CN108152511A - Coxsack A16 virus antigen polypeptides and its IgM antibody detection kit - Google Patents

Coxsack A16 virus antigen polypeptides and its IgM antibody detection kit Download PDF

Info

Publication number
CN108152511A
CN108152511A CN201711409686.XA CN201711409686A CN108152511A CN 108152511 A CN108152511 A CN 108152511A CN 201711409686 A CN201711409686 A CN 201711409686A CN 108152511 A CN108152511 A CN 108152511A
Authority
CN
China
Prior art keywords
coxsack
solution
virus
virus antigen
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201711409686.XA
Other languages
Chinese (zh)
Other versions
CN108152511B (en
Inventor
李有生
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Rhfay Biotechnology Co Ltd
Original Assignee
Guangzhou Rhfay Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Rhfay Biotechnology Co Ltd filed Critical Guangzhou Rhfay Biotechnology Co Ltd
Priority to CN201711409686.XA priority Critical patent/CN108152511B/en
Publication of CN108152511A publication Critical patent/CN108152511A/en
Application granted granted Critical
Publication of CN108152511B publication Critical patent/CN108152511B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • 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
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention provides a kind of Coxsack A16 virus antigen polypeptides to have such as SEQ ID NO：Amino acid sequence shown in 1.The antigen polypeptide carries out multiple Coxsack A16 viral prevalences strains genetics row 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 it is Coxsack A16 virus antigen polypeptides to use enzyme-labelled antigen, can be directed to the Coxsack A16 Strain of a variety of hypotypes, identify the serum virus of a variety of Coxsack A16 viruses infection.The detection kit of the present invention is reproducible, high specificity, high sensitivity, effectively detects the situation of Coxsack A16 viruses infection, plays the role of in terms of prevention and control Coxsack A16 virus infection active and effective.

Description

Coxsack A16 virus antigen polypeptides and its IgM antibody detection kit
Technical field
The invention belongs to immuno-biology technical fields, and in particular to a kind of Coxsack A16 virus antigen polypeptides and its IgM Antibody assay kit.
Background technology
Coxsackie virus belongs to non-poliovirus in Picornaviridae, is broadly divided into two kinds of hypotype A types And Type B.Coxsack A16 viruses belong to A type Coxsackie virus, it is to cause the main disease of hand-foot-and-mouth disease poison prevalence with EV71 viruses Substance is the highest enterovirus of China's Mainland prevalence frequency.RNA segment of the Coxsack A16 viruses containing one section of single-stranded positive, Length substantially 7400bp, the coat protein of package RNA nucleic acid is 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.The children of less than 5 years old are easier infection Coxsack A16 viruses due to developing immune system prematurity.General Ke's Sa Strange A16 viruses infection incubation period is 3-6 days, is mainly broadcast by excrement oral instructions, 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 of object control patient and pain.Therefore, carry out Coxsack A16 viruses early diagnosis be control and prevention of disease emphasis, early every From the possibility that patient reduces transmission.
The early diagnosis of Coxsack A16 viruses 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 variations are very fast, virus Hypotype also compare it is more, be mainly reflected in VP1 protein sequences and easily change.Immunology diagnosis mainly selects Coxsack A16 virus VP 1s 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 kits, the sensitivity with high detection can solve the problems, such as clinically existing missing inspection, It is suitble to be widely popularized.
Invention content
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 antigens are more Peptide, a kind of Coxsack A16 virus antigen polypeptide conjugates and preparation method thereof and Coxsack A16 virus IgM antibody detection reagents Box.Coxsack A16 virus antigen polypeptides are the high antigen fragments of homology of a variety of Coxsack A16 viruses；Detection kit is to Sa Strange A16 viruses have the sensitivity of high detection, can solve the problems, such as clinically existing missing inspection, be suitble to be widely popularized.
To solve the above problems, the technical solution used is as follows：
Present invention firstly provides a kind of Coxsack A16 virus antigen polypeptides to have such as SEQID N0：Amino acid shown in 1 Sequence.The antigen polypeptide carries out multiple Coxsack A16 viral prevalences strains genetics row comparison 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 viruses Antigen polypeptide is formed with carrier protein；Chemical coupling agent is EDC；The carrier protein includes BSA albumen, OVA albumen, KLH eggs In vain.
Preferably, the carrier protein is BSA albumen.
The present invention provides a kind of preparation methods of Coxsack A16 virus antigen polypeptide conjugates, which is characterized in that including Following steps：
Coxsack A16 virus antigen polypeptides and EDC.HCL is taken to distinguish soluble in water, mixing, in the condition that temperature is 4 DEG C Under, it is stirred to react 30 minutes, is then slowly added dropwise into mixed liquor into carrier protein solution, pH value is adjusted to be stirred to react to 7.4 16-24 hours, reactant was dialysed 20-30 hours again, and a dialyzate was changed every 3-5 hours, changed liquid altogether 6 times, and Coxsack A16 is made Virus antigen polypeptide conjugate.
Preferably, a concentration of 2-20mg/ml of the carrier protein solution.
It is particularly preferred, the carrier protein solution be BSA protein solutions, a concentration of 2- of the BSA protein solutions 20mg/ml。
Preferably, the mass ratio of the Coxsack A16 virus antigen polypeptides and EDC.HCl are (1-3): (1-10)；It is described The mass volume ratio g/L of Coxsack A16 virus antigen polypeptides 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 dilutions of enzyme label.
Preferably, the Coxsack A16 virus antigen polypeptides conjugate of the enzyme label is by Coxsack A16 virus antigen polypeptides Conjugate is obtained with horseradish peroxidase by Over-voltage protection crosslinking.
Particularly preferred, the preparation method of the Coxsack A16 virus antigen polypeptide conjugates of the enzyme label is including following Step：
S1：Coxsack A16 virus antigen polypeptide conjugates are diluted to 1-5mg/ml, with carbonate buffer solution dialysis 10- 16 hours；The carbonate buffer solution molar concentration be 50mM, pH value 9.6；
S2：Horseradish peroxidase is configured to the horseradish peroxidase solution of a concentration of 10mg/ml, adds in NaIO4 Solution reacts at room temperature 10-45 minutes；After reaction, ethylene glycol is added in, is reacted 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 polypeptides that horseradish peroxidase reaction solution is added in after step S1 dialysis are coupled In object, under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；Coxsack A16 virus antigen polypeptides conjugate and horseradish peroxide The volume ratio 1 of compound enzyme reaction solution: (2-10)；
S4：By NaBH4It is made into the NaBH of a concentration of 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, rocked once per half an hour；NaBH4Solution and horseradish peroxidating The volume ratio 1 of object enzyme reaction solution: (1-10)；
S5：It is dialysed 24-30 hours with PBS buffer solution in the reaction solution of step S4, liquid is changed during dialysis 5-8 times, add in etc. The glycerine of volume is measured, is uniformly mixed, the Coxsack A16 virus antigen polypeptide conjugates of enzyme label are made；The PBS buffer solution Molar concentration is 10mM, pH value 7.2.
Preferably, the Coxsack A16 virus antigen polypeptide conjugates dilution of the enzyme label is dilute with enzyme-labelled antigen It releases liquid and the Coxsack A16 virus antigen polypeptide conjugates 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 The molar concentration of woods and ProClin300, wherein PBS buffer solution are 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 Be worth is 7.2.
Preferably, the Coxsack A16 virus antigen polypeptides conjugate is Coxsack A16 virus antigen polypeptide-BSA eggs In vain.
Preferably, the preparation method for being coated with the ELISA Plate of goat-anti human IgM antibody includes the following steps：
1) it is coated with：Goat-anti human IgM antibody is diluted 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 in, adds 95-105 μ L per hole, is coated with 16-24 hours under conditions of being 4 DEG C in temperature, Ran Houyong PBST is washed 1 time；
2) it closes：Add 150 μ L of confining liquid per hole, closed 16-20 hours under conditions of being 4 DEG C in temperature, get 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 It spends 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.
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 kits further include 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 antibodies.
Particularly preferred, the negative control sera is Healthy Human Serum；Positive control serum is Coxsack A16 virus senses 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 solution 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 contains the citrate buffer that mass concentration is 0.2% hydrogen peroxide urea；Institute State the TMB that substrate B is a concentration of 0.2mg/ml.
Particularly preferred, the terminate liquid is the sulfuric acid solution that molar concentration is 2M.
Particularly preferred, the cleaning solution includes mole of phosphate buffer and Tween-20, wherein phosphate buffer A concentration of 0.2M, 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.
Application method the present invention provides Coxsack A16 virus IgM antibody detection kits includes the following steps：
(1) it balances and with liquid：
It takes out kit and places 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, carries out Loading sequence record.2 are at least set per plate 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 serums.1 hole of blank control is vacant.Remaining micropore adds in 100ul sample diluting liquids add sample 10ul to be checked, and reaction plate, which is gently shaken, makes sample blending.
(4) it incubates：
After sealing plate film sealing plate, put in 37 DEG C of incubators or water-bath and react 30 minutes.
(5) board-washing：
Sealing plate film is taken off, with board-washing machine washing plate 5 times, finally buckle and does on blotting paper.
(6) enzyme mark antigen liquid：
100ul enzyme-labelled antigen liquid is added in per hole, except blank well.
(7) it incubates：
After sealing plate film sealing plate, put in 37 DEG C of incubators or water-bath and react 30 minutes.
(8) board-washing：
Sealing plate film is taken off, with board-washing machine washing plate 5 times, finally buckle and does on blotting paper.
(9) it develops the color：
Substrate A liquid is first added in, 50ul is added in per hole, adds substrate B liquid, 50ul is added in per hole, gently shakes mixing, is used 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 per hole, gently shakes mixing.
(11) it measures：
It sets microplate reader Detection wavelength (to detect preferably with dual wavelength 450/630) as 450nm, measures each hole A values.Unicast Long detection：It is returned to zero with blank well, tests the A values of each hole 450nm.Double UV check：Blank well can not be set, tests each hole 450nm/ The A values of 630nm.
(12) result judgement：
Critical value：Cut off (C.0)=0.10+ negative controls are averaged (NC) A values.
Feminine gender judgement：Sample A values<It is determined as Coxsack A16 virus IgM antibodies feminine gender during critical value (Cut off values).
Positive judgement：It is determined as the Coxsack A16 virus IgM antibodies positive during sample A values >=critical value (Cut off values).
The present invention also provides Coxsack A16 virus IgM antibodies detection kit, Coxsack A16 is viral in sample is detected The application of IgM antibody, so as to provide a kind of approach for detecting Coxsack A16 viruses.
Preferably, the sample is human serum or human plasma.
Beneficial effects of the present invention：
1st, Coxsack A16 virus antigen polypeptides of the invention are viral to multiple Coxsack A16 using bioinformatics method Epidemic strain carries out genetics row and compares design synthesis, can be directed to the Coxsack A16 Strain of a variety of hypotypes；
2nd, detection kit of the invention is Coxsack A16 virus antigen polypeptides 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 viruses infection；
3rd, detection kit of the invention is reproducible, high specificity, high sensitivity, effectively detects Coxsack A16 viruses The situation of infection plays the role of active and effective in terms of prevention and control Coxsack A16 virus infection；
4th, detection kit of the invention is easy to operate, and detection time is short, can meet the medical institutions of different stage and make With, Coxsack A16 viruses infection early diagnosis in play the role of preferable monitoring.
Specific embodiment
The present invention proposes that a kind of Coxsack A16 virus antigen polypeptides have such as SEQ ID NO：Amino acid sequence shown in 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 row, and by its synthesis polypeptide, which can identify the serum of a variety of hypotype Coxsack A16 viruses infection Antibody.Gill biochemistry Shanghai Co., Ltd is supplied to synthesize Coxsack A16 virus antigen polypeptide sequences, purity 98% More than, pass through mass spectroscopy qualification.
The present invention proposes a kind of Coxsack A16 virus antigen polypeptide conjugates, by Coxsack A16 virus antigen polypeptides with carrying Body protein forms.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.
Technical scheme of the present invention is described further below by specific preferred embodiment combination effete test embodiment, but The present invention is not limited in following embodiment.
Embodiment 1
(1) Coxsack A16 virus antigen polypeptides
The sequence such as SEQ ID NO of Coxsack A16 virus antigen polypeptide segments：Shown in 1,19 amino acid, is entrusted in total Gill biochemistry Shanghai Co., Ltd is responsible for synthesis, and purity is more than 98%, and freeze-drying preserves.
(2) Coxsack A16 virus antigen polypeptides conjugate (Coxsack A16 virus antigen polypeptide-BSA albumen)
The preparation method of Coxsack A16 virus antigen polypeptide-BSA albumen includes the following steps：
20mg Coxsack A16 virus antigen polypeptides and 50mg EDC.HCL is taken to be dissolved in respectively in 0.5ml ultra-pure waters, is mixed, Under conditions of temperature is 4 DEG C, it is stirred to react 30 minutes, the BSA into a concentration of 10mg/ml is then slowly added dropwise into mixed liquor Protein solution adjusts pH value to be stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary to 7.4 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 kits
A kind of Coxsack A16 virus IgM antibodies detection kit includes antibody coated elisa plate, enzyme-labelled antigen liquid, the moon 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 gauge lattice or 4 × 12 hole gauge lattice.
Enzyme-labelled antigen solution (the Coxsack A16 virus antigen polypeptide-BSA diluted protein solutions of enzyme label)：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 solutions, 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 solutions)：6ml/ bottles or 3ml/ bottles, 1 bottle.
Cleaning solution (0.2M phosphate buffers, 0.5% Tween-20, pH 7.2)：50ml/ bottles or 30ml/ bottles, 1 bottle.
Specification：1 part.
Specifically, the preparation method for being coated with the ELISA Plate of goat-anti human IgM antibody includes the following steps：
1) it is coated with：Goat-anti human IgM antibody is diluted to a concentration of 5 μ g/mL with carbonate buffer solution (50mM, pH 9.6), is added Enter ELISA Plate, add 100 μ L per hole, be coated with 16-24 hours under conditions of being 4 DEG C in temperature, then washed 1 time with PBST；
2) it closes：Add 150 μ L of confining liquid per hole, closed 18 hours under conditions of being 4 DEG C in temperature, get rid of deblocking liquid (10mMPBS buffer solutions, 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, the preparation method of the Coxsack A16 virus antigen polypeptide-BSA albumen of enzyme label includes the following steps：
S1：Coxsack A16 virus antigen polypeptide-BSA albumen is diluted to 1-5mg/ml, 1ml Coxsack A16 viruses is taken to resist Former polypeptide-BSA diluted protein solutions are packed into bag filter, place carbonate buffer solution (50mM, pH 9.6) and dialyse 10-16 hours；
S2：Horseradish peroxidase (HRP) is configured to the HRP solution of a concentration of 10mg/ml, in the HRP solution of 4ml Add in the NaIO of 1ml4Solution reacts at room temperature 30 minutes；After reaction, 40ul ethylene glycol is added in, is reacted at room temperature 30 minutes, is made HRP reaction solutions；
S3：The HRP reaction solutions of 4ml are 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 of a concentration of 10mg/ml4Solution, by the NaBH of 1500ul4Solution is added in step S3 Mixed liquor in, in temperature under conditions of 4 DEG C, to react 1-3 hour, per half an hour rock once；
S5：It is dialysed 24-30 hours with PBS buffer solution (10mM, pH 7.2) in the reaction solution of step S4, liquid is changed during dialysis 5-8 times, the glycerine of equivalent volumes is added in, 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 solutions of enzyme label is dilute 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 conjugates that enzyme marks are diluted to 1000-5000 times.
Embodiment 2
(1) Coxsack A16 virus antigen polypeptides
The sequence such as SEQ ID NO of Coxsack A16 virus antigen polypeptide segments：Shown in 1,19 amino acid, is entrusted in total Gill biochemistry Shanghai Co., Ltd is responsible for synthesis, and purity is more than 98%, and freeze-drying preserves.
(2) Coxsack A16 virus antigen polypeptides conjugate (Coxsack A16 virus antigen polypeptide-OVA albumen)
The preparation method of Coxsack A16 virus antigen polypeptide-OVA albumen includes the following steps：
20mg Coxsack A16 virus antigen polypeptides and 50mg EDC.HCL is taken to be dissolved in respectively in 0.5ml ultra-pure waters, is mixed, Under conditions of temperature is 4 DEG C, it is stirred to react 30 minutes, the OVA into a concentration of 10mg/ml is then slowly added dropwise into mixed liquor Protein solution adjusts pH value to be stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary to 7.4 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 kits
A kind of Coxsack A16 virus IgM antibodies detection kit includes antibody coated elisa plate, enzyme-labelled antigen liquid, the moon 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 gauge lattice or 4 × 12 hole gauge lattice.
Enzyme-labelled antigen solution (the Coxsack A16 virus antigen polypeptide-OVA diluted protein solutions of enzyme label)：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 solutions)：6ml/ bottles or 3ml/ bottles, 1 bottle.
Cleaning solution (0.2M phosphate buffers, 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, the preparation method of the Coxsack A16 virus antigen polypeptide-OVA albumen of enzyme label includes the following steps：
S1：Coxsack A16 virus antigen polypeptide-OVA albumen is diluted to 1-5mg/ml, 1ml Coxsack A16 viruses is taken to resist Former polypeptide-OVA diluted protein solutions are packed into bag filter, place carbonate buffer solution (50mM, pH 9.6) and dialyse 10-16 hours；
S2：Horseradish peroxidase (HRP) is configured to the HRP solution of a concentration of 10mg/ml, in the HRP solution of 4ml Add in the NaIO of 1ml4Solution reacts at room temperature 30 minutes；After reaction, 40ul ethylene glycol is added in, is reacted at room temperature 30 minutes, is made HRP reaction solutions；
S3：The HRP reaction solutions of 4ml are 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 of a concentration of 10mg/ml4Solution, by the NaBH of 1500ul4Solution is added in step S3 Mixed liquor in, in temperature under conditions of 4 DEG C, to react 1-3 hour, per half an hour rock once；
S5：It is dialysed 24-30 hours with PBS buffer solution (10mM, pH 7.2) in the reaction solution of step S4, liquid is changed during dialysis 5-8 times, the glycerine of equivalent volumes is added in, 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 solutions of enzyme label is dilute with enzyme-labelled antigen Liquid (10mMPBS buffer solutions, 10% calf serum, 0.25% collagen, 0.1% aminopyrine, 0.1%ProClin300) will The Coxsack A16 virus antigen polypeptide conjugates of enzyme label are diluted to 1000-5000 times.
Embodiment 3
(1) Coxsack A16 virus antigen polypeptides
The sequence such as SEQ ID N0 of Coxsack A16 virus antigen polypeptide segments：Shown in 1,19 amino acid, is entrusted in total Gill biochemistry Shanghai Co., Ltd is responsible for synthesis, and purity is more than 98%, and freeze-drying preserves.
(2) Coxsack A16 virus antigen polypeptides conjugate (Coxsack A16 virus antigen polypeptide-KLH albumen)
The preparation method of Coxsack A16 virus antigen polypeptide-KLH albumen includes the following steps：
20mg Coxsack A16 virus antigen polypeptides and 50mg EDC.HCL is taken to be dissolved in respectively in 0.5ml ultra-pure waters, is mixed, Under conditions of temperature is 4 DEG C, it is stirred to react 30 minutes, the KLH into a concentration of 10mg/ml is then slowly added dropwise into mixed liquor Protein solution adjusts pH value to be stirred to react 16-24 hours, reactant is dialysed 20-30 hours again, is changed every 3-5 hours primary to 7.4 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 kits
A kind of Coxsack A16 virus IgM antibodies detection kit includes antibody coated elisa plate, enzyme-labelled antigen liquid, the moon 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 gauge lattice or 4 × 12 hole gauge lattice.
Enzyme-labelled antigen solution (the Coxsack A16 virus antigen polypeptide-KLH diluted protein solutions of enzyme label)：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 solutions)：6ml/ bottles or 3ml/ bottles, 1 bottle.
Cleaning solution (0.2M phosphate buffers, 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, the preparation method of the Coxsack A16 virus antigen polypeptide-KLH albumen of enzyme label includes the following steps：
S1：Coxsack A16 virus antigen polypeptide-KLH albumen is diluted to 1-5mg/ml, 1ml Coxsack A16 viruses is taken to resist Former polypeptide-K LH diluted protein solutions are packed into bag filter, place carbonate buffer solution (50mM, pH 9.6) and dialyse 10-16 hours；
S2：Horseradish peroxidase (HRP) is configured to the HRP solution of a concentration of 10mg/ml, in the HRP solution of 4ml Add in the NaIO of 1ml4Solution reacts at room temperature 30 minutes；After reaction, 40ul ethylene glycol is added in, is reacted at room temperature 30 minutes, is made HRP reaction solutions；
S3：The HRP reaction solutions of 4ml are 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 of a concentration of 10mg/ml4Solution, by the NaBH of 1500ul4Solution is added in step S3 Mixed liquor in, in temperature under conditions of 4 DEG C, to react 2 hours, rocked once per half an hour；
S5：It is dialysed 24-30 hours with PBS buffer solution (10mM, pH 7.2) in the reaction solution of step S4, liquid is changed during dialysis 5-8 times, the glycerine of equivalent volumes is added in, 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 solutions of enzyme label is dilute 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 conjugates that enzyme marks are 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 viruses, Coxsack A10 viruses, Coxsack A8 patients with viral infections.
Diagnose confirmation method：Fluorescence PCR method.
Experimental method：Each patients with viral infections chooses 2, acquires 14 parts of virus infection serum specimens altogether, selects and implement Coxsack A16 virus IgM antibody detection kits prepared by example 1 are tested.
The application method of Coxsack A16 virus IgM antibody detection kits includes the following steps：
(1) it balances and with liquid：
It takes out kit and places 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, carries out Loading sequence record.2 are at least set per plate 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 serums.1 hole of blank control is vacant.Remaining micropore adds in 100ul sample diluting liquids add sample 10ul to be checked, and reaction plate, which is gently shaken, makes sample blending.
(4) it incubates：
After sealing plate film sealing plate, put in 37 DEG C of incubators or water-bath and react 30 minutes.
(5) board-washing：
Sealing plate film is taken off, with board-washing machine washing plate 5 times, finally buckle and does on blotting paper.
(6) enzyme mark antigen liquid：
100ul enzyme-labelled antigen liquid is added in per hole, except blank well.
(7) it incubates：
After sealing plate film sealing plate, put in 37 DEG C of incubators or water-bath and react 30 minutes.
(8) board-washing：
Sealing plate film is taken off, with board-washing machine washing plate 5 times, finally buckle and does on blotting paper.
(9) it develops the color：
Substrate A liquid is first added in, 50ul is added in per hole, adds substrate B liquid, 50ul is added in per hole, gently shakes mixing, is used 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 per hole, gently shakes mixing.
(11) it measures：
It sets microplate reader Detection wavelength (to detect preferably with dual wavelength 450/630) as 450nm, measures each hole A values.Unicast Long detection：It is returned to zero with blank well, tests the A values of each hole 450nm.Double UV check：Blank well can not be set, tests each hole 450nm/ The A values of 630nm.
(12) result judgement：
Critical value：Cut off (C.0)=0.10+ negative controls are averaged (NC) A values.
Feminine gender judgement：Sample A values<It is determined as Coxsack A16 virus IgM antibodies feminine gender during critical value (Cut off values).
Positive judgement：It is determined as the Coxsack A16 virus IgM antibodies positive during sample A values >=critical value (Cut off values).
As a result：Coxsack A16 virus IgM antibodies detection kit cannot identify influenza A virus, influenza B virus, Respiratory Syncytial Virus(RSV), Respiratory Tract Adenovirus, EV71 viruses, Coxsack A10 viruses, 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.
By the above results as it can be seen that Coxsack A16 virus IgM antibody detection kits specificity is 100%.
Effete test embodiment 2：
Coxsack A16 virus IgM antibodies detection kit and Coxsack A16 viral nucleic acid fluorescence PCR method contrast experiments
Clinical doubtful patients with viral infections 100 is chosen, acquires the blood preparation and oropharyngeal swab specimen of patient respectively, respectively With Coxsack A16 virus IgM antibodies detection kit and Coxsack A16 viral nucleic acids fluorescence PCR method prepared by embodiment 1 into Row test, as a result such as table 1：
1 Coxsack A16 virus IgM antibodies detection kit of table and fluorescence PCR method comparison result
Coxsack A16 virus IgM antibodies detection kit prepared by the embodiment of the present invention 1 and Coxsack A16 viral nucleic acids Fluorescence PCR method compares, and sensitivity reaches 95.12%, specificity 94.92%.
Embodiment 2,3 and the 1 basic phase of result and implementation column of Coxsack A16 viral nucleic acid fluorescence PCR method contrast experiments Together, details are not described herein.
By the above results as it can be seen that Coxsack A16 virus IgM antibodies 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 is not done to the present invention, therefore All contents without departing from technical solution of the present invention, technical spirit according to the present invention is made to the above embodiment any simply to repair Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.
<110>Guangzhou Rui Hui biotech inc
<120>Coxsack A16 virus antigen polypeptides 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 (10)

1. a kind of Coxsack A16 virus antigen polypeptides, it is characterised in that：With such as SEQ ID NO：Amino acid sequence shown in 1.
2. a kind of Coxsack A16 virus antigen polypeptide conjugates, it is characterised in that：The conjugate is resisted by Coxsack A16 viruses Former polypeptide is formed with carrier protein；Chemical coupling agent is EDC；The carrier protein includes BSA albumen, OVA albumen, KLH albumen.
3. a kind of preparation method of Coxsack A16 virus antigen polypeptide conjugates, which is characterized in that include the following steps：
Coxsack A16 virus antigen polypeptides and EDC.HCL is taken to distinguish soluble in water, mixing, under conditions of temperature is 4 DEG C, is stirred Reaction 30 minutes is mixed, is then slowly added dropwise into mixed liquor into carrier protein solution, adjusts pH value that it is small to be stirred to react 16-24 to 7.4 When, reactant is dialysed 20-30 hours again, and a dialyzate was changed every 3-5 hours, changes liquid altogether 6 times, and Coxsack A16 viruses are made and resist Former polypeptide coupling.
4. preparation method according to claim 3, it is characterised in that：The Coxsack A16 virus antigen polypeptides and The mass ratio of EDC.HCl is (1-3): (1-10)；The mass volume ratio g/L of the Coxsack A16 virus antigen polypeptides and water is (20-60): the mass volume ratio g/L of 1, the EDC.HCl and water is (20-200): 1；The carrier protein solution it is a concentration of 2-20mg/ml。
5. a kind of Coxsack A16 virus IgM antibody detection kits, 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 enzyme The Coxsack A16 virus antigen polypeptide conjugate dilutions of label.
6. Coxsack A16 virus IgM antibody detection kits according to claim 5, it is characterised in that：The enzyme label Coxsack A16 virus antigen polypeptides conjugate led to by Coxsack A16 virus antigen polypeptides conjugate with horseradish peroxidase Cross what Over-voltage protection crosslinking obtained.
7. Coxsack A16 virus IgM antibody detection kits according to claim 6, it is characterised in that：The enzyme label The preparation methods of Coxsack A16 virus antigen polypeptide conjugates include the following steps：
S1：Coxsack A16 virus antigen polypeptide conjugates are diluted to 1-5mg/ml, it is small with carbonate buffer solution dialysis 10-16 When；The carbonate buffer solution molar concentration be 50mM, pH value 9.6；
S2：Horseradish peroxidase is configured to the horseradish peroxidase solution of a concentration of 10mg/ml, adds in NaIO4Solution, Room temperature reaction 10-45 minutes；After reaction, ethylene glycol is added in, is reacted 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 conjugates after step S1 dialysis, Under conditions of temperature is 4 DEG C, hemodialysis reaction 4 hours；Coxsack A16 virus antigen polypeptides conjugate and horseradish peroxidase The volume ratio 1 of reaction solution: (2-10)；
S4：By NaBH4It is made into the NaBH of a concentration of 2-100mg/ml4Solution, by NaBH4Solution adds in the mixed liquor in step S3 In, it under conditions of temperature is 4 DEG C, reacts 1-3 hours, is rocked once per half an hour；NaBH4Solution and horseradish peroxidase The volume ratio 1 of reaction solution: (1-10)；
S5：It is dialysed 24-30 hours with PBS buffer solution in the reaction solution of step S4, liquid is changed during dialysis 5-8 times, add in equivalent body Long-pending glycerine is uniformly mixed, and the Coxsack A16 virus antigen polypeptide conjugates of enzyme label are made；Mole of the PBS buffer solution A concentration of 10mM, pH value 7.2.
8. Coxsack A16 virus IgM antibody detection kits according to claim 5, it is characterised in that：The enzyme label Coxsack A16 virus antigen polypeptide conjugates dilution be that the Coxsack A16 for mark enzyme with enzyme-labelled antigen dilution is sick Malicious antigen polypeptide conjugate is diluted to 1000-5000 times；The enzyme-labelled antigen dilution include PBS buffer solution, calf serum, The molar concentration of collagen, aminopyrine and ProClin300, wherein PBS buffer solution are 10mM, and the volume of calf serum is dense It is 10% to spend, and the mass concentration of collagen is 0.25%, and the mass concentration of aminopyrine is the matter of 0.1%, ProClin300 A concentration of 0.1% is measured, pH value 7.2.
9. Coxsack A16 virus IgM antibody detection kits according to claim 5, it is characterised in that：It is coated with goat-anti The preparation method of the ELISA Plate of human IgM antibody includes the following steps：
1) it is coated with：Goat-anti human IgM antibody is diluted to a concentration of 5 μ with molar concentration 50mM, the carbonate buffer solution that pH value is 9.6 G/mL adds in ELISA Plate, adds 95-105 μ L per hole, is coated with 16-24 hours under conditions of being 4 DEG C in temperature, is then washed with PBST It washs 1 time；
2) it closes：Add 150 μ L of confining liquid per hole, closed 16-20 hours under conditions of being 4 DEG C in temperature, get rid of deblocking liquid, do It is dry；The confining liquid includes the molar concentration of PBS buffer solution, calf serum, sucrose and ProClin300, wherein 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.
10. Coxsack A16 virus IgM antibody detection kits according to claim 5, it is characterised in that：Further include the moon Property 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 It states sample diluting liquid and includes PBS buffer solution, BSA, casein and ProClin300, the molar concentration of wherein 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 contains the citrate buffer that mass concentration is 0.2% hydrogen peroxide urea；The bottom Object B is the TMB of a concentration of 0.2mg/ml；The terminate liquid is the sulfuric acid solution that molar concentration is 2M；The cleaning solution includes phosphorus The molar concentration of phthalate buffer and Tween-20, wherein phosphate buffer is 0.2M, and the mass concentration of Tween-20 is 0.5%, pH value 7.2.
CN201711409686.XA 2017-12-20 2017-12-20 Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit Active CN108152511B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201711409686.XA CN108152511B (en) 2017-12-20 2017-12-20 Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201711409686.XA CN108152511B (en) 2017-12-20 2017-12-20 Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit

Publications (2)

Publication Number Publication Date
CN108152511A true CN108152511A (en) 2018-06-12
CN108152511B CN108152511B (en) 2019-01-04

Family

ID=62465254

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201711409686.XA Active CN108152511B (en) 2017-12-20 2017-12-20 Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit

Country Status (1)

Country Link
CN (1) CN108152511B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110045105A (en) * 2018-11-09 2019-07-23 广州市妇女儿童医疗中心 Coxsack A16 virus IgA antibody quantum dot immune fluorescent chromatograph test strip and kit
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
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
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
CN113671175A (en) * 2021-09-07 2021-11-19 普十生物科技(北京)有限公司 Reagent and kit for detecting thromboxane B2
CN117783524A (en) * 2024-02-26 2024-03-29 中国医学科学院医学生物学研究所 Establishment and application of double-antibody sandwich method for indirect quantitative detection of coxsackie A10 type virus antigen

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1609874A2 (en) * 2004-05-21 2005-12-28 Bioanalisi Centro Sud S.n.c. di Perseu Sinibaldo e C. System for searching for and identifying pathogenic agents
TWI261618B (en) * 2000-11-29 2006-09-11 Dr Chip Biotechnology Inc Method for detecting and differentiating enteroviruses and the primers and probes therefor
CN101402674A (en) * 2008-10-20 2009-04-08 中国人民解放军第三军医大学 Functional peptide segment of epididymis protease inhibitors and uses thereof
CN101988924A (en) * 2009-07-31 2011-03-23 上海科新生物技术股份有限公司 Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method
CN102590504A (en) * 2012-01-31 2012-07-18 上海尧浩生物技术有限公司 CoxB IgG/Ig/M test kit using enzyme linked immunosorbent assay
WO2013098655A2 (en) * 2011-11-03 2013-07-04 Sentinext Therapeutics Sdn Bhd Antigens and vaccines directed against human enteroviruses
CN103833830A (en) * 2012-11-22 2014-06-04 中国科学院上海巴斯德研究所 Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof
CN104569392A (en) * 2013-10-28 2015-04-29 广州市康润生物科技有限公司 ELISA kit for detecting syphilis specific IgM antibody and preparation method of ELISA kit
CN104710537A (en) * 2015-04-09 2015-06-17 汪运山 Coxsackie virus CA16 VP1 recombinant antigens, and monoclonal antibodies and application thereof
CN105044362A (en) * 2015-04-04 2015-11-11 吉林双正医疗科技有限公司 IgM antibody joint detection device and method for coxsackievirus A16 and enterovirus 71
CN105085625A (en) * 2014-05-22 2015-11-25 中国科学院上海巴斯德研究所 Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16
CN106046124A (en) * 2016-07-19 2016-10-26 兰州雅华生物技术有限公司 Preparation method of coxsackievirus antigen and rapid detection kit prepared by utilizing antigen and used for detecting coxsackievirus antibody
CN106353502A (en) * 2016-08-25 2017-01-25 山西医科大学 ELISA kit for detecting human cytomegalovirus (HCMV) IgG and IgM antibodies

Patent Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI261618B (en) * 2000-11-29 2006-09-11 Dr Chip Biotechnology Inc Method for detecting and differentiating enteroviruses and the primers and probes therefor
EP1609874A2 (en) * 2004-05-21 2005-12-28 Bioanalisi Centro Sud S.n.c. di Perseu Sinibaldo e C. System for searching for and identifying pathogenic agents
CN101402674A (en) * 2008-10-20 2009-04-08 中国人民解放军第三军医大学 Functional peptide segment of epididymis protease inhibitors and uses thereof
CN101988924A (en) * 2009-07-31 2011-03-23 上海科新生物技术股份有限公司 Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method
WO2013098655A2 (en) * 2011-11-03 2013-07-04 Sentinext Therapeutics Sdn Bhd Antigens and vaccines directed against human enteroviruses
CN102590504A (en) * 2012-01-31 2012-07-18 上海尧浩生物技术有限公司 CoxB IgG/Ig/M test kit using enzyme linked immunosorbent assay
CN103833830A (en) * 2012-11-22 2014-06-04 中国科学院上海巴斯德研究所 Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof
CN104569392A (en) * 2013-10-28 2015-04-29 广州市康润生物科技有限公司 ELISA kit for detecting syphilis specific IgM antibody and preparation method of ELISA kit
CN105085625A (en) * 2014-05-22 2015-11-25 中国科学院上海巴斯德研究所 Genetically engineered vaccine against both Enterovirus 71 and Coxsackie virus A16
CN105044362A (en) * 2015-04-04 2015-11-11 吉林双正医疗科技有限公司 IgM antibody joint detection device and method for coxsackievirus A16 and enterovirus 71
CN105548538A (en) * 2015-04-04 2016-05-04 吉林双正医疗科技有限公司 IgM antibody combined detection device of Coxsackie virus A16 and enterovirus 71 and making method thereof
CN205301333U (en) * 2015-04-04 2016-06-08 吉林双正医疗科技有限公司 Detection device is united to coxsackie virus A16 and enterovirus 71's igM antibody
CN104710537A (en) * 2015-04-09 2015-06-17 汪运山 Coxsackie virus CA16 VP1 recombinant antigens, and monoclonal antibodies and application thereof
CN106046124A (en) * 2016-07-19 2016-10-26 兰州雅华生物技术有限公司 Preparation method of coxsackievirus antigen and rapid detection kit prepared by utilizing antigen and used for detecting coxsackievirus antibody
CN106353502A (en) * 2016-08-25 2017-01-25 山西医科大学 ELISA kit for detecting human cytomegalovirus (HCMV) IgG and IgM antibodies

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
DONG ZHU等: "A new factor influencing pathogen detection by molecular assay in children with both mild and severe hand, foot, and mouth disease", 《DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE》 *
FEIHAI XU等: "Development of an IgM-capture ELISA for Coxsackievirus A16 infection", 《JOURNAL OF VIROLOGICAL METHODS》 *
GORDON C. BROWN等: "Fluorescent Antibody Responses of Cases and Contacts of Hand, Foot, and Mouth Disease", 《INFECTION AND IMMUNITY》 *
JING ZHANG等: "Development and evaluation of rapid point-of-care tests for detection of Enterovirus 71 and Coxsackievirus A16 specific immunoglublin M antibodies", 《JOURNAL OF VIROLOGICAL METHODS》 *
NAN YU等: "Evaluation of human enterovirus 71 and coxsackievirus A16 specific immunoglobulin M antibodies for diagnosis of hand-foot-and-mouth disease", 《VIROLOGY JOURNAL》 *
OTFRIED MARQUARDT等: "Differential diagnosis and genetic analysis of the antigenically related swine vesicular disease virus and coxsackie viruses", 《JOURNAL OF VIROLOGICAL METHODS》 *
任庆杰等: "柯萨奇病毒A组16型研究进展", 《微生物学免疫学进展》 *
宋建等: "合肥市0-14岁人群柯萨奇病毒A组16型和肠道病毒71型的感染状况调查", 《安徽医科大学学报》 *
朱托夫等: "人血清和脑脊液中柯萨奇A病毒IgG抗体的检测", 《病毒学报》 *
朱托夫等: "抗体捕捉ELISA法在诊断柯萨奇A16病毒感染中的应用", 《病毒学报》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110045105A (en) * 2018-11-09 2019-07-23 广州市妇女儿童医疗中心 Coxsack A16 virus IgA antibody quantum dot immune fluorescent chromatograph test strip and kit
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
CN113671175A (en) * 2021-09-07 2021-11-19 普十生物科技(北京)有限公司 Reagent and kit for detecting thromboxane B2
CN117783524A (en) * 2024-02-26 2024-03-29 中国医学科学院医学生物学研究所 Establishment and application of double-antibody sandwich method for indirect quantitative detection of coxsackie A10 type virus antigen
CN117783524B (en) * 2024-02-26 2024-05-03 中国医学科学院医学生物学研究所 Establishment and application of double-antibody sandwich method for indirect quantitative detection of coxsackie A10 type virus antigen

Also Published As

Publication number Publication date
CN108152511B (en) 2019-01-04

Similar Documents

Publication Publication Date Title
CN108152511B (en) Coxsack A16 virus antigen polypeptide and its IgM antibody detection kit
RU2730897C1 (en) Method of using recombinant proteins sars-cov-2 as part of a test system for elisa test with determining igm, igg, iga class antibody levels in blood serum/plasma of covid-19 patients
CN109627293B (en) Seikaga valley virus structural protein epitope polypeptide and application thereof
CN105527437B (en) A kind of detection kit and its application
CN103864906B (en) Foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent
Yang et al. Development of a quick and simple detection methodology for foot-and-mouth disease virus serotypes O, A and Asia 1 using a generic RapidAssay Device
KR102071539B1 (en) A Method of Detecting Anti-Zika Virus Antibodies Using Monoclonal Antibody Specific to the Zika Envelope and Non-Structural Protein 1 and Rapid Diagnostic Kit for Detecting Anti-Zika Virus Antibodies
CN106432434B (en) Foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent
CN105837686B (en) Monoclonal antibody and application thereof
CN104090101A (en) Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof
CN108169484B (en) EV71 virus antigen polypeptide and its IgM antibody detection kit
CN103408641B (en) Foot and mouth disease virus structural protein antibody enzyme-linked immunosorbent assay kit
CN106771182A (en) Brucella abortus IgM subclass antibodies indirect ELISA testing kits
WO2005106483A1 (en) Detection of west nile virus
CN104478998A (en) Pig foot-and-mouth disease virus non-structural protein 3ABC antibody enzyme-linked immunosorbent assay kit
CN111398585B (en) Immunodiagnosis kit for specifically detecting chikungunya virus NSP1 antigen
CN106645714B (en) EV71 virus IgA antibody test strips and its application
CN104961809A (en) Foot and mouth disease O-type structure protein VP1 antibody enzyme-linked immune detection reagent kit
CN104833804A (en) Helicobacter pylori IgG antibody ELISA semi-quantitative detection kit and application thereof
CN109580945B (en) Enzyme linked immunosorbent assay kit for detecting O-type Guangxi strain antigen of foot-and-mouth disease and application thereof
CN107219365A (en) A kind of chemiluminescence detection kit based on foot and mouth disease virus 3B neoepitope Westerns
CN106771218A (en) A kind of kit of p24 antigens of detection HIV 1 and its application
CN111458498A (en) Hand-foot-mouth EV71 antigen detection kit
CN107727856A (en) Detect the test strips of HIV antibody, detection line coating buffer and preparation method thereof in urine
CN103833830A (en) Conservative neutralizing epitope polypeptide of Coxsackievirus A16 and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant