CN108152511B - Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit - Google Patents
Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit Download PDFInfo
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- CN108152511B CN108152511B CN201711409686.XA CN201711409686A CN108152511B CN 108152511 B CN108152511 B CN 108152511B CN 201711409686 A CN201711409686 A CN 201711409686A CN 108152511 B CN108152511 B CN 108152511B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
Abstract
The present invention provides a kind of Coxsack A16 virus antigen polypeptides to have the amino acid sequence as shown in SEQ ID NO:1.The antigen polypeptide carries out genetics column to multiple Coxsack A16 viral prevalence strains using bioinformatics method and compares design synthesis, can be directed to the Coxsack A16 Strain of a variety of hypotypes.The present invention also provides a kind of Coxsack A16 virus IgM antibody detection kits, and using enzyme-labelled antigen is Coxsack A16 virus antigen polypeptide, can be directed to the Coxsack A16 Strain of a variety of hypotypes, identify the serum virus of a variety of Coxsack A16 virus infections.The case where detection kit of the invention is reproducible, high specificity, high sensitivity, effectively detects Coxsack A16 virus infection, plays the role of active and effective in terms of prevention and control Coxsack A16 virus infection.
Description
Technical field
The invention belongs to immuno-biology technical fields, and in particular to a kind of Coxsack A16 virus antigen polypeptide and its IgM
Antibody assay kit.
Background technique
Coxsackie virus belongs to non-poliovirus in Picornaviridae, is broadly divided into two kinds of hypotype A types
And Type B.Coxsack A16 virus belongs to A type Coxsackie virus, it and EV71 virus are the main diseases for causing hand-foot-and-mouth disease poison popular
Substance is the highest enterovirus of China's Mainland prevalence frequency.RNA segment of the Coxsack A16 virus containing one section of single-stranded positive,
The coat protein of length substantially 7400bp, package RNA nucleic acid are mainly VP1, VP2, VP3, VP4.Wherein, in structural proteins
The function that VP1 albumen plays absorption infected cell is that most important virus neutralizes antigen, and usually vaccine and diagnosing main is ground
Study carefully object.5 years old children below are easier infection Coxsack A16 virus due to developing immune system prematurity.General Ke's Sa
Odd A16 virus infection incubation period is 3-6 days, is mainly broadcast by excrement oral instructions, and propagation mainly occurs in kindergarten, nursery.
Due to there is no effective drug and vaccine before Coxsack A16 virales, the mode of clinical symptomatic treatment, general medication are mainly taken
The fever and pain of object control patient.Therefore, carry out Coxsack A16 virus early diagnosis be control and prevention of disease emphasis, early every
A possibility that reducing transmission from patient.
The early diagnosis of Coxsack A16 virus includes fluorescent quantitative PCR technique and immunology serologic diagnosis, due to exempting from
Epidemiology diagnostic operation is simple and quick to be applied in different medical unit than wide.But Coxsack A16 virus variation is very fast, virus
Hypotype also compare it is more, be mainly reflected in VP1 protein sequence and be easy to happen variation.Immunology diagnosis mainly selects Coxsack
A16 virus VP 1 albumen carries out diagnostic design as target position, since the sequence of VP1 albumen is changeable, causes detection leakage phenomenon tighter
Weight.
Therefore, the present invention develops the high antigenic polypeptide fragments of homology and Coxsack of one kind of multiple Coxsack A16 viruses
A16 virus IgM antibody detection kit, the sensitivity with high detection can solve clinically existing missing inspection problem,
It is suitble to be widely popularized.
Summary of the invention
Of the existing technology in order to overcome the problems, such as, the object of the present invention is to provide a kind of Coxsack A16 viral antigen is more
Peptide, a kind of Coxsack A16 virus antigen polypeptide conjugate and preparation method thereof and Coxsack A16 virus IgM antibody detection reagent
Box.Coxsack A16 virus antigen polypeptide is the high antigen fragment of homology of a variety of Coxsack A16 viruses；Detection kit is to Sa
Odd A16 virus has the sensitivity of high detection, can solve clinically existing missing inspection problem, is suitble to be widely popularized.
To solve the above problems, the technical solution adopted is as follows:
Present invention firstly provides a kind of Coxsack A16 virus antigen polypeptides to have the amino acid as shown in SEQID N0:1
Sequence.The antigen polypeptide carries out genetics column comparison to multiple Coxsack A16 viral prevalence strains using bioinformatics method and sets
Meter synthesis, the Coxsack A16 Strain of a variety of hypotypes can be directed to.
The present invention provides a kind of Coxsack A16 virus antigen polypeptide conjugates, and the conjugate is by Coxsack A16 virus
Antigen polypeptide and carrier protein form；Chemical coupling agent is EDC；The carrier protein includes BSA albumen, OVA albumen, KLH egg
It is white.
Preferably, the carrier protein is BSA albumen.
The present invention provides a kind of preparation methods of Coxsack A16 virus antigen polypeptide conjugate, which is characterized in that including
Following steps:
Coxsack A16 virus antigen polypeptide and EDC.HCL is taken to distinguish soluble in water, mixing, the condition for being 4 DEG C in temperature
Under, it is stirred to react 30 minutes, is then slowly added dropwise into mixed liquor into carrier protein solution, adjusts pH value to 7.4, be stirred to react
16-24 hours, reactant was dialysed 20-30 hours again, and a dialyzate was changed every 3-5 hours, was changed altogether liquid 6 times, and Coxsack A16 is made
Virus antigen polypeptide conjugate.
Preferably, the concentration of the carrier protein solution is 2-20mg/ml.
Particularly preferred, the carrier protein solution is BSA protein solution, and the concentration of the BSA protein solution is 2-
20mg/ml。
Preferably, the mass ratio of the Coxsack A16 virus antigen polypeptide and EDC.HCl are (1-3): (1-10)；It is described
The mass volume ratio g/L of Coxsack A16 virus antigen polypeptide and water is (20-60): the quality volume of 1, the EDC.HCl and water
It is (20-200) than g/L: 1.
Preferably, the dialyzate is PBS buffer solution, and the molar concentration of the PBS buffer solution is 10mM, pH value 7.2.
The present invention provides a kind of Coxsack A16 virus IgM antibody detection kits, including antibody coated elisa plate and enzyme
Labelled antigen liquid；The antibody coated elisa plate is the ELISA Plate for being coated with goat-anti human IgM antibody, and the enzyme-labelled antigen liquid is
The Coxsack A16 virus antigen polypeptide conjugate dilution of enzyme label.
Preferably, the Coxsack A16 virus antigen polypeptide conjugate of the enzyme label is by Coxsack A16 virus antigen polypeptide
Conjugate is crosslinked acquisition by Over-voltage protection with horseradish peroxidase.
Particularly preferred, the preparation method of the Coxsack A16 virus antigen polypeptide conjugate of the enzyme label includes following
Step:
S1: being diluted to 1-5mg/ml for Coxsack A16 virus antigen polypeptide conjugate, with carbonate buffer solution dialysis 10-
16 hours；The carbonate buffer solution molar concentration is 50mM, pH value 9.6；
S2: horseradish peroxidase is configured to the horseradish peroxidase solution that concentration is 10mg/ml, NaIO is added4
Solution reacts at room temperature 10-45 minutes；After reaction, ethylene glycol is added, reacts at room temperature 20-60 minutes, horseradish peroxidase is made
Reaction solution；The horseradish peroxidase solution and NaIO4The volume ratio of solution is 1: 2-10, and the horseradish peroxidase is molten
The volume ratio of liquid and ethylene glycol is 1: (5-20)；
S3: the Coxsack A16 virus antigen polypeptide that horseradish peroxidase reaction solution is added after step S1 dialysis is coupled
In object, under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；Coxsack A16 virus antigen polypeptide conjugate and horseradish peroxide
The volume ratio 1 of compound enzyme reaction solution: (2-10)；
S4: by NaBH4It is made into the NaBH that concentration is 2-100mg/ml4Solution, by NaBH4Solution is added in the mixed of step S3
It closes in liquid, under conditions of temperature is 4 DEG C, reacts 1-3 hours, per half an hour rocks once；NaBH4Solution and horseradish peroxidating
The volume ratio 1 of object enzyme reaction solution: (1-10)；
S5: being dialysed 24-30 hours with PBS buffer solution in the reaction solution of step S4, changed during dialysis liquid 5-8 times, be added etc.
The glycerol of volume is measured, is uniformly mixed, the Coxsack A16 virus antigen polypeptide conjugate of enzyme label is made；The PBS buffer solution
Molar concentration is 10mM, pH value 7.2.
Preferably, the Coxsack A16 virus antigen polypeptide conjugate dilution of the enzyme label is dilute with enzyme-labelled antigen
It releases liquid and the Coxsack A16 virus antigen polypeptide conjugate that enzyme marks is diluted to 1000-5000 times.
Particularly preferred, the enzyme-labelled antigen dilution includes PBS buffer solution, calf serum, collagen, amino ratio
Woods and ProClin300, wherein the molar concentration of PBS buffer solution is 10mM, and the volumetric concentration of calf serum is 10%, collagen egg
White mass concentration is 0.25%, and the mass concentration that the mass concentration of aminopyrine is 0.1%, ProClin300 is 0.1%, pH
Value is 7.2.
Preferably, the Coxsack A16 virus antigen polypeptide conjugate is Coxsack A16 virus antigen polypeptide-BSA egg
It is white.
Preferably, be coated with the preparation method of the ELISA Plate of goat-anti human IgM antibody the following steps are included:
1) it is coated with: diluting goat-anti human IgM antibody to concentration with molar concentration 50mM, the carbonate buffer solution that pH value is 9.6
For 5 μ g/mL, ELISA Plate is added, every hole adds 95-105 μ L, is coated with 16-24 hours under conditions of temperature is 4 DEG C, then uses
PBST is washed 1 time；
2) close: every hole adds 150 μ L of confining liquid, closes 16-20 hours under conditions of temperature is 4 DEG C, gets rid of deblocking liquid,
It is dry；The confining liquid includes PBS buffer solution, calf serum, sucrose and ProClin300, and wherein PBS buffer solution is mole dense
Degree is 10mM, and the volumetric concentration of calf serum is 5%, and the mass concentration that the mass concentration of sucrose is 3%, ProClin300 is
0.05%, pH value 7.2.
Particularly preferred, the ELISA Plate is detachable ELISA Plate.
Most preferably, the ELISA Plate is the high adsorption capacity enzyme mark of the high adsorption capacity ELISA Plate in dismountable 96 hole or 48 holes
Plate.
Preferably, a kind of Coxsack A16 virus IgM antibody detection kit further includes negative control sera, positive control
Serum, sample diluting liquid substrate A, substrate B, terminate liquid and cleaning solution.This kit is to detect human serum using prize law principle
Middle Coxsack A16 virus IgM antibody.
Particularly preferred, the negative control sera is Healthy Human Serum；Positive control serum is Coxsack A16 virus sense
Contaminate the serum of patient.
Particularly preferred, the sample diluting liquid includes PBS buffer solution, BSA, casein and ProClin300, wherein PBS
The molar concentration of buffer is 10mM, and the mass concentration of BSA is 0.6%, and the mass concentration of casein is 0.2%,
The mass concentration of ProClin300 is 0.1%, pH value 7.2.
Particularly preferred, substrate A is the citrate buffer for being 0.2% hydrogen peroxide urea containing mass concentration；Institute
Stating substrate B is the TMB that concentration is 0.2mg/ml.
Particularly preferred, the terminate liquid is the sulfuric acid solution that molar concentration is 2M.
Particularly preferred, the cleaning solution includes phosphate buffer and Tween-20, wherein mole of phosphate buffer
Concentration is 0.2M, and the mass concentration of Tween-20 is 0.5%, pH value 7.2.It is 1: 20 dilute by volume when the cleaning solution uses
It releases.
The present invention provides the application method of Coxsack A16 virus IgM antibody detection kit the following steps are included:
(1) it balances and matches liquid:
It takes out kit placement equilibrium at room temperature 30 minutes or more；Cleaning solution distilled water or deionized water are diluted 20 times.
(2) it numbers:
Serum specimen is numbered, and is added on corresponding ELISA Plate, Loading sequence record is carried out.Every plate at least sets 2
Hole negative control, 1 hole positive control and 1 hole blank control.
(3) it is loaded:
2 hole of negative control, 1 hole of positive control, each 100ul control serum.1 hole of blank control is vacant.Remaining micropore is added
100ul sample diluting liquid adds sample 10ul to be checked, and reaction plate, which is gently shaken, makes sample blending.
(4) it incubates:
After sealing plate film sealing plate, sets in 37 DEG C of incubators or water-bath and react 30 minutes.
(5) board-washing:
Sealing plate film is taken off, is machine-washed plate 5 times with board-washing, is finally buckled on blotting paper dry.
(6) enzyme mark antigen liquid:
Every hole is added 100ul enzyme-labelled antigen liquid, except blank well.
(7) it incubates:
After sealing plate film sealing plate, sets in 37 DEG C of incubators or water-bath and react 30 minutes.
(8) board-washing:
Sealing plate film is taken off, is machine-washed plate 5 times with board-washing, is finally buckled on blotting paper dry.
(9) it develops the color:
Substrate A liquid is first added, 50ul is added in every hole, adds substrate B liquid, and 50ul is added in every hole, and gently concussion mixes, and uses
After sealing plate film sealing plate, 37 DEG C are protected from light colour developing 15 minutes.
(10) it terminates:
Terminate liquid 50ul is added in every hole, and gently concussion mixes.
(11) it measures:
It sets microplate reader Detection wavelength (to detect preferably with dual wavelength 450/630) as 450nm, measures each hole A value.Unicast
Long detection: it is returned to zero with blank well, tests the A value of each hole 450nm.Double UV check: can not set blank well, test each hole 450nm/
The A value of 630nm.
(12) result judgement:
Critical value: Cut off (C.0)=0.10+ negative control is averaged (NC) A value.
Feminine gender determines: being determined as that Coxsack A16 virus IgM antibody is negative when sample A value < critical value (Cut off value).
The positive determines: being determined as that Coxsack A16 virus IgM antibody is positive when sample A value >=critical value (Cut off value).
Coxsack A16 virus that the present invention also provides Coxsack A16 virus IgM antibody detection kits in test sample
The application of IgM antibody, to provide a kind of approach for detecting Coxsack A16 virus.
Preferably, the sample is human serum or human plasma.
Beneficial effects of the present invention:
1, Coxsack A16 virus antigen polypeptide of the invention is using bioinformatics method to multiple Coxsack A16 viruses
Epidemic strain carries out genetics column and compares design synthesis, can be directed to the Coxsack A16 Strain of a variety of hypotypes；
2, detection kit of the invention is Coxsack A16 virus antigen polypeptide using enzyme-labelled antigen, can be directed to more
The Coxsack A16 Strain of kind hypotype, identifies the serum virus of a variety of Coxsack A16 virus infections；
3, detection kit of the invention is reproducible, high specificity, high sensitivity, effectively detects Coxsack A16 virus
The case where infection, plays the role of active and effective in terms of prevention and control Coxsack A16 virus infection；
4, detection kit of the invention is easy to operate, and detection time is short, and the medical institutions that can satisfy different stage make
With playing the role of preferable monitoring in the early diagnosis of Coxsack A16 virus infection.
Specific embodiment
The present invention proposes that a kind of Coxsack A16 virus antigen polypeptide has the amino acid sequence as shown in SEQ ID NO:1.
The present invention carries out alignment's research to the Coxsack A16 Strain genome of 18 plants of different subtypes, screens a gene sequence
The relatively conservative segment of column, and by its synthesis polypeptide, which can identify the serum of a variety of hypotype Coxsack A16 virus infections
Antibody.Coxsack A16 virus antigen polypeptide sequence is supplied to gill biochemistry Shanghai Co., Ltd to synthesize, purity 98%
More than, pass through mass spectroscopy qualification.
The present invention proposes a kind of Coxsack A16 virus antigen polypeptide conjugate, by Coxsack A16 virus antigen polypeptide and carries
Body protein composition.Chemical coupling agent is EDC, and carrier protein includes BSA albumen, OVA albumen, KLH albumen.
The commercially available acquisition of each raw material, reagent in following embodiment.
Below by specific preferred embodiment combination effete test embodiment, technical scheme is described further, but
The present invention is not limited in embodiment below.
Embodiment 1
(1) Coxsack A16 virus antigen polypeptide
The sequence of Coxsack A16 virus antigen polypeptide segment is as shown in SEQ ID NO:1, and 19 amino acid, is entrusted in total
Gill biochemistry Shanghai Co., Ltd is responsible for synthesis, and purity is greater than 98%, and freeze-drying saves.
(2) Coxsack A16 virus antigen polypeptide conjugate (Coxsack A16 virus antigen polypeptide-BSA albumen)
The preparation method of Coxsack A16 virus antigen polypeptide-BSA albumen the following steps are included:
It takes 20mg Coxsack A16 virus antigen polypeptide and 50mg EDC.HCL to be dissolved in 0.5ml ultrapure water respectively, mixes,
It under conditions of temperature is 4 DEG C, is stirred to react 30 minutes, the BSA for being 10mg/ml into concentration is then slowly added dropwise into mixed liquor
Protein solution is adjusted pH value to 7.4, is stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary
Dialyzate (10mM PBS buffer solution, pH7.2) changes liquid 6 times altogether, and Coxsack A16 virus antigen polypeptide-BSA albumen is made.
(3) a kind of Coxsack A16 virus IgM antibody detection kit
A kind of Coxsack A16 virus IgM antibody detection kit includes antibody coated elisa plate, enzyme-labelled antigen liquid, yin
Property control serum, positive control serum, sample diluting liquid substrate A, substrate B, terminate liquid, cleaning solution and specification.
In the present embodiment:
Antibody coated elisa plate (is coated with goat-anti human IgM antibody ELISA Plate): 8 × 12 hole specifications or 4 × 12 hole specifications.
The enzyme-labelled antigen solution Coxsack A16 virus antigen polypeptide-BSA diluted protein solution of label (enzyme): 12ml/ bottles or
6ml/ bottles of person.1 bottle.
Negative control sera (Healthy Human Serum): 1ml/ bottles, 1 bottle.
Positive control serum (serum of Coxsack A16 patients with viral infections): 1ml/ bottles, 1 bottle.
Sample diluting liquid (10mMPBS buffer, 0.6%BSA, 0.2% casein, 0.1%ProClin300, pH
7.2): 12ml/ bottles or 6ml/ bottles, 1 bottle.
Substrate A liquid (citrate buffer, 0.2% hydrogen peroxide urea): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Substrate B liquid (0.2mg/ml TMB): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Terminate liquid (2M sulfuric acid solution): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Cleaning solution (0.2M phosphate buffer, 0.5% Tween-20, pH 7.2): 50ml/ bottles or 30ml/ bottles, 1 bottle.
Specification: 1 part.
Specifically, be coated with the preparation method of the ELISA Plate of goat-anti human IgM antibody the following steps are included:
1) be coated with: diluting goat-anti human IgM antibody to concentration with carbonate buffer solution (50mM, pH 9.6) is 5 μ g/mL, is added
Enter ELISA Plate, every hole adds 100 μ L, is coated with 16-24 hours under conditions of temperature is 4 DEG C, is then washed 1 time with PBST；
2) close: every hole adds 150 μ L of confining liquid, closes 18 hours under conditions of temperature is 4 DEG C, gets rid of deblocking liquid
(10mMPBS buffer, 5% calf serum, 3% sucrose, 0.05%ProClin300, pH 7.2), it is dry.Preferably, enzyme mark
Plate is the high adsorption capacity ELISA Plate of the high adsorption capacity ELISA Plate in dismountable 96 hole or 48 holes.
Specifically, enzyme label Coxsack A16 virus antigen polypeptide-BSA albumen preparation method the following steps are included:
S1: being diluted to 1-5mg/ml for Coxsack A16 virus antigen polypeptide-BSA albumen, takes 1ml Coxsack A16 virus anti-
Former polypeptide-BSA diluted protein solution is packed into bag filter, places carbonate buffer solution (50mM, pH 9.6) and dialyses 10-16 hours；
S2: horseradish peroxidase (HRP) is configured to the HRP solution that concentration is 10mg/ml, in the HRP solution of 4ml
The NaIO of 1ml is added4Solution reacts at room temperature 30 minutes；After reaction, 40ul ethylene glycol is added, reacts at room temperature 30 minutes, is made
HRP reaction solution；
S3: the HRP reaction solution of 4ml is added in the Coxsack A16 virus antigen polypeptide-BSA albumen after step S1 dialysis,
Under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；
S4: by NaBH4It is made into the NaBH that concentration is 10mg/ml4Solution, by the NaBH of 1500ul4Solution is added in step S3
Mixed liquor in, temperature be 4 DEG C under conditions of, react 1-3 hours, per half an hour rocks once；
S5: it is dialysed 24-30 hours in the reaction solution of step S4 with PBS buffer solution (10mM, pH 7.2), liquid is changed during dialysis
5-8 times, the glycerol of equivalent volumes is added, is uniformly mixed, the Coxsack A16 virus antigen polypeptide-BSA albumen of enzyme label is made.
Specifically, the Coxsack A16 virus antigen polypeptide-BSA diluted protein solution of enzyme label is to be diluted with enzyme-labelled antigen
Liquid (10mM PBS buffer solution, 10% calf serum, 0.25% collagen, 0.1% aminopyrine, 0.1%ProClin300)
The Coxsack A16 virus antigen polypeptide conjugate that enzyme marks is diluted to 1000-5000 times.
Embodiment 2
(1) Coxsack A16 virus antigen polypeptide
The sequence of Coxsack A16 virus antigen polypeptide segment is as shown in SEQ ID NO:1, and 19 amino acid, is entrusted in total
Gill biochemistry Shanghai Co., Ltd is responsible for synthesis, and purity is greater than 98%, and freeze-drying saves.
(2) Coxsack A16 virus antigen polypeptide conjugate (Coxsack A16 virus antigen polypeptide-OVA albumen)
The preparation method of Coxsack A16 virus antigen polypeptide-OVA albumen the following steps are included:
It takes 20mg Coxsack A16 virus antigen polypeptide and 50mg EDC.HCL to be dissolved in 0.5ml ultrapure water respectively, mixes,
It under conditions of temperature is 4 DEG C, is stirred to react 30 minutes, the OVA for being 10mg/ml into concentration is then slowly added dropwise into mixed liquor
Protein solution is adjusted pH value to 7.4, is stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary
Dialyzate (10mM PBS buffer solution, pH7.2) changes liquid 6 times altogether, and Coxsack A16 virus antigen polypeptide-OVA albumen is made.
(3) a kind of Coxsack A16 virus IgM antibody detection kit
A kind of Coxsack A16 virus IgM antibody detection kit includes antibody coated elisa plate, enzyme-labelled antigen liquid, yin
Property control serum, positive control serum, sample diluting liquid substrate A, substrate B, terminate liquid, cleaning solution and specification.
In the present embodiment:
Antibody coated elisa plate (is coated with goat-anti human IgM antibody ELISA Plate): 8 × 12 hole specifications or 4 × 12 hole specifications.
The enzyme-labelled antigen solution Coxsack A16 virus antigen polypeptide-OVA diluted protein solution of label (enzyme): 12ml/ bottles or
6ml/ bottles of person.1 bottle.
Negative control sera (Healthy Human Serum): 1ml/ bottles, 1 bottle.
Positive control serum (serum of Coxsack A16 patients with viral infections): 1ml/ bottles, 1 bottle.
Sample diluting liquid (10mM PBS buffer solution, 0.6%BSA, 0.2% casein, 0.1%ProClin300, pH
7.2): 12ml/ bottles or 6ml/ bottles, 1 bottle.
Substrate A liquid (citrate buffer, 0.2% hydrogen peroxide urea): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Substrate B liquid (0.2mg/ml TMB): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Terminate liquid (2M sulfuric acid solution): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Cleaning solution (0.2M phosphate buffer, 0.5% Tween-20, pH 7.2): 50ml/ bottles or 30ml/ bottles, 1 bottle.
Specification: 1 part.
Specifically, being coated with the ELISA Plate of goat-anti human IgM antibody, the preparation method is the same as that of Example 1.
Specifically, enzyme label Coxsack A16 virus antigen polypeptide-OVA albumen preparation method the following steps are included:
S1: being diluted to 1-5mg/ml for Coxsack A16 virus antigen polypeptide-OVA albumen, takes 1ml Coxsack A16 virus anti-
Former polypeptide-OVA diluted protein solution is packed into bag filter, places carbonate buffer solution (50mM, pH 9.6) and dialyses 10-16 hours；
S2: horseradish peroxidase (HRP) is configured to the HRP solution that concentration is 10mg/ml, in the HRP solution of 4ml
The NaIO of 1ml is added4Solution reacts at room temperature 30 minutes；After reaction, 40ul ethylene glycol is added, reacts at room temperature 30 minutes, is made
HRP reaction solution；
S3: the HRP reaction solution of 4ml is added in the Coxsack A16 virus antigen polypeptide-OVA albumen after step S1 dialysis,
Under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；
S4: by NaBH4It is made into the NaBH that concentration is 10mg/ml4Solution, by the NaBH of 1500ul4Solution is added in step S3
Mixed liquor in, temperature be 4 DEG C under conditions of, react 1-3 hours, per half an hour rocks once；
S5: it is dialysed 24-30 hours in the reaction solution of step S4 with PBS buffer solution (10mM, pH 7.2), liquid is changed during dialysis
5-8 times, the glycerol of equivalent volumes is added, is uniformly mixed, the Coxsack A16 virus antigen polypeptide-OVA albumen of enzyme label is made.
Specifically, the Coxsack A16 virus antigen polypeptide-OVA diluted protein solution of enzyme label is to be diluted with enzyme-labelled antigen
Liquid (10mMPBS buffer, 10% calf serum, 0.25% collagen, 0.1% aminopyrine, 0.1%ProClin300) will
The Coxsack A16 virus antigen polypeptide conjugate of enzyme label is diluted to 1000-5000 times.
Embodiment 3
(1) Coxsack A16 virus antigen polypeptide
The sequence of Coxsack A16 virus antigen polypeptide segment is as shown in SEQ ID N0:1, and 19 amino acid, is entrusted in total
Gill biochemistry Shanghai Co., Ltd is responsible for synthesis, and purity is greater than 98%, and freeze-drying saves.
(2) Coxsack A16 virus antigen polypeptide conjugate (Coxsack A16 virus antigen polypeptide-KLH albumen)
The preparation method of Coxsack A16 virus antigen polypeptide-KLH albumen the following steps are included:
It takes 20mg Coxsack A16 virus antigen polypeptide and 50mg EDC.HCL to be dissolved in 0.5ml ultrapure water respectively, mixes,
It under conditions of temperature is 4 DEG C, is stirred to react 30 minutes, the KLH for being 10mg/ml into concentration is then slowly added dropwise into mixed liquor
Protein solution is adjusted pH value to 7.4, is stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary
Dialyzate (10mM PBS buffer solution, pH7.2) changes liquid 6 times altogether, and Coxsack A16 virus antigen polypeptide-KLH albumen is made.
(3) a kind of Coxsack A16 virus IgM antibody detection kit
A kind of Coxsack A16 virus IgM antibody detection kit includes antibody coated elisa plate, enzyme-labelled antigen liquid, yin
Property control serum, positive control serum, sample diluting liquid substrate A, substrate B, terminate liquid, cleaning solution and specification.
In the present embodiment:
Antibody coated elisa plate (is coated with goat-anti human IgM antibody ELISA Plate): 8 × 12 hole specifications or 4 × 12 hole specifications.
The enzyme-labelled antigen solution Coxsack A16 virus antigen polypeptide-KLH diluted protein solution of label (enzyme): 12ml/ bottles or
6ml/ bottles of person.1 bottle.
Negative control sera (Healthy Human Serum): 1ml/ bottles, 1 bottle.
Positive control serum (serum of Coxsack A16 patients with viral infections): 1ml/ bottles, 1 bottle.
Sample diluting liquid (10mM PBS buffer solution, 0.6%BSA, 0.2% casein, 0.1%ProClin300, pH
7.2): 12ml/ bottles or 6ml/ bottles.1 bottle.
Substrate A liquid (citrate buffer, 0.2% hydrogen peroxide urea): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Substrate B liquid (0.2mg/ml TMB): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Terminate liquid (2M sulfuric acid solution): 6ml/ bottles or 3ml/ bottles, 1 bottle.
Cleaning solution (0.2M phosphate buffer, 0.5% Tween-20, pH 7.2): 50ml/ bottles or 30ml/ bottles, 1 bottle.
Specification: 1 part.
Specifically, being coated with the ELISA Plate of goat-anti human IgM antibody, the preparation method is the same as that of Example 1.
Specifically, enzyme label Coxsack A16 virus antigen polypeptide-KLH albumen preparation method the following steps are included:
S1: being diluted to 1-5mg/ml for Coxsack A16 virus antigen polypeptide-KLH albumen, takes 1ml Coxsack A16 virus anti-
Former polypeptide-K LH diluted protein solution is packed into bag filter, places carbonate buffer solution (50mM, pH 9.6) and dialyses 10-16 hours；
S2: horseradish peroxidase (HRP) is configured to the HRP solution that concentration is 10mg/ml, in the HRP solution of 4ml
The NaIO of 1ml is added4Solution reacts at room temperature 30 minutes；After reaction, 40ul ethylene glycol is added, reacts at room temperature 30 minutes, is made
HRP reaction solution；
S3: the HRP reaction solution of 4ml is added in the Coxsack A16 virus antigen polypeptide-KLH albumen after step S1 dialysis,
Under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；
S4: by NaBH4It is made into the NaBH that concentration is 10mg/ml4Solution, by the NaBH of 1500ul4Solution is added in step S3
Mixed liquor in, temperature be 4 DEG C under conditions of, react 2 hours, per half an hour rocks once；
S5: it is dialysed 24-30 hours in the reaction solution of step S4 with PBS buffer solution (10mM, pH 7.2), liquid is changed during dialysis
5-8 times, the glycerol of equivalent volumes is added, is uniformly mixed, the Coxsack A16 virus antigen polypeptide-KLH albumen of enzyme label is made.
Specifically, the Coxsack A16 virus antigen polypeptide-KLH diluted protein solution of enzyme label is to be diluted with enzyme-labelled antigen
Liquid (10mM PBS buffer solution, 10% calf serum, 0.25% collagen, 0.1% aminopyrine, 0.1%ProClin300)
The Coxsack A16 virus antigen polypeptide conjugate that enzyme marks is diluted to 1000-5000 times.
Effete test embodiment 1:
Coxsack A16 virus IgM antibody detection kit specificity experiments
Sample: the serum specimen of clinical Virus patients, respectively influenza A virus, influenza B virus, respiratory tract close
Cellular virus, Respiratory Tract Adenovirus, EV71 virus, Coxsack A10 virus, Coxsack A8 patients with viral infections.
Diagnose confirmation method: fluorescence PCR method.
Experimental method: every kind of patients with viral infections chooses 2, acquires 14 parts of virus infection serum specimens altogether, selects and implements
Coxsack A16 virus IgM antibody detection kit prepared by example 1 is tested.
The application method of Coxsack A16 virus IgM antibody detection kit the following steps are included:
(1) it balances and matches liquid:
It takes out kit placement equilibrium at room temperature 30 minutes or more；Cleaning solution distilled water or deionized water are diluted 20 times.
(2) it numbers:
Serum specimen is numbered, and is added on corresponding ELISA Plate, Loading sequence record is carried out.Every plate at least sets 2
Hole negative control, 1 hole positive control and 1 hole blank control.
(3) it is loaded:
2 hole of negative control, 1 hole of positive control, each 100ul control serum.1 hole of blank control is vacant.Remaining micropore is added
100ul sample diluting liquid adds sample 10ul to be checked, and reaction plate, which is gently shaken, makes sample blending.
(4) it incubates:
After sealing plate film sealing plate, sets in 37 DEG C of incubators or water-bath and react 30 minutes.
(5) board-washing:
Sealing plate film is taken off, is machine-washed plate 5 times with board-washing, is finally buckled on blotting paper dry.
(6) enzyme mark antigen liquid:
Every hole is added 100ul enzyme-labelled antigen liquid, except blank well.
(7) it incubates:
After sealing plate film sealing plate, sets in 37 DEG C of incubators or water-bath and react 30 minutes.
(8) board-washing:
Sealing plate film is taken off, is machine-washed plate 5 times with board-washing, is finally buckled on blotting paper dry.
(9) it develops the color:
Substrate A liquid is first added, 50ul is added in every hole, adds substrate B liquid, and 50ul is added in every hole, and gently concussion mixes, and uses
After sealing plate film sealing plate, 37 DEG C are protected from light colour developing 15 minutes.
(10) it terminates:
Terminate liquid 50ul is added in every hole, and gently concussion mixes.
(11) it measures:
It sets microplate reader Detection wavelength (to detect preferably with dual wavelength 450/630) as 450nm, measures each hole A value.Unicast
Long detection: it is returned to zero with blank well, tests the A value of each hole 450nm.Double UV check: can not set blank well, test each hole 450nm/
The A value of 630nm.
(12) result judgement:
Critical value: Cut off (C.0)=0.10+ negative control is averaged (NC) A value.
Feminine gender determines: being determined as that Coxsack A16 virus IgM antibody is negative when sample A value < critical value (Cut off value).
The positive determines: being determined as that Coxsack A16 virus IgM antibody is positive when sample A value >=critical value (Cut off value).
As a result: Coxsack A16 virus IgM antibody detection kit cannot identify influenza A virus, influenza B virus,
Respiratory Syncytial Virus(RSV), Respiratory Tract Adenovirus, EV71 virus, Coxsack A10 virus, Coxsack A8 patients with viral infections's serum.
Embodiment 2, the result of 3 specificity experiments and implementation column 1 are essentially identical, and details are not described herein.
As can be seen from the above results Coxsack A16 virus IgM antibody detection kit specificity is 100%.
Effete test embodiment 2:
Coxsack A16 virus IgM antibody detection kit and Coxsack A16 viral nucleic acid fluorescence PCR method comparative experiments
Clinical doubtful patients with viral infections 100 are chosen, acquires the blood preparation and oropharyngeal swab specimen of patient respectively, respectively
With embodiment 1 prepare Coxsack A16 virus IgM antibody detection kit and Coxsack A16 viral nucleic acid fluorescence PCR method into
Row test, as a result such as table 1:
1 Coxsack A16 virus IgM antibody detection kit of table and fluorescence PCR method comparison result
Coxsack A16 virus IgM antibody detection kit and Coxsack A16 viral nucleic acid prepared by the embodiment of the present invention 1
Fluorescence PCR method compares, and sensitivity reaches 95.12%, specificity 94.92%.
The result and the basic phase of implementation column 1 of embodiment 2,3 and Coxsack A16 viral nucleic acid fluorescence PCR method comparative experiments
Together, details are not described herein.
As can be seen from the above results Coxsack A16 virus IgM antibody detection kit of the present invention can meet clinical detection
Condition.
The above described is only a preferred embodiment of the present invention, limitation in any form not is done to the present invention, therefore
All contents without departing from technical solution of the present invention, it is made to the above embodiment according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
<110>biotech inc Guangzhou Rui Hui
<120>Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> PRT
<213> Amino Acid Sequence
<400> 1
Thr Val Gly Thr Glu Lys Ser Pro His Ser
5 10
Ile Thr Leu Arg Val Tyr Met Arg Ile
15 19
Claims (8)
1. a kind of Coxsack A16 virus antigen polypeptide conjugate, it is characterised in that: the conjugate is by having such as SEQ ID NO:
The Coxsack A16 virus antigen polypeptide and carrier protein of amino acid sequence shown in 1 form；Chemical coupling agent is EDC.HCl；Institute
Stating carrier protein includes BSA albumen, OVA albumen, KLH albumen；
The Coxsack A16 virus antigen polypeptide conjugate is prepared by the following method to obtain:
It takes Coxsack A16 virus antigen polypeptide and EDC.HCl to distinguish soluble in water, mixing, under conditions of temperature is 4 DEG C, stirs
Reaction 30 minutes is mixed, is then slowly added dropwise into mixed liquor into carrier protein solution, is adjusted pH value to 7.4, it is small to be stirred to react 16-24
When, reactant is dialysed 20-30 hours again, and a dialyzate was changed every 3-5 hours, is changed altogether liquid 6 times, and it is anti-that Coxsack A16 virus is made
Former polypeptide coupling；
The mass ratio of the Coxsack A16 virus antigen polypeptide and EDC.HCl are (1-3): (1-10)；The Coxsack A16
The mass volume ratio g/L of virus antigen polypeptide and water is (20-60): the mass volume ratio g/L of 1, the EDC.HCl and water is
(20—200):1；The concentration of the carrier protein solution is 2-20mg/ml.
2. a kind of preparation method of Coxsack A16 virus antigen polypeptide conjugate, which comprises the following steps:
Coxsack A16 virus antigen polypeptide and EDC.HCL with the amino acid sequence as shown in SEQ ID NO:1 is taken to distinguish molten
Yu Shuizhong, mixing are stirred to react 30 minutes, are then slowly added dropwise into mixed liquor into carrier egg under conditions of temperature is 4 DEG C
White solution is adjusted pH value to 7.4, is stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary saturating
Liquid is analysed, is changed altogether liquid 6 times, Coxsack A16 virus antigen polypeptide conjugate is made；
The mass ratio of the Coxsack A16 virus antigen polypeptide and EDC.HCl are (1-3): (1-10)；The Coxsack A16
The mass volume ratio g/L of virus antigen polypeptide and water is (20-60): the mass volume ratio g/L of 1, the EDC.HCl and water is
(20—200):1；The concentration of the carrier protein solution is 2-20mg/ml.
3. a kind of Coxsack A16 virus IgM antibody detection kit, it is characterised in that: including antibody coated elisa plate and enzyme mark
Remember antigen liquid；The antibody coated elisa plate is the ELISA Plate for being coated with goat-anti human IgM antibody, and the enzyme-labelled antigen liquid is to use
The Coxsack A16 virus antigen polypeptide conjugate of the enzyme label of enzyme-labelled antigen diluted, the Coxsack A16 virus are anti-
Former polypeptide coupling is conjugate described in claim 1.
4. Coxsack A16 virus IgM antibody detection kit according to claim 3, it is characterised in that: the enzyme label
Coxsack A16 virus antigen polypeptide conjugate led to by Coxsack A16 virus antigen polypeptide conjugate with horseradish peroxidase
Cross what Over-voltage protection crosslinking obtained.
5. Coxsack A16 virus IgM antibody detection kit according to claim 4, it is characterised in that: the enzyme label
Coxsack A16 virus antigen polypeptide conjugate preparation method the following steps are included:
S1: being diluted to 1-5mg/ml for Coxsack A16 virus antigen polypeptide conjugate, small with carbonate buffer solution dialysis 10-16
When；The carbonate buffer solution molar concentration is 50mM, pH value 9.6；
S2: horseradish peroxidase is configured to the horseradish peroxidase solution that concentration is 10mg/ml, NaIO is added4Solution,
Room temperature reaction 10-45 minutes；After reaction, ethylene glycol is added, reacts at room temperature 20-60 minutes, horseradish peroxidase reaction is made
Liquid；The horseradish peroxidase solution and NaIO4The volume ratio of solution is 1:(2-10), the horseradish peroxidase solution
Volume ratio with ethylene glycol is 1:(5-20)；
S3: horseradish peroxidase reaction solution is added in the Coxsack A16 virus antigen polypeptide conjugate after step S1 dialysis,
Under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；Coxsack A16 virus antigen polypeptide conjugate and horseradish peroxidase
The volume ratio 1:(2-10 of reaction solution)；
S4: by NaBH4It is made into the NaBH that concentration is 2-100mg/ml4Solution, by NaBH4The mixed liquor in step S3 is added in solution
In, under conditions of temperature is 4 DEG C, react 1-3 hours, per half an hour rocks once；NaBH4Solution and horseradish peroxidase
The volume ratio 1:(1-10 of reaction solution)；
S5: being dialysed 24-30 hours with PBS buffer solution in the reaction solution of step S4, change liquid 5-8 times during dialysis, and equivalent body is added
Long-pending glycerol is uniformly mixed, and the Coxsack A16 virus antigen polypeptide conjugate of enzyme label is made；Mole of the PBS buffer solution
Concentration is 10mM, pH value 7.2.
6. Coxsack A16 virus IgM antibody detection kit according to claim 3, it is characterised in that: the enzyme label
Coxsack A16 virus antigen polypeptide conjugate dilution be enzyme mark with enzyme-labelled antigen dilution Coxsack A16 sick
Malicious antigen polypeptide conjugate is diluted to 1000-5000 times；The enzyme-labelled antigen dilution include PBS buffer solution, calf serum,
Collagen, aminopyrine and ProClin300, wherein the molar concentration of PBS buffer solution is 10mM, and the volume of calf serum is dense
Degree is 10%, and the mass concentration of collagen is 0.25%, and the mass concentration of aminopyrine is the matter of 0.1%, ProClin300
Measuring concentration is 0.1%, pH value 7.2.
7. Coxsack A16 virus IgM antibody detection kit according to claim 3, it is characterised in that: be coated with goat-anti
The preparation method of the ELISA Plate of human IgM antibody the following steps are included:
1) be coated with: it is 5 μ that the carbonate buffer solution for being 9.6 with molar concentration 50mM, pH value, which dilutes goat-anti human IgM antibody to concentration,
ELISA Plate is added in g/mL, and every hole adds 95-105 μ L, is coated with 16-24 hours under conditions of temperature is 4 DEG C, is then washed with PBST
It washs 1 time；
2) close: every hole adds 150 μ L of confining liquid, closes 16-20 hours under conditions of temperature is 4 DEG C, gets rid of deblocking liquid, does
It is dry；The confining liquid includes PBS buffer solution, calf serum, sucrose and ProClin300, wherein the molar concentration of PBS buffer solution
For 10mM, the volumetric concentration of calf serum is 5%, and the mass concentration that the mass concentration of sucrose is 3%, ProClin300 is
0.05%, pH value 7.2.
8. Coxsack A16 virus IgM antibody detection kit according to claim 3, it is characterised in that: further include feminine gender
Control serum, positive control serum, sample diluting liquid substrate A, substrate B, terminate liquid and cleaning solution；
The negative control sera is Healthy Human Serum；Positive control serum is the serum of Coxsack A16 patients with viral infections；Institute
Stating sample diluting liquid includes PBS buffer solution, BSA, casein and ProClin300, and wherein the molar concentration of PBS buffer solution is
The mass concentration of 10mM, BSA are 0.6%, and the mass concentration that the mass concentration of casein is 0.2%, ProClin300 is
0.1%, pH value 7.2；Substrate A is the citrate buffer for being 0.2% hydrogen peroxide urea containing mass concentration；The bottom
Object B is the TMB that concentration is 0.2mg/ml；The terminate liquid is the sulfuric acid solution that molar concentration is 2M；The cleaning solution includes phosphorus
Phthalate buffer and Tween-20, wherein the molar concentration of phosphate buffer is 0.2M, and the mass concentration of Tween-20 is
0.5%, pH value 7.2.
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| CN110687291A (en) * | 2019-10-30 | 2020-01-14 | 中国食品药品检定研究院 | Coxsackie virus A16 type virus antigen detection kit |
| CN113495157A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Enzyme conjugate diluent, total thyroxine quantitative detection kit and use method thereof |
| CN111999495A (en) * | 2020-08-18 | 2020-11-27 | 北京贝尔生物工程股份有限公司 | Kit for detecting Coxsackie group B virus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof |
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| CN103833830B (en) * | 2012-11-22 | 2018-09-04 | 中国科学院上海巴斯德研究所 | Conservative neutralizing epitope polypeptide of coxsackie virus A 16-type and application thereof |
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