CN104961809B - The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa - Google Patents
The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa Download PDFInfo
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- CN104961809B CN104961809B CN201510402149.7A CN201510402149A CN104961809B CN 104961809 B CN104961809 B CN 104961809B CN 201510402149 A CN201510402149 A CN 201510402149A CN 104961809 B CN104961809 B CN 104961809B
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of hostis pecoris structural proteins antibody ELISA immunity detection reagents.A kind of Foot-and-mouth disease antibody ELISA immunity detection reagent, including the coated enzyme-linked reaction plate of hostis pecoris Structural protein VP1 antigen epitope polypeptide and enzyme label antiantibody；The Foot-and-mouth disease VP1 antigen epitope polypeptides are polypeptide in sequence table shown in sequence 1, the polypeptide in the polypeptide in sequence table shown in sequence 2 and sequence table shown in sequence 3.This kit is coated with reaction plate using chemical synthesis VP1 Antigenic Peptides, and antigen dosage is few, sensitivity and specificity are high, can efficiently detect whether that there are mouth disease virus infections.Kit specificity of the present invention is good, sensitive, efficient, has good market prospects.
Description
Technical field
The present invention relates to a kind of Foot-and-mouth disease antibody ELISA immunity detection reagents, particularly ox aftosa
O-shaped virus structural protein VP1 antibody ELISA immunity detection reagents.
Background technology
Aftosa (Foot and Mouth Disease, FMD) is by foot and mouth disease virus (Foot-and-mouth
Disease virus, FMDV) caused by infect acute, hot, high degree in contact infectiousness based on artiodactyl and quick remote
The animal epidemic of propagation, with infectiousness is strong, spread speed is fast, harm is serious and famous.World Organization for Animal Health (OIE)
The many animals that being classified as must be notified to suffer from one of infectious disease altogether, and China is classified as a kind of zoonosis.
Aftosa infectiousness is high, propagates rapidly, and the livestocks such as infected pigs, ox, sheep often result in cub death, adults production
Ability drastically declines.The disease propagate it is rapid, can cause it is global be very popular, huge economic damage is not only caused to animal husbandry
It loses, and seriously affects international trade and the reputation of animals and animal product.Due to the significant damage of FMD, U.S.'s authority
《Livestock contagious disease》Early in just statement in version in 1981：FMD is not only economy disease, while is also political epidemic disease.
Foot and mouth disease virus belongs to Picornaviridae, Hostis.By the 20 symmetrical capsid of face body of "false" and
Viral nucleic acid is formed, and the entire shape of the virion is spherical, is not stringent positive 20 face body.Complete virion includes
Capsid and RNA two parts, capsid is by each 60 molecular compositions of 4 kinds of structural proteins such as VP1, VP2, VP3 and VP4.Structural protein VP1
Virion surface is largely exposed to, is to determine that the main component of virus antigenicity and stimulation body generate neutralizing antibody
Major protein.VP1 variability is maximum in 4 kinds of structural proteins, and VP2 is more conservative, and VP4 is most conservative.The work(of viral capsid proteins
Can have：RNA is protected to degrade from nuclease；It identifies particular cellular receptors, determines host range and tissue tropism；Determine virus
Antigenicity；Release and transmission viral RNA are entered by cell membrane in permissive cell；Guidance selection and packaging virus RNA.
The aftosa serum detection method that OIE in 2004 is announced has 3 kinds：Virus neutralization tests (VNT), liquid phase block
ELISA (LPB-ELISA), solid phase competitive ELISA (SPC-ELISA), wherein virus neutralization tests are the most classical in 3 kinds of methods
Method, be evaluate other methods gold standard.1996, aftosa world reference laboratory established liquid phase blocking
ELISA method by constantly improve and perfects, it has also become the method for the extensive Serologic detection in various countries is exempted from for detecting animal
Epidemic disease antibody, carries out serosurvey etc. at assessment animal immune situation, and the sensibility, specificity and stability of this method exist always
It gains public acceptance in the world, is to use most wide aftosa serum detection method at present.2001, aftosa world reference experiment
Room establishes solid phase competitive ELISA method, and this method is special other than the traditional advantage with Liquid-phase blocking ELISA method
Property it is more preferable, operate more simple (its operating time is only 3.5h), 2004 by OIE be asserted aftosa serum investigate most
It is new to recommend method.
Aftosa is to be related to the great animal epidemic of economic security of the country.At present, most developing countries and area
The prevalence of aftosa is all controlled by the method for vaccine inoculation.China prevention and control FMD mainly takes vaccine immunity and antibody detection
Based on Synthetical prevention policy, drove entirety antibody level is improved by vaccine inoculation and prevents the infection of aftosa and propagation.
Therefore quick diagnosis, ox Efficacy evaluation, natural infection and the antidiastole of vaccine immunity of infection of foot-and-mouth disease become prevention and control
The Some Key Technologies of aftosa.
Invention content
The object of the present invention is to provide a kind of new for diagnosing whether aftosa susceptible animal ox infects the O-shaped disease of aftosa
The structural proteins antibody ELISA immunity detection reagent of poison.
The present invention provides also a kind of Foot-and-mouth disease VP1 antigen epitope polypeptides, for shown in sequence 1 in sequence table
Polypeptide, the polypeptide in the polypeptide in sequence table shown in sequence 2 or sequence table shown in sequence 3.
The present invention provides also a kind of Foot-and-mouth disease VP1 antigen epitope polypeptide compositions, is by sequence table
The mixture of polypeptide composition in polypeptide and sequence table in polypeptide, sequence table shown in sequence 1 shown in sequence 2 shown in sequence 3.
The Foot-and-mouth disease antibody ELISA immunity detection reagent of the present invention, including foot and mouth disease virus structure egg
The coated enzyme-linked reaction plate of VP1 antigen epitope polypeptides and enzyme label antiantibody in vain；The O-shaped virus structural protein VP1 of aftosa
Antigen epitope polypeptide is polypeptide in sequence table shown in sequence 1, sequence 3 in the polypeptide in sequence table shown in sequence 2 and sequence table
Shown polypeptide.
3 institute of sequence in polypeptide and sequence table in polypeptide, sequence table in the sequence table shown in sequence 1 shown in sequence 2
The mass ratio of the polypeptide shown is (0.5~2.0): (0.5~2.0): 1, preferably 1: 1: 1.Involved envelope antigen is using solid
The method production of phase chemical synthesis, envelope antigen contain the major antigenic sites of Foot-and-mouth disease VP1, can be with mouth hoof
The Structural protein VP1 antibody specific bond generated after the infection of epidemic disease poison or after immune.The marker enzyme of enzyme label antiantibody is
Horseradish peroxidase or alkaline phosphatase；The enzyme label antiantibody marks goat-anti ox IgG for enzyme；The enzyme marks antiantibody
The preferably goat-anti ox IgG of horseradish peroxidase-labeled.
The enzyme-linked reaction plate is detachable 96 hole elisa Plates；The Foot-and-mouth disease VP1 epitopes are more
Peptide, which is that chemistry is artificial synthesized, to be obtained.
The optimum preparating condition of the coated enzyme-linked reaction plate of Foot-and-mouth disease VP1 antigen epitope polypeptides
It is：The Foot-and-mouth disease VP1 antigen epitope polypeptides are dissolved in the carbonate solution of the pH9.6 of 100 μ l, Ran Houjia
To 96 hole polystyrene enzyme-linked reaction plates, per hole 150ng, placed at 37 DEG C and stand overnight that (8-12 is small at 2~4h, then 4~8 DEG C
When), polypeptide antigen is made fully to be combined with enzyme-linked reaction plate, is then added according to 300 μ l/ holes pure containing 1% (g/ml) ox blood
The PBS buffer solution (pH=7.4) of albumen (BSA), 37 DEG C of 2~3h of Seal treatment, with washing buffer, (concentrated cleaning solution distills
The 20 times of dilutions of water or deionized water obtain washing buffer) washing after dry, after enzyme-linked reaction plate drying after 4 DEG C be sealed.
Contain developing solution and terminate liquid in the kit；When marker enzyme be horseradish peroxidase when developing solution by developing the color
Liquid A liquid and developing solution B liquid composition, the developing solution A liquid are the citrate-phosphate buffer of the hydrogen peroxide urea containing 0.6mg/ml
Liquid；The developing solution B liquid is tetramethyl benzidine (TMB) solution of 0.2mg/ml, is mixed both during use with 1: 1 ratio；
When marker enzyme is alkaline phosphatase, developing solution is 4- nitrophenols phosphate buffers；The terminate liquid is the sulfuric acid of 2mol/L
Solution.
The kit further includes negative control sera, positive control serum；The negative control sera is with no mouth hoof
The normal calf serum of epidemic disease antibody；The positive control serum is with the Foot-and-mouth disease VP1 antigen epitope polypeptides
The serum obtained for immunogen immune ox；
The positive control serum is specifically envelope antigen (the foot and mouth disease virus structure egg synthesized with above-mentioned artificial chemistry
White VP1 antigen epitope polypeptides) as immunogene, 6 monthly age health oxes (antibodies against foot-and-mouth disease virus is negative) ox is immunized, prepares height and exempts from
Serum adds in 1000U/ml streptomysins and 1000U/ml penicillin, crosses 0.2 μm of filter membrane degerming, the positive control as kit
Serum.
The negative control sera is after being quarantined 7 with 6 monthly age health oxes (antibodies against foot-and-mouth disease virus negative),
Jugular vein aseptic collection blood adds in 1000U/ml streptomysins and 1000U/ml penicillin, 0.2 μm of filter membrane degerming is crossed, as examination
Agent box negative control sera.
The kit further includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid is contains 0.5% (g/ml) junket
The PBS buffer solution (crossing 0.45 μm of filter membrane) that 0.01M, pH of albumen are 7.4；Concentrated cleaning solution：0.01M, pH 7.4, contains
The phosphate buffer of 0.5% (ml/ml) Tween-20 and 0.05% (g/ml) Sodium azide preservative (is removed by 0.2 μm of filter membrane
Bacterium).
The detection program of kit of the present invention is：
1st, it balances：Kit from cold storage environment is taken out, it is spare to put equilibrium at room temperature 30min；The preceding mixing of liquid reagent.
2nd, with liquid：The 20 times of dilutions of concentrated cleaning solution distilled water or deionized water are obtained into washing buffer；
3rd, it sets：1 blank well, 3 negative control holes and 2 Positive control wells are stayed, remaining is sample to be tested hole.
4th, sample pre-dilution to be measured：Using sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to 1: 21 dilution proportion；Add 100 μ l Sample dilutions per hole in dilution plate hole, be separately added into 5 μ l samples to be tested.
5th, it is loaded：Blank well is not loaded；3 negative control holes respectively add 100 μ l negative controls, and 2 Positive control wells respectively add
100 μ l positive controls；Remaining hole is respectively by presetting plus the 100 diluted samples to be tested of μ l.Sample-adding process time span should be use up
It measures short.
6th, it incubates：Mixing is shaken, is put in 37 DEG C of incubators or water-bath, reacts 30min.
7th, board-washing：Reaction solution is discarded, the washing buffer after 300 μ l dilutions is added per hole, 15s is impregnated, gets rid of and abandon washing lotion.Continuously
It is patted dry after board-washing 4 times.
8th, it is enzyme：Blank well is not enzyme；Remaining each hole adds the goat-anti ox IgG of 100 μ l horseradish peroxidase-labeleds.
9th, it incubates：It puts in 37 DEG C of incubators or water-bath, reacts 30min.
10th, board-washing：Reaction solution is discarded, the 300 μ l of washing buffer after dilution are added in per hole, 15s is impregnated, gets rid of and abandon washing
Liquid.It is patted dry after continuous board-washing 5 times.
11st, it develops the color：Being separately added into the substrate developing solution of 100 μ l per hole, (wherein solution A and solution B are using volume ratio as 1: 1
Ratio mixes).It is capped, reacts 15min in 37 DEG C of insulating boxs；
12nd, 50 μ l terminate liquids are separately added into per hole and terminate reaction, mixing；
13rd, the OD per hole is measured450Value (adds the reaction plate of terminate liquid OD should be read in 15min450Value).
The judgement of testing result：
1st, negative control OD450Average value should≤0.15, otherwise in vain.
2nd, each detected value of positive control should be between 0.9-1.9, otherwise in vain.
3rd, the calculating of critical value：Critical value=0.17 × positive control OD450It is worth average value.
Serum to be checked measures OD450Value >=critical value person is judged to the positive；Serum to be checked measures OD450Value ＜ critical value persons are judged to
It is negative.
The kit of the present invention, available for detecting aftosa structural proteins antibody, to judge whether tested animal infects mouth
Aphtovirus or inoculation aftosa vaccine, wherein, animal aftosa viral disease is hostis pecoris disease, is specifically as follows ox mouthful
Hostis pecoris disease caused by the O-shaped virus of fever aphthous.
Foot and mouth disease virus structure described in above-mentioned Foot-and-mouth disease VP1 antigen epitope polypeptides or claim 2
Albumen VP1 antigen epitope polypeptides composition is preparing the application in detecting whether the kit of infection animal hoof-and-mouth disease viral disease
It belongs to the scope of protection of the present invention.
The positive effect of the present invention is：
The present invention carries out Accurate Analysis using bioinformatics method to the epitope of structural proteins, from VP1 albumen
Main Antigenic on filter out the peptide fragments of suitable ELISA detections.The peptide fragment has concentrated epitope, has sensitivity
The advantages of height, high specificity.
Meanwhile it is tried using advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen for being coated with enzyme reaction plate and preparing
Positive control serum in agent box.
In addition, since the envelope antigen used in kit is chemically synthesized polypeptide, without foreign protein, purity is high, into one
Step improves the efficiency of detection aftosa structural proteins antibody, to judge whether tested animal infects foot and mouth disease virus or inoculation epidemic disease
Seedling.
In short, the present invention establishes a species specificity, sensibility and reproducible indirect ELISA using VP1 structural proteins
Method provides a kind of simple and effective new method for the detection of ox aftosa immune antiboidy and the diagnosis of infection antibody, is mouth hoof
Epidemic disease Synthetical prevention provides technical support.The Foot-and-mouth disease ELISA diagnostic kit of the present invention is used
Foot and mouth disease virus or inoculation aftosa vaccine whether are infected in detection animal, is advantageously reduced caused by China's aftosa outburst
Loss, is also beneficial to the foundation of China's aftosa prevention and control system.
Kit of the present invention is coated with integrated enzyme reaction using the Antigenic Peptide of chemical synthesis Structural protein VP1 major antigenic sites
Plate, antigen dosage is few, high sensitivity, high specificity, after mouth disease virus infection or inoculation inactivated vaccine can be effectively detected
The Structural protein VP1 antibody of generation, to judge whether tested animal infects foot and mouth disease virus or vaccine inoculation, with ox hoof-and-mouth disease
Malicious non-structural protein antibody assay kit combination, can distinguish mouth disease virus infection animal and vaccine immunity animal.Experiment knot
Fruit shows that kit of the invention is reproducible, high specificity, high sensitivity.The needs of different levels personnel can be met, had
Vast market prospect and good economical, societal benefits.
Specific embodiment
Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is conventional method unless otherwise specified.Medicine used in following embodiments
Material raw material, reagent material etc. unless otherwise specified, are commercially available products.
The preparation of the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent envelope antigen of embodiment 1, ox aftosa
This experiment is directed to the difference of domestic aftosa prevalence strain, using bioinformatics method to aftosa O-shaped virus
The multiple hypotypes of VP1 structural proteins carry out Accurate Analysis, filter out suitable peptide fragment, are respectively synthesized out with full-automatic polypeptide synthetic instrument
According to 3 peptides of popular strain sequence design, sequence is respectively shown in sequence 1 in sequence table, sequence 2 and sequence 3, and purity is made
The about 90% more complete envelope antigen of update, can adapt to the characteristic that foot and mouth disease virus is constantly mutated, improves the inspection of antibody positive
Extracting rate.Polypeptide synthesis method can be conventional method, and the present invention synthesizes three polypeptides of the present invention with the following method, as this hair
The coating antigen of bright kit.
Applied Biosystem full-automatic polypeptide synthetic instruments (model 433A) can be used to make for the envelope antigen of the present invention
It is standby.With Merrifield solid-phase synthesis, using Fmoc (9-fluorenylmethyloxycarbonyl, 9- fluorenes first
Oxygen carbonyl) modification amino acid, using Rink Amide MBHA resins as solid phase carrier.Production process includes polypeptide antigen
Synthesis in solid state, polypeptide cleavage and identification, five parts of antigen purification, freeze-drying and preservation.It illustrates individually below：
First, envelope antigen synthesis in solid state
1st, the preparation of synthetic agent
Synthesize envelope antigen amino acid sequence such as SEQ ID NO：1、SEQ ID NO：2 and SEQ ID NO：Shown in 3.
Prepare the amino acid (being purchased from NOVA companies) of suitable Fmoc modifications according to envelope antigen sequence and synthesis scale,
It adds in into corresponding Cartridge.Equally synthesis scale claims resin 5g as required, is put into reaction chamber, upper and lower lid is tightened,
The weight of labelling, the title of peptide synthesized by record, lot number, the TARE of reaction chamber and alleged resin.Reaction chamber is packed into and is synthesized
Instrument.It prepares suitable synthetic agent and includes 100% NMP, 3% AIM (hexanoyl imidazoles), 35% PIP (piperidines), 100%
MeOH (methanol) etc. be placed into corresponding reagent bottle.
2nd, the detection of synthesizer state
It checks 433A Peptide systhesis instrument whether normal operation, after booting, runs Run Self Test programs, instrument is certainly
Whether normal examine indices.In addition check whether nitrogen is sufficient, whether normal (the normal gauge pressures of 433A of system gauge pressure
10.2psi).The performance of reply instrument is had gained some understanding before synthesis, so to be measured to the flow velocity of each synthetic agent.433A
Synthesizer：Flow Rate1-18 are sent to synthesizer, Main Menu-Module Test- is selected to be looked for by Prer or next
Module A, ModuleD, ModuleI, ModuleI, Module A)-measured or observed by more by Start-, if stream
It measures improper, then adjusts lower valve pressure, until reaching requirement (specific testing requirements see the table below 1).
1. Peptide synthesizer flow rate detection standard scale of table
| Reagent | Bottle number | Module | Critical field |
| 35%Piperidine | 1 | A | 1.0~1.2ml |
| 3%AIM | 4 | D | 1.0~1.2ml |
| 100%MeOH | 9 | I | 3.5~4.0ml |
| DIC | 8 | I | 0.45~0.55g |
| 100%NMP | 10 | A | 2.6~2.8ml |
3rd, envelope antigen synthesis starts
The amino acid sequence that will be synthesized in the program of 433A synthesizers send 1.0 Sol DIC90 of Std Fmoc to
On synthesizer.The sequence of File-New-Sequence- Edit and Compose peptides preserves.File-New-Run checks that Chemistry is
No is 1.0 Sol DIC 90 of Std Fmoc；Whether Sequence is to be deposited name；Set Cycles；It preserves.It is finally sent to
On synthesizer.
Main Menu-Cycle Monitor-begin, bring into operation.
4th, envelope antigen synthesis carries out
The removing of Fmoc groups, the electron attraction of the fluorenes ring system of Fmoc groups make 9-H have acidity, are easily removed compared with weak base
It goes, when reaction is eliminated to form hexichol fluorenes alkene with piperidines (PIP) attack 9-H, β, it is easy to be formed by two level cyclammonium attack stable
Addition product."-NH is exposed after the removing of Fmov groups2" group to be to carry out synthetic reaction.Then the next of activation is added in
In the amino acid and I-hydroxybenzotriazole (1-hydroxybenzotriazole, HOBT) to reactor of Fmoc radical protections.
Peptide sequence as described above, synthesis when are to N-terminal, according to specific sequence, successively constantly since C-terminal
Repeat synthesis step (synthesizer is automatically performed by program, specific circulation step such as the following table 2).Period observe and record reagent dosage and
Operating condition.
2. envelope antigen of table synthesizes circulation step
5th, envelope antigen synthesis terminates
Synthesizer will be automatically stopped, and peptide resin (peptide is additionally attached on resin now) base after envelope antigen synthesizes
This washes clean.Then reactor is removed from Peptide synthesizer, then after washing peptide resin 3 times with 100% methanol, in draught cupboard
Then polypeptide resin is fully transferred in the polyethylene bottle of brown by interior drying, be put into -20 DEG C of refrigerators, and sealed membrane sealing is standby
With.
2nd, the cracking and identification of envelope antigen
1st, the cracking of polypeptide antigen
It is chemically bound together through the obtained polypeptide of above-mentioned reaction with solid phase carrier, it is necessary to by specific
The acidolysis of organic acid polypeptide is detached with solid phase carrier.Also the guarantor on each amino acid functional group is eliminated while acidolysis
Protect base.Step is as follows：
The polypeptide resin (referring to peptide to be additionally attached on resin) of synthesis is taken out out of refrigerator, is put into the round-bottomed flask of a 2L
It is interior, the tripropyl of 90ml trifluoroacetic acids (Trifluoroacetic acid, TFA), 10ml are added in into flask in draught cupboard
Flask, is then steadily placed on magnetic stirring apparatus by silane (TIS) and magnetic stick, and persistently stirring 1h extremely reacts at room temperature
Completely.After reaction, the TFA in 30~120min removing crude products is persistently evaporated using the Rotary Evaporators with cold-trap.So
The crude product of polypeptide antigen is cleaned multiple times with dimethylformamide (DMF) afterwards, finally by the resin mixed sand core funnel
It filters out, both obtains envelope antigen.
2nd, the identification of envelope antigen
With substance assistant laser desorpted winged examination time mass spectrum method after polypeptide antigen synthesis
(MODAL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis, with common amino
Acid analysis identifies synthesized peptide.
3rd, envelope antigen purifies
The polypeptide antigen after cyclisation is carried out ultrafiltration using circulating tangential flow filtration film packet (to be produced with PALL companies
The circulating tangential flow filtration film packets of Tangential Flow Device and the peristaltic pump mating with it), polypeptide antigen is as big
Molecule cannot be by the filter membrane of certain pore size, and the small molecule that building-up process early period and later stage cyclization are formed or introduced is miscellaneous
Matter can then pass through filter membrane.Then again by aperture be 0.2 μm of filter degerming, last acquired solution is dispensed into aseptic plastic
It is labelled in bottle.Title, number, product batch number, concentration, date of manufacture, pot-life and the preservation of polypeptide are indicated on label
Condition, after packing, be stored in -20 DEG C or -40 DEG C it is spare.
4th, envelope antigen is freeze-dried
For the ease of long-term storage and transport, need to be freeze-dried envelope antigen to obtain the more of solid state
Peptide.The envelope antigen freezed in advance is positioned on the freeze drier of Labconco and is dried, obtains solid state
Envelope antigen.It is labelled after packaging.The title of dated polypeptide, number, product batch number, concentration, date of manufacture, preservation on label
Time limit and preservation condition.
The preparation of the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of embodiment 2, ox aftosa
Foot-and-mouth disease antibody ELISA immunity detection reagent includes：
(1) it is coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of foot-and-mouth disease virus antigen；2 × 96 holes.
(2) the goat-anti ox IgG polyclonal antibodies of horseradish peroxidase-labeled are (purchased from SouthernBiotech companies, goods
Number 6030-05), 2 bottles (each 12ml).
(3) positive control serum：It is using the envelope antigen that artificial chemistry in embodiment 1 synthesizes as immunogene, June is immunized
Hyper-immune serum prepared by age health ox (antibodies against foot-and-mouth disease virus is negative), adds in 1000U/ml streptomysins and 1000U/ml moulds
Element crosses 0.2 μm of filter membrane degerming, the positive control serum as kit.1 pipe (1ml).
(4) negative control sera：It is neck after being quarantined 7 with 6 monthly age health oxes (antibodies against foot-and-mouth disease virus is negative)
Vein aseptic collection blood adds in 1000U/ml streptomysins and 1000U/ml penicillin, 0.2 μm of filter membrane degerming is crossed, as reagent
Box negative control sera.1 pipe (1.5ml).
(5) substrate developing solution：It is made of two kinds of solution mixing, solution A is the lemon of the hydrogen peroxide urea containing 0.6mg/ml
Acid phosphoric acid salt buffer (1 bottle (12ml))；Solution B is tetramethyl benzidine (TMB) solution (1 bottle (12ml)) of 0.2mg/ml；
It is mixed both during use by 1: 1 ratio of volume ratio.
(6) concentrated cleaning solution (20 ×)：Contain 0.5% (ml/ml) Tween-20 and 0.05% (g/ml) Sodium azide anti-corrosion
The 0.01M phosphate buffers of agent, pH 7.4, after by 0.2 μm of filter membrane degerming.50ml/ bottles, 2 bottles.
(7) terminate liquid：The sulfuric acid solution of 2mol/L.1 bottle of (12ml)
(8) sample diluting liquid：0.01M, pH containing 0.5% (g/ml) casein are 7.4 PBS buffer solution, cross 0.45 μ
M filter membranes.2 bottles (each 12ml).
As needed, serum can also dilutes 2 pieces of plate (96 hole) in kit, for the gradient dilution of blood serum sample.
Wherein, the preparation method for being coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of foot-and-mouth disease virus antigen is：It will
Polypeptide antigen prepared by embodiment 1 is dissolved in the carbonate solution of pH 9.6, is then added to 96 hole polystyrene enzyme-linked reaction plates, often
Hole 150ng (3 institutes of sequence in the polypeptide and sequence table in the polypeptide and sequence table wherein in sequence table shown in sequence 1 shown in sequence 2
The polypeptide shown, the mass ratio of three kinds of polypeptides is 1: 1: 1), being stood overnight at 37 DEG C of placements 2~4h, then 4~8 DEG C resists polypeptide
Original is fully combined with enzyme-linked reaction plate.Then the PBS containing 1% (g/ml) bovine serum albumin(BSA) (BSA) is added according to 300 μ l/ holes
Buffer solution (pH=7.4), 37 DEG C of 2~3h of closing, with washing buffer, (20 times of concentrated cleaning solution distilled water or deionized water are dilute
Release to obtain washing buffer) washing after dry, after enzyme-linked reaction plate drying after 4 DEG C be sealed.
The coincidence rate experiment of the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of embodiment 3, ox aftosa
First, the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent application method of ox aftosa
1st, it balances：The kit for being stored in 4 DEG C is taken out, is balanced spare to room temperature；The preceding mixing of liquid reagent.
2nd, with liquid：The 20 times of dilutions of concentrated cleaning solution distilled water or deionized water are obtained into washing buffer；
3rd, it sets：1 blank well, 3 negative control holes and 2 Positive control wells are stayed, remaining is sample to be tested hole.
4th, sample pre-dilution to be measured：Using sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to 1: 21 dilution proportion；Add 100 μ l Sample dilutions per hole in dilution plate hole, be separately added into 5 μ l samples to be tested.
5th, it is loaded：Blank well is not loaded；3 negative control holes respectively add 100 μ l negative controls, and 2 Positive control wells respectively add
100 μ l positive controls；Remaining hole is respectively by presetting plus the 100 diluted samples to be tested of μ l.Sample-adding process time span should be use up
It measures short.
6th, it incubates：Mixing is shaken, is put in 37 DEG C of incubators or water-bath, reacts 30min.
7th, board-washing：Reaction solution is discarded, the washing buffer obtained in 300 μ l steps 2 is added per hole, 15s is impregnated, gets rid of to abandon and wash
Liquid.It is patted dry after continuous board-washing 4 times.
8th, it is enzyme：Blank well is not enzyme；Remaining each hole adds more grams of the goat-anti ox IgG of 100 μ l horseradish peroxidase-labeleds
Grand antibody.
9th, it incubates：It puts in 37 DEG C of incubators or water-bath, reacts 30min.
10th, board-washing：Reaction solution is discarded, the 300 μ l of washing buffer after dilution are added in per hole, 15s is impregnated, gets rid of and abandon washing
Liquid.It is patted dry after continuous board-washing 5 times.
11st, it develops the color：Being separately added into the substrate developing solution of 100 μ l per hole, (wherein solution A and solution B are using volume ratio as 1: 1
Ratio mixes).It is capped, reacts 15min in 37 DEG C of insulating boxs；
12nd, 50 μ l terminate liquids are separately added into per hole and terminate reaction, mixing；
13rd, the OD per hole is measured450Value (adds the reaction plate of terminate liquid OD should be read in 15min450Value).
The judgement of testing result：
1st, negative control OD450Average value should≤0.15, otherwise in vain.
2nd, each detected value of positive control should be between 0.9-1.9, otherwise in vain.
3rd, the calculating of critical value：Critical value=0.17 × positive control OD450It is worth average value.
Serum to be checked measures OD450Value >=critical value person is judged to the positive；Serum to be checked measures OD450Value ＜ faces value person and is judged to the moon
Property.
2nd, suckling mouse serum neutralization test (MSN) method
One of domestic common aftosa serum detection method is suckling mouse neutralization test (MSN), therefore tried with this at present
Agent box carries out coincidence rate experiment with suckling mouse neutralization test.
Material
1. 50 parts of Positive Seras of immune serum (ox mouthful hoof inactivated vaccine be immunized after positive serum), industry is herded in
Limited company Lanzhou biology pharmaceutical factory provides.
2. healthy 50 parts of cow's serum is provided by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd..
3. experiment with virus be aftosa OM II viruses (the O-shaped weak poison OMII strains of mouseization, purchased from Lanzhou veterinary institute,
Preserved by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. and used) virus was passed into for 2 generations through 6~8 age in days suckling mouse balb/c
Afterwards, virus is collected.It is measured with 3~4 age in days suckling mouses and adjusts its malicious valency to 1000LD50/ 0.2ml is placed in -20 DEG C and preserves for use.
Suckling mouse serum neutralization test method is as follows：Serum PBS is made into 2 times of dilutions, takes 1ml and equivalent 100LD50/ ml's
The mixing of OM II systems virus liquids, is incubated 1 hour in 37 DEG C, is then inoculated with 3 age in days suckling mouses (0.2ml is subcutaneously injected in neck), every group
(that is, each serum to be checked) 4, while virus control, normal healthy controls and standard positive serum control group are set up, every group 3,
7d is observed, if record suckling mouse fatality ratio and judgement are as a result, the strong work of suckling mouse 4/4 or 3/4, serum to be checked is positive (R), if newborn
Mouse 4/4 or 3/4 is dead, then serum to be checked is negative (-)；If suckling mouse 2/4 is strong living or 2/4 is dead, judge that result is suspicious (±),
Replication need to be doubled, is repeated in testing, is had 1 group to be positive, be then determined as the positive.
Sensibility=judgement positive serum sample number/test serum sample number
3rd, coincidence rate result of the test：
In the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa prepared using embodiment 2 and suckling mouse
100 parts of cow's serums, wherein 50 parts of positive serum (above-mentioned immune serum) are detected respectively with experiment (MSN), and negative serum is (above-mentioned strong
Health cow's serum) 50 parts, as a result display such as table 3, hostis pecoris Structural protein VP1 antibody ELISA immunity detection reagent it is quick
Perception is 98%, and the sensibility of suckling mouse serum neutralization test only has 90%, in 50 parts of positive serums, two methods detection knot
Fruit is unanimously 46 parts, and therefore, the coincidence rate of this kit and suckling mouse serum neutralization test is 92%, therefore this kit has
Higher sensibility.The coincidence rate of both tests to negative serum is 100%, and testing result is negative (being shown in Table 3).
Hostis pecoris Structural protein VP1 antibody ELISA immunity detection reagent detects altogether with suckling mouse neutralization test
100 parts of cow's serums, wherein 96 parts of cow's serum two methods testing results are consistent, 4 parts of ox serum detection results are variant, coincidence rate
It is 96%.
The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent (ELISA) of 3. Ns of aftosas of table and suckling mouse serum neutralize
Test method(s) (MSN) is to the testing result of positive serum and negative serum
The sensitivity tests of the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of embodiment 4, ox aftosa
Sensitivity tests is using the O-shaped Structural protein VP1 antibody ELISA immune detection of ox aftosa prepared in embodiment 2
(cow's serum is provided kit detection cattle infected serum by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd., is identified as ox
Aftosa it is O-shaped virus infection serum) and vaccine immunity after serum (ox mouth hoof inactivated vaccine be immunized after positive serum, herded in
Baoshan, Yunnan factory of Industry Co., Ltd provide) result.The three batches of Foot-and-mouth diseases prepared using embodiment 2
Antibody ELISA immunosorbent adsorption test diagnostic kit (batch NOZM001, NOZM002, NOZM003), according to ox mouthful in embodiment 3
The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent application method of fever aphthous virus infection blood O-shaped to 200 parts of ox aftosas
Positive serum detection sensitivity after clear and 200 parts of ox mouthful hoof inactivated vaccines are immunized is tested.
Lot number is that the testing result of NOZM001 kits shows that this kit is to the sensibility of 200 parts of infection cow's serums
195/200 × 100%=97.5%；The sensibility that 200 parts of ox aftosa vaccines are immunized with cow's serum is 198/200 × 100%
=99%.Lot number is that the testing result of NOZM002 kits shows that this kit is to the sensibility of 200 parts of infection cow's serums
197/200 × 100%=98.5%；The sensibility that 200 parts of aftosa vaccines are immunized with cow's serum is 197/200 × 100%=
98.5%.Lot number is that the testing result visualizingre agent box of NOZM003 kits is 197/ to the sensibility of 200 parts of infection cow's serums
200 × 100%=98.5%；The sensibility that 200 parts of aftosa vaccines are immunized with cow's serum is 195/200 × 100%=
97.5%.
The specific test of the O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of embodiment 5, ox aftosa
Using three batches of kits in embodiment 4 according to the aftosa synthetic peptide structural proteins antibody described in embodiment 3
The application method of enzyme-linked immunologic detecting kit healthy cow's serum (Baoshan factory of Zhongmu Industry Co., Ltd and orchid to 300 parts
State biology pharmaceutical factory provides), (the Zhongmu Industry Co., Ltd's Lanzhou life of 10 parts of ox aftosa Asia I type (Asia1) positive serums
Object pharmaceutical factory provide), 10 parts of ox foot-and-mouth disease a type (A) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer)
With 10 parts of bovine viral diarrhea virus (BVDV) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer) point
It is not detected.
Specific detection result such as following table (table 4) display of kit, to 300 parts, the testing result of healthy cow's serum is shown
Show, the specificity of NOZM001 kits is that the specificity of 99.0%, NOZM002 kits is 98.6%, NOZM003 kits
Specificity be 99.3%.It is positive to 10 parts of ox aftosa Asia I type (Asia I) positive serums, 10 parts of ox foot-and-mouth disease a types (A)
Serum and 10 parts of bovine viral diarrhea virus positive serum testing results are illustrated as feminine gender, therefore three kits are to this 30 parts
The specificity of positive serum detection is 100%.
4. aftosa synthetic peptide structural proteins antibody ELISA immunity detection reagent specific detection result of table
The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of embodiment 6, ox aftosa tries compared with comparable product
It tests
Resisted using three batches of kits in embodiment 4 according to the O-shaped Structural protein VP1 of ox aftosa described in embodiment 3
The application method of body enzyme-linked immunologic detecting kit to 276 parts of clinical serum samples, (protect by Zhongmu Industry Co., Ltd Yunnan
Mountain factory, Lanzhou biology pharmaceutical factory provide) it is detected, while ox, the sheep produced using Shanghai Unitewin biomedical company (UBI)
FMDV VP1 structural proteins antibody ELISA immunosorbent adsorption test diagnostic kit carries out 276 parts of clinical serums parallel
Detection, operating method are carried out with criterion according to the operating procedure of (UBI) specification and criterion respectively.Shanghai Unitewin
The judgement of the ox, sheep FMDV VP1 structural proteins antibody ELISA immunosorbent adsorption test diagnostic kit of biomedical company
Standard：(1) negative control hole mean OD value answers≤0.15, and each Positive control wells OD values answer >=0.9, and≤1.9. is otherwise, examination
It is invalid to test.Critical value=0.23 × Positive control wells mean OD value.(2) during test sample hole OD values ＜ critical values, feminine gender is judged to,
As foot-and-mouth disease virus resistant VP1 structural proteins negative antibody.(3) test sample hole OD values ＞ critical values, are judged to the positive.
276 parts of serum are detected using the kit in embodiment 4, positive rate is 37.0 (102/276), and negative rate is
63.0% (174/276).The ox of Shanghai Unitewin biomedical company (UBI), sheep FMDV VP1 structural proteins abzyme
The positive rate of linked immunosorbent adsorption test diagnostic kit detection is 36.2% (100/276), and negative rate is 63.8% (176/
276).The positive coincidence rate of the two is 98.0% (100/102), and negative match-rate is 98.9% (174/176).Two kits
276 parts of serum samples are detected, the testing result for sharing 258 parts of serum is consistent, and total coincidence rate is 93.5% (258/
276)。
Claims (13)
1. Foot-and-mouth disease VP1 antigen epitope polypeptides are the polypeptide in sequence table shown in sequence 1.
2. Foot-and-mouth disease VP1 antigen epitope polypeptide compositions, be as shown in sequence in sequence table 1 polypeptide, sequence
The mixture of polypeptide composition in polypeptide and sequence table in list shown in sequence 2 shown in sequence 3；Sequence 1 in the sequence table
The mass ratio of polypeptide in polypeptide and sequence table in shown polypeptide and sequence table shown in sequence 2 shown in sequence 3 for (0.5~
2.0):(0.5~2.0):1.
3. a kind of Foot-and-mouth disease antibody ELISA immunity detection reagent, including Foot-and-mouth disease VP1
The coated enzyme-linked reaction plate of antigen epitope polypeptide and enzyme label antiantibody；The Foot-and-mouth disease VP1 epitopes
Polypeptide 2 institute of sequence in the polypeptide shown in sequence 1 in the polypeptide shown in sequence in sequence table 1 or sequence table and sequence table
The mixing that polypeptide shown in sequence 3 forms in sequence 1 and sequence table in the mixed polypeptide of the polypeptide composition shown or sequence table is more
It is more shown in sequence 3 in polypeptide and sequence table in polypeptide, sequence table in peptide or sequence table shown in sequence 1 shown in sequence 2
The mixed polypeptide of peptide composition.
4. kit according to claim 3, it is characterised in that：When the Foot-and-mouth disease VP1 antigen tables
Position polypeptide is as shown in sequence 3 in the polypeptide shown in the polypeptide shown in sequence in sequence table 1, sequence 2 in sequence table and sequence table
During the mixed polypeptide of polypeptide composition, polypeptide and sequence in the polypeptide and sequence table in the sequence table shown in sequence 1 shown in sequence 2
The mass ratio of polypeptide in list shown in sequence 3 is (0.5~2.0):(0.5~2.0):1.
5. kit according to claim 4, it is characterised in that：Polypeptide, sequence table in the sequence table shown in sequence 1
The mass ratio of polypeptide shown in sequence 3 is 1 in polypeptide and sequence table shown in middle sequence 2:1:1.
6. according to the kit of any one in claim 3-5, it is characterised in that：The marker enzyme of enzyme label antiantibody is
Horseradish peroxidase or alkaline phosphatase；The enzyme label antiantibody marks goat-anti ox IgG for enzyme.
7. kit according to claim 6, it is characterised in that：The enzyme label antiantibody is preferably horseradish peroxidase
The goat-anti ox IgG of enzyme label.
8. according to the kit of any one in claim 3-5, it is characterised in that：The enzyme-linked reaction plate is detachable 96 hole
ELISA Plate；The Foot-and-mouth disease VP1 antigen epitope polypeptides, which are that chemistry is artificial synthesized, to be obtained.
9. kit according to claim 6, it is characterised in that：The Foot-and-mouth disease VP1 epitopes
The preparation method of the coated enzyme-linked reaction plate of polypeptide is to be dissolved in the Foot-and-mouth disease VP1 antigen epitope polypeptides
The carbonate solution of the pH 9.6 of 100 μ l is then added to 96 hole polystyrene enzyme-linked reaction plates, per hole 150ng, in 37 DEG C of placements
It is placed 8-12 hours at 2-4 hours, then 4-8 DEG C, polypeptide antigen is made fully to be combined with enzyme-linked reaction plate, then according to 300 μ l/ holes
The PBS buffer solution of the pH7.4 containing 1g/100ml bovine serum albumin(BSA)s is added in, 37 DEG C of Seal treatments 2-3 hours are got rid of after washing
It is dry, it is sealed for 4 DEG C after enzyme-linked reaction plate drying.
10. kit according to claim 6, it is characterised in that：Also contain developing solution and terminate liquid in the kit；
When marker enzyme be horseradish peroxidase when developing solution be made of developing solution A liquid and developing solution B liquid, the developing solution A liquid be containing
The citrate phosphate buffer of 0.6mg/ml hydrogen peroxide ureas, the developing solution B liquid are the tetramethyl biphenyl of 0.2mg/ml
Amine aqueous solution；When marker enzyme is alkaline phosphatase, developing solution is 4- nitrophenols phosphate buffers；The terminate liquid is 2mol/L
Sulfuric acid solution.
11. kit according to claim 6, it is characterised in that：The kit further includes negative control sera, the positive
Control serum；The negative control sera is the normal calf serum with no foot-and-mouth disease antibody；The positive control serum is with institute
It is the serum that immunogen immune ox obtains to state Foot-and-mouth disease VP1 antigen epitope polypeptides；The kit further includes
Sample diluting liquid and concentrated cleaning solution；The PBS that sample diluting liquid is 7.4 for 0.01M, pH containing 0.005g/100ml caseins
Buffer solution；Concentrated cleaning solution：0.01M, pH 7.4, containing concentration expressed in percentage by volume be 0.8%~1.2% Tween-20 and
The phosphate buffer of 0.05g/100ml Sodium azide preservatives.
12. the mouth hoof described in Foot-and-mouth disease VP1 antigen epitope polypeptides described in claim 1 or claim 2
Epidemic disease poison Structural protein VP1 antigen epitope polypeptide composition detects whether the kit of infection animal hoof-and-mouth disease viral disease preparing
In application；Wherein, animal aftosa viral disease is hostis pecoris disease.
13. application according to claim 12, it is characterised in that：The animal aftosa poison is the O-shaped disease of ox aftosa
Hostis pecoris disease caused by poison.
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| CN105807052B (en) * | 2016-05-13 | 2017-12-15 | 郑州中道生物技术有限公司 | O-shaped FMDV antibody direct competive ELISA detection kit |
| CN109485703B (en) * | 2016-08-29 | 2021-07-06 | 中牧实业股份有限公司 | Foot-and-mouth disease A-type structural protein VP1 antigen epitope polypeptide and application thereof |
| CN109870570A (en) * | 2018-12-29 | 2019-06-11 | 广东云天抗体生物科技有限公司 | A kind of enzyme linked immunological kit detecting monkey IL-18 |
| CN109870571A (en) * | 2018-12-29 | 2019-06-11 | 广东云天抗体生物科技有限公司 | A kind of enzyme linked immunological kit detecting monkey G-CSF |
| CN115902233A (en) * | 2022-08-19 | 2023-04-04 | 中国医学科学院北京协和医院 | Kit for detecting anti-Ri antibody and application thereof |
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