detected, while carrying out Parallel testing using HI test.It is compared to HI test, this method detects clinical sample
Positive coincidence rate be 98.8%；Negative match-rate is 97%；The total coincidence rate of yin and yang attribute is 98.7%.This method has good
Sensibility and specificity, testing determined yin and yang attribute with HI has higher coincidence rate, being capable of effective monitoring and evaluation newcastle disease epidemic disease
The prevention and control of ND are of great significance and are worth by the chicken serum antibody level after seedling is immune.
Detailed description of the invention
Fig. 1 is the canonical plotting of NDV standard positive serum.
Specific embodiment
The present invention is described in further detail combined with specific embodiments below, so as to those skilled in the art understand that.
The polypeptide analysis of the detection newcastle disease virus antibody of embodiment 1
According to the amino acid sequence of newcastle disease virus HN albumen, its opposite conserved positions is analyzed by blast software,
And hydrophilicity analysis (hydrophilicity), flexibility (flexibility), non-obstructing property of binding protein sequence
(accessibility) B cell epitope (the http://crdd.osdd.net/raghava/ of comprehensive analysis albumen such as
Bcepred/), 4 kinds of sequences are had chosen as candidate polypeptide sequence, it may be assumed that NDV-H1, NDV-H2, NDV-H3 and NDV-H4.
According to the amino acid sequence of newcastle disease virus F protein, its opposite conserved positions is analyzed by blast software,
And hydrophilicity analysis (hydrophilicity), flexibility (flexibility), non-obstructing property of binding protein sequence
(accessibility) B cell epitope (the http://crdd.osdd.net/raghava/ of comprehensive analysis albumen such as
Bcepred/), a kind of sequence is had chosen as candidate polypeptide sequence, it may be assumed that NDV-F.
These candidate polypeptide sequences are done into sequence-specific analysis on the website NCBI, find these types of polypeptide sequence in addition to
Outside existing in fowl newcastle disease virus, in addition only discovery has homologous sequence in Xanthomonas citri, does not interfere this more
Specificity of the peptide as newcastle disease virus antibody test.
Therefore, by the synthesis of these polypeptides commission hundred Yi Xin Bioisystech Co., Ltd of Wuhan, and it is carried out by chromatography
Purifying.Polypeptide sequence is as follows:
SEQ ID NO.1:LNKLEESNRKLDKVN, is named as NDV-F, as shown in SEQ ID NO.1；
SEQ ID NO.2:SAVFDSTSRSRITRVSSSSTKAAYTTSTCFK, is named as NDV-H1, such as SEQ ID
Shown in NO.2；
SEQ ID NO.3:PDEQDYQIR, is named as NDV-H2, as shown in SEQ ID NO.3；
SEQ ID NO.4:PDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, is named as NDV-H3, such as SEQ ID
Shown in NO.4；
SEQ ID NO.5:RYNDTCPDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, is named as NDV-H4, such as SEQ
Shown in ID NO.5；
Embodiment 2 detects the screening of the polypeptide of newcastle disease virus antibody
By above-mentioned artificial synthesized polypeptide using glutaraldehyde method by itself and BSA carrier protein couplet, to improve polypeptide coating
Efficiency.Method particularly includes: 2mg carrier B SA is dissolved in 2mL PBS buffer solution (0.01mol/L, pH 7.4), in PBS buffer solution
Middle room temperature dialysed overnight；1mg/mL polypeptide is taken to add in the carrier B SA solution of 2mL, it is stirring while adding, it is dripped in Xiang Shangshu mixed liquor
Add 0.2% glutaraldehyde solution of 1mL, it is stirring while adding.4h is stirred under room temperature, and 400 μ L are added in Xiang Shangshu mixed liquor
1mol/L glycerol continues to stir 1h, is allowed to terminate reaction；Above-mentioned mixed liquor is packed into bag filter, is placed in PBS buffer solution 4 DEG C
Dialysed overnight；Collect peptide carrier conjugate, -80 DEG C of preservations.
It is added in ELISA Plate after using coating buffer to dilute by 1:40 respectively polypeptide after coupling obtained above, every polypeptide
Respectively plus 1 column, every 100 μ L of hole set and act on 15h at 2~8 DEG C.Coating buffer is discarded, every hole is added 200 μ L confining liquids, sets 37 DEG C of incubations
1h abandons hole inner sealing liquid.1:40,1:80,1:160,1 are done respectively to NDV positive serum and negative serum by sample diluting liquid:
Negative, each dilution of positive serum is added in different antigen polypeptide coatings hole respectively, measures each hole OD by 320 dilutions630nm
Value, and P/N value is calculated, select the maximum polypeptide of P/N value as optimum response polypeptide, for detecting the antibody indirect ELISA side NDV
The foundation of method, the results are shown in Table 1.
Table 1 detects the polypeptide of NDV antibody and the selection result (OD of BSA conjugate630nmValue)
Using 4 kinds of coupled peptides derived above as antigen, detects it and reacted with negative serum with NDV positive serum
Property, optimum response polypeptide is selected by P/N value.It is found by detection, the P/N value of NDV-H4 polypeptide is maximum, therefore selects NDV-
H4 polypeptide is used to detect the foundation of NDV antibody ELISA method.
The best peridium concentration of antigen and serum of 3 newcastle disease virus ELISA antibody assay kit of embodiment most preferably dilute
The determination of degree
The determination that antigen best peridium concentration and serum optimum dilution degree are carried out by square matrix titration, with coating buffer by NDV-
After H4 polypeptide and BSA conjugate are diluted by 1:100,1:200,1:400,1:800,1:1600, it is added separately in ELISA Plate, often
A dilution adds 1 column, and every 100 μ L of hole sets and acts on 15 hours at 2~8 DEG C.Coating buffer is discarded, 200 μ L confining liquids are added in every hole
(5% skimmed milk) is set and is incubated 1 hour at 37 DEG C, and hole inner sealing liquid is abandoned.Newcastle disease virus positive serum is made of sample diluting liquid
1:320,1:640,1:1280,1:2560,1:5120 dilution, negative serum does 1:40,1:80 dilution, by positive serum, feminine gender
Each dilution of serum is added separately to carry out ELISA detection in the coating hole of different antigen concentrations, measures each hole OD630nm
Value.Best antigen coat concentration and best serum diluted concentration are selected according to each hole readings.The results are shown in Table 2.
Table 2 determines the square matrix titration results (OD of best antigen coat concentration and best serum dilution630nmValue)
Square matrix titration results are shown, when antigen diluent degree is 1:800, and positive serum dilution is 1:40, HI potency
For 10log2, it is 4log that positive serum, which is diluted to HI potency,2When OD630nmValue for 0.4736 (clinically usually with HI potency <
4log2When determine the serum for feminine gender), hence it is evident that greater than the OD of the diluted negative serum of 1:40630nmValue 0.088.Therefore it selects anti-
Former best diluted concentration is 1:800 dilution, and serum optimum dilution degree is 1:40 dilution.
Therefore, the antigen coated microplate of the ELISA kit using be added on ELISA Plate above-mentioned NDV-H4 polypeptide with
BSA conjugate solution after 2~8 DEG C are incubated for 15 hours, discards the coating buffer in hole, and every hole adds 200 μ L of confining liquid, and 37 DEG C incubate 1
Hour, -20 DEG C of preservations.The diluted concentration for detecting antibody is 1:40.
The determination of 4 yin and yang attribute criterion of embodiment and sensibility, specificity, the detection with HI yin and yang attribute coincidence rate
It is measured by the NDV positive serum to known HI potency, is drawn and marked according to the OD value of its doubling dilution serum
Directrix curve, and carry out OD value and the conversion of HI potency.This method is to HI potency in 4log2~8log2Between positive serum detection
In obvious linear relationship, R2=0.9902, show that the linear relationship is good, as a result as shown in Figure 1.According to positive serum HI potency
In 4log2OD value determine yin and yang attribute criterion, converted by OD value and HI potency, when detection serum conversion HI effect
Valence >=4log2When determine the serum for the positive；Detect conversion HI potency < 4log of serum2When determine the serum for feminine gender.
5 parts of NDV negative serums are detected using the ELISA kit, OD630nmValue is followed successively by 0.0918,
0.1685,0.055,0.0206,0.1332, the HI potency of conversion is respectively less than 4log2, illustrate that this method testing result and HI are tried
It is consistent to test testing result, i.e., 5 parts of serum are feminine gender.
In order to determine the sensibility of ELISA kit, the positive clinical serum of HI potency known to 5 parts selected at random is done
1:40~1:2560 doubling dilution is detected, and the results are shown in Table 3, shows that the ELISA kit can be detected accurately
HI potency is 4log2Or more serum.HI can be distinguished and test distinguished yin and yang attribute serum, the sensibility with HI test
It is almost the same.
Result (the OD of table 3ELISA method detection doubling dilution positive clinical serum630nmValue)
In order to determine the specificity of ELISA kit, using the ELISA method to H_5 subtype (AIV-
H5), avian influenza virus H7 hypotype (AIV-H7), avian influenza virus H9 hypotype (AIV-H9), avian infectious bronchitis virus
(IBV), infections chicken cloacal bursa virus (IBDV) positive serum is detected, OD630nmValue be respectively 0.116,0.1716,
0.2134,0.0969,0.1612, the HI potency of conversion is below 4log2, illustrate that NDV-H4 polypeptid specificity is good, with it
Cross reaction is not present by main infectious diseases of chicken positive serum in he.
In order to further determine coincidence rate when kit clinical detection with HI test, tested using the kit and HI flat
449 parts of chicken serum samples of row detection clinical source, testing result are as shown in table 4.It is compared to HI test, kit detection
The positive coincidence rate of clinical sample is (411 ÷ 416) × 100%=98.8%；Negative match-rate is (32 ÷ 33) × 100%=
97%；The total coincidence rate of yin and yang attribute is (443 ÷ 449) × 100%=98.7%.
The yin and yang attribute coincidence rate of 4 kit of table and HI testing inspection clinical sample
Other unspecified parts are the prior art.Although above-described embodiment is made that the present invention and retouches in detail
State, but it is only a part of the embodiment of the present invention, rather than whole embodiments, people can also according to the present embodiment without
Other embodiments are obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.
Claims (6)
1. it is a kind of detect newcastle disease virus antibody polypeptide, it is characterised in that: the polypeptide be NDV-F, NDV-H1, NDV-H2,
Any one in NDV-H3 and NDV-H4, wherein
The amino acid sequence of NDV-F is LNKLEESNRKLDKVN, as shown in SEQ ID NO.1；
The amino acid sequence of NDV-H1 is SAVFDSTSRSRITRVSSSSTKAAYTTSTCFK, as shown in SEQ ID NO.2；
The amino acid sequence of NDV-H2 is PDEQDYQIR, as shown in SEQ ID NO.3；
The amino acid sequence of NDV-H3 is PDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, as shown in SEQ ID NO.4；
The amino acid sequence of NDV-H4 is RYNDTCPDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, such as SEQ ID NO.5 institute
Show.
2. detecting the polypeptide of newcastle disease virus antibody according to claim 1, it is characterised in that: the polypeptide is NDV-H4,
Its amino acid sequence is RYNDTCPDEQDYQIRMAKSSYKPGRFGGKRIQQAILS, as shown in SEQ ID NO.5.
3. a kind of ELISA detection kit for detecting newcastle disease virus antibody, it is characterised in that: the ELISA detection kit
It include the ELISA Plate with the polypeptide of bovine serum albumin(BSA) coupling.
4. detecting the ELISA detection kit of newcastle disease virus antibody according to claim 3, it is characterised in that: as excellent
Scheme is selected, the ELISA detection kit further includes the goat-anti chicken Ig γ of horseradish peroxidase (HRP) label, serum dilution
Liquid, cleaning solution, standard positive control, negative control, substrate solution A, substrate solution B and terminate liquid.
5. detecting the ELISA detection kit of newcastle disease virus antibody according to claim 4, it is characterised in that: described to wash
It washs and contains KH in every liter of liquid2PO40.2g, Na2HPO4·12H2O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20
0.5mL；
The confining liquid: skimmed milk 5g adds washing buffer to 100mL；
The serum dilution: bovine serum albumin(BSA) 2g, skimmed milk 5g add washing buffer to 100mL；
The enzyme labelled antibody: the goat-anti chicken Ig γ of horseradish peroxidase-labeled is diluted into 8000 times of systems using serum dilution
It is standby；
The substrate solution A:0.2M Na2HPO425.7mL 0.1M citric acid 24.3mL adds distilled water to 50mL, uses preceding addition
3%H2O2150μL；
The substrate solution B:0.2M Na2HPO425.7mL, 0.1M citric acid 24.3mL add distilled water to 50mL, use preceding addition
The TMB ethanol solution 2.5mL of 2mg/mL；
The substrate solution: substrate solution A and substrate solution B equal proportion mix, ready-to-use；
The terminate liquid: 2.5mL hydrofluoric acid is added in 900mL water for injection, and is settled to 1000mL；
The standard positive control: the serum of SPF chicken preparation is immunized in newcastle disease vaccine, and it is 10log that blood clotting, which inhibits potency,2；
The negative control: the serum of monthly age SPF chicken acquisition.
6. the side of newcastle disease virus antibody in a kind of ELISA detection kit detection chicken serum for detecting newcastle disease virus antibody
Method, it is characterised in that: the following steps are included:
1) preparation of sample to be tested: taking animal's whole blood, and after blood sufficiently solidifies, 4000r/min is centrifuged 10min, collects supernatant；
Obtain sample to be tested.
2) dilution of each sample:
A. the dilution of measuring samples: taking sample to be tested, is diluted with sample diluting liquid by 1:40, and mix well；
B. the dilution of negative serum: taking 1 part of negative serum, is diluted with sample diluting liquid by 1:40, and mix well；
C. the dilution of standard positive serum: taking 1 part of NDV standard positive serum of known HI potency, presses 1:40 with sample diluting liquid
~1:2560 doubling dilution, sequentially adds after mixing well into ELISA Plate hole；
3) ELISA Plate for taking -20 DEG C of preservations, is first washed antigen coated microplate 1 time with 1 × cleaning solution, takes 100 μ L to dilute to be checked
Sample adds in antigen coated microplate, 37 DEG C of incubation 45min；
4) liquid in hole is discarded, 1 × cleaning solution, 200 μ L is added in every hole, washes repeatedly 3 times, stands 3min every time, and last time is clapped
It is dry；
5) goat-anti chicken Ig γ secondary antibody 100 μ L, 37 DEG C of incubation 30min is added in every hole；
6) liquid in hole is discarded, is repeated board-washing 5 times with 1 × cleaning solution, is stood 3min every time, pat dry for the last time；
7) 100 μ L substrate developing solutions are added in every hole, and 37 DEG C are protected from light colour developing 15min；
8) every hole is added 50 μ L terminate liquids and terminates reaction after the completion of developing the color, and OD is surveyed in microplate reader in 10min630nmValue；
9) result judgement: the OD value as obtained by standard positive serum doubling dilution draws standard curve, and carries out OD value and HI effect
Whether valence conversion reaches the criterion of clinical HI test yin and yang attribute by comparing the conversion HI potency for detecting serum, thus really
The yin and yang attribute of regular inspection survey serum:
As HI potency >=4log that the serum testing result to be checked is conversion2When, it is determined as in serum to be checked that there are anti-newcastle diseases
The antibody of virus, i.e., it is positive；
Conversely, as HI potency < 4log that the serum testing result to be checked is conversion2When, it is determined as in serum to be checked that there is no anti-
The antibody of newcastle disease virus, i.e., it is negative.
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| 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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