CN102590504A - CoxB IgG/Ig/M test kit using enzyme linked immunosorbent assay - Google Patents
CoxB IgG/Ig/M test kit using enzyme linked immunosorbent assay Download PDFInfo
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Abstract
The invention discloses a CoxB IgG/Ig/M test kit using an enzyme linked immunosorbent assay. The test kit comprises a porous enzyme linked board, a reagent bottle containing a sample diluent, a reagent bottle containing positive control, a reagent bottle containing negative control, a reagent bottle containing an IgG enzyme labeled antibody, a reagent bottle containing a substrate A, a reagent bottle containing a substrate B, a reagent bottle containing a washing concentrate, and a reagent bottle containing a stop buffer, and the inner walls of micro reaction holes of the porous enzyme linked board are coated with CoxB antigens. The CoxB IgG/Ig/M test kit using the enzyme linked immunosorbent assay is high in specificity and sensitivity and short in operation time, and can be used for qualitatively testing CoxB IgG/Ig/M for multiple users.
Description
Technical field
The present invention relates to CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit.
Background technology
Coxsackie B group (CoxB) virus is a kind of of enterovirus, after CBV infects, aseptic meningitis, flesh fiber crops low, herpangina, myocarditis and pericarditis, acute hemorrhagic conjunctivitis etc. can occur.The coxsackie B group has 6 serotypes.The mensuration of CoxB IgG/IgM can detect the patient early and whether suffer the CoxB virus infections recently; Especially the myocarditis of acute stage is carried out the generaI investigation of CoxB IgG/IgM; To diagnosis basis be provided to cardiomyopathies, particularly the positive of CoxB IgM will point out the patient to be infected in early days by cardiac muscle; The favourable index that can cooperate simultaneously clinical diagnosis and observation has great significance to patient's treatment and rehabilitation.
Change of coxsackie b virus diagnosis of infection method mainly contains three kinds, viral isolation identification, Serological testing and Protocols in Molecular Biology at present.Virus separation and Culture and molecular biology method are complicated bothersome; Traditional isolation of virus length consuming time; And that PCR method exist to detect cost is expensive and high to the risk of environmental cross-contamination, can't satisfy the demand of handling great amount of samples during the viral prevalence simultaneously, fast. accurately.Serological progress (express method) opened up should virus the quick diagnosis technology, it has the advantage of high sensitivity and high specific, in the diagnosis of virus infections, brought into play more and more important effect.
Summary of the invention
The objective of the invention is to overcome defective of the prior art, a kind of CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit is provided.
The present invention has adopted following technical proposals to solve above-mentioned technical matters:
A kind of CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit; Comprise the porous elisa plate; The reagent bottle of sample diluting liquid, positive control, negative control, IgG enzyme labelled antibody or IgM enzyme labelled antibody, substrate A, substrate B, concentrated cleaning solution and stop buffer is housed respectively, and the inwall of little reacting hole of said porous elisa plate is coated with CoxB group antigen.
CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit is CoxB IgG or CoxB IgM enzyme-linked immunosorbent assay (ELISA) kit.
Further, said porous elisa plate is 48 hole elisa plates or 96 hole elisa plates.
Sample diluting liquid in the kit also can adopt conventional ELISA sample diluting liquid, as contains the sample diluting liquid of phosphate buffer, perhaps also can (PBS is pH7.2-7.5) with＜0.1wt%NaN in order to contain the 0.01M phosphate buffer
3The 10wt% calf serum.
Positive control in the kit is the positive serum that contains CoxB group IgG antibody or CoxB group IgM antibody through inactivation of virus.
The negative serum of negative control in the kit.
IgG enzyme labelled antibody in the kit is the IgG antibody of horseradish peroxidase (HRP) mark, and IgG antibody can be various types of IgG antibody, like mouse-anti human IgG, goat anti-human igg or rabbit anti-human igg.
IgM enzyme labelled antibody in the kit is the μ chain IgM antibody of HRPO (HRP) mark.
Substrate A in the kit, substrate B are conventional ELISA substrate A and substrate B.
Concentrated cleaning solution in the kit can adopt the conventional ELISA cleansing solution that concentrates, as contains the phosphate buffer of tween, perhaps also can be the concentrated 0.01MPBS of 20X.
Stop buffer in the kit can adopt conventional ELISA stop buffer.
The principal ingredient of the CoxB group antigen that encapsulates on the porous elisa plate is the mixing of Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and the Cox B6 virus of purifying and complete inactivation.Each viral blending ratio can be adjusted arbitrarily according to the detection needs.Generally speaking, be the basis in viral total amount, each viral percentage by weight is 6-20%.
The method of envelope antigen adopts conventional on the porous elisa plate.
Further, said CoxB group antigen adopts the method that comprises the following steps to prepare:
1) virus amplification: will be selected to cultivate in the Hela cell that arbitrary seed culture of viruses in Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and the Cox B6 virus is seeded to individual layer and obtain the cytopathy venom;
2) virus discharges: the cytopathy venom is repeated freeze thawing more than three times, then ultrasonic disruption cell in ice-water bath;
3) inactivation of virus: will repeat freeze thawing more than three times the cytopathy venom or through the cytopathy venom complete inactivation of ultrasonic disruption cell, the cytopathy venom of deactivation need be negative through the inactivation of virus experimental examination;
4) viral antigen purifying:
4.1 will after virus discharge cytopathy venom with inactivation of virus and places 18-20 ℃ to melt appropriate time, add formalin and mix, the final concentration of formaldehyde in mixed liquor is 0.12-0.20v%;
4.2 mixed liquor gradient centrifugation isolated viral:
4.3 adding protection liquid, isolated virus obtains viral antigen liquid.
5) utilize step 1-4 to prepare the viral antigen liquid of Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 respectively, the viral antigen liquid of the Cox B1 that makes, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 is mixed obtaining CoxB group antigen.
In the step 1, the condition that can adopt routine to be suitable for the growth of Hela cell behind the viral seed culture of viruses of Hela cell inoculation CoxB group is cultivated.More excellent, can be cited like embodiment, put 37 ℃ of absorption 1 hour earlier, behind the adding cell maintenance medium (RPMI-1640 that contains the 2wt% calf serum), put 5%CO
2Incubator is cultured to 100% cell generation pathology for 36 ℃.
In the step 2, the ice-water bath temperature is generally 0 ℃.
In the step 3, cytopathy venom complete inactivation can adopt the method for conventional complete inactivation virus, like ultraviolet inactivation.
In the step 4, the said formalin of step 4.1 can be formalin.
In the step 4.1, the time of melting is 15-20min.
In the step 4.2, the gradient centrifugation isolated viral specifically comprises the following steps:
4.2.1 mixed liquor low-speed centrifugal at least 20 minutes is got supernatant;
4.2.2 centrifugal at least 60 minutes of supernatant 35000-45000rpm/min gets deposition;
4.2.3 deposition places on the 13-17wt% glycerine water solution after dissolving with PBS (phosphate buffer), 50000-65000rpm/min is centrifugal to get deposition more than 60 minutes, and deposition is isolated virus.
Further, among the step 4.2.1, the centrifugal speed of low-speed centrifugal is 5000-8000rpm/min.
Used PBS is 0.01-0.05M among the step 4.2.3, pH7.2-7.5.Deposition is 1 with the volume ratio of PBS: (2-3).
The liquid of protection described in the step 4.3 can be glycerine or dimethyl sulfoxide, and virus can be 1 with the volume ratio of protection liquid: (1-2).
Further, the preparation of said CoxB group antigen also comprises:
Step 5: will utilize the multiple mixing in the antigen liquid of Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 that step 1-4 prepares respectively.
The method of above-mentioned purified virus adopts appropriate method processing cytopathy venom before concentrating in that virus is centrifugal; Especially the processing of debita spissitudo formalin; On the one hand between the protein terminal group, form cross linked chain (bridged bond), the degradation speed of the albumen that slowed down does not allow virion in the cell by other organic solvents destruction of degrading rapidly; Thereby play effect effectively fixing, stable, the protected protein agent; On the other hand, the formalin of debita spissitudo is handled and has also been improved the viral centrifugal preceding power that removes slag that concentrates, and cooperates suitable concentrated centrifugal method again; Finally effectively improve the viral purification recovery, strengthened the accuracy in detection of viral antigen specificity and kit.The virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output surpasses 2.0mg/100g.
In the kit, the porous elisa plate that is coated with CoxB group antigen layer can adopt conventional method will be antigen coated to the elisa plate after, the mode of then sealing with confining liquid makes.
Except that comprising above-mentioned substance, also can comprise needed reagent, instrument or container when conventional ELISA tests, in the kit like distilled water or deionized water, washing lotion drop bottle, test tube and instructions etc.
CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit specificity of the present invention and highly sensitive, the running time is short, can be used for many person-portions qualitative detection CoxB IgG/IgM.
Embodiment
Below enumerate specific embodiment and further set forth the present invention, should be understood that instance is not to be used to limit protection scope of the present invention.
The preparation of embodiment 1CoxB group antigen
Reagent:
Cell maintenance medium: 2% calf serum 1640 is kept liquid (1640 powder, NaHCO
3, antibiotic, calf serum, H
2O)
Cell culture fluid: 10% calf serum RPMI-1640 (1640 powder, NaHCO
3, antibiotic, calf serum, H
2O)
Preparation process:
1, gets 0.5ml Cox B1 virus seed culture of viruses and be seeded in the Hela cell bottle (the Hela cell is with 36 ℃ of cultivations of cell culture fluid) that grows into individual layer, put 37 ℃ of absorption 1 hour, add cell maintenance medium (RPMI-1640 that contains 2% calf serum) 10ml again, put 5%CO
2Incubator was cultivated about 48-72 hour for 36 ℃, observed 100% cell generation pathology, obtained the cytopathy venom.
2, get that the cytopathy venom repeats freeze thawing three times after the 0.5ml freeze thawing, purpose is to guarantee that intracellular virus all discharges.
3, viral complete inactivation: ultra violet lamp cytopathy venom complete inactivation virus.Get the viral liquid 1ml of deactivation, conventional method is cultivated and is gone down to posterity three times, cytopathy do not occur, shows that viral complete inactivation promptly can be used for making antigen.
4, cytopathy venom that inactivation of virus test the is negative ultrasonic disruption cell (300W/3 second/stop 3 seconds * 30 circulations) in ice-water bath of learning from else's experience, to guarantee clasmatosis, intracellular virus all discharges.
5, viral antigen purifying
5.1 be the viral recovery that guarantees to discharge in the cell; To all discharge rearmounted 18-20 ℃ in clasmatosis, virus melted 15 minutes; Adding formaldehyde saturated aqueous solution (formalin) to the final concentration of formaldehyde in mixed liquor is 0.16v%, with the stabilized cell crushed material.
5.2 gradient centrifugation
With centrifugal 20 minutes of crushed material 5000rpm/min, get supernatant, the 40000rpm/min high speed centrifugation is 60 minutes again, gets deposition.
Deposition adds the PBS dissolving of the pH7.2 0.01M of 2 times of volume ratios, places above the 10ml 15wt% glycerite, and 50000rpm/min high speed centrifugation 60 minutes is got deposition.
Sediment adds the glycerine of 1 times of volume, obtains Cox B1 viral antigen liquid.
6, adopt step 1-5 to prepare Cox B2, Cox B3, Cox B4, Cox B5, Cox B6 viral antigen liquid respectively.
Each viral antigen liquid equal-volume that 7, will prepare obtains CoxB group antigen than mixing.
Through detecting, each virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output reaches as high as 2.45mg virus/100g cytopathy venom all above 2.0mg virus/100g cytopathy venom.
The preparation of embodiment 2CoxB group antigen
Reagent: with embodiment 1
Preparation process:
1, gets 0.5ml Cox B1 virus seed culture of viruses and be seeded in the Hela cell bottle (the Hela cell is with 36 ℃ of cultivations of cell culture fluid) that grows into individual layer, put 37 ℃ of absorption 1 hour, add cell maintenance medium (RPMI-1640 that contains 2% calf serum) 10ml again, put 5%CO
2Incubator was cultivated about 48-72 hour for 36 ℃, observed 100% cell generation pathology, obtained the cytopathy venom.
2, get that the cytopathy venom repeats freeze thawing three times after the 0.5ml freeze thawing, purpose is to guarantee that intracellular virus all discharges.
3, viral complete inactivation: ultra violet lamp cytopathy venom complete inactivation virus.Get the viral liquid 1ml of deactivation, conventional method is cultivated and is gone down to posterity three times, cytopathy do not occur, shows that viral complete inactivation promptly can be used for making antigen.
4, cytopathy venom that inactivation of virus test the is negative ultrasonic disruption cell (300W/3 second/stop 3 seconds * 30 circulations) in ice-water bath of learning from else's experience, to guarantee clasmatosis, intracellular virus all discharges.
5, viral antigen purifying
5.1 be the viral recovery that guarantees to discharge in the cell, will all discharge rearmounted 18-20 ℃ in clasmatosis, virus and melt 20 minutes, adding formaldehyde saturated aqueous solution (formalin) to the final concentration of formaldehyde in mixed liquor is 0.12v%,
With the stabilized cell crushed material.
5.2 gradient centrifugation
With centrifugal 20 minutes of crushed material 8000rpm/min, get supernatant, the 35000rpm/min high speed centrifugation is 60 minutes again, gets deposition.
Deposition adds the pH7.4 of 3 times of volume ratios, and the PBS dissolving of 0.02M places above the 10ml 13wt% glycerite, and 65000rpm/min high speed centrifugation 60 minutes is got deposition.
Sediment adds the dimethyl sulfoxide of 1 times of volume, obtains Cox B1 viral antigen liquid.
6, adopt step 1-5 to prepare Cox B2, Cox B3, Cox B4, Cox B5, Cox B6 viral antigen liquid respectively.
Each viral antigen liquid that 7, will prepare is respectively got the 1-3 parts by volume and is mixed acquisition CoxB group antigen.
Through detecting, each virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output is all above 2.0mg virus/100g cytopathy venom.
Embodiment 3
Reagent: with embodiment 1
Preparation process:
1, gets 0.5ml Cox B1 virus seed culture of viruses and be seeded in the Hela cell bottle (the Hela cell is with 36 ℃ of cultivations of cell culture fluid) that grows into individual layer, put 37 ℃ of absorption 1 hour, add cell maintenance medium (RPMI-1640 that contains 2% calf serum) 10ml again, put 5%CO
2Incubator was cultivated about 48-72 hour for 36 ℃, observed 100% cell generation pathology, obtained the cytopathy venom.
2, get that the cytopathy venom repeats freeze thawing three times after the 0.5ml freeze thawing, purpose is to guarantee that intracellular virus all discharges.
3, inactivation of virus: ultra violet lamp cytopathy venom complete inactivation virus.Get the viral liquid 1ml of deactivation, conventional method is cultivated and is gone down to posterity three times, cytopathy do not occur, shows that viral complete inactivation promptly can be used for making antigen.
4, cytopathy venom that inactivation of virus test the is negative ultrasonic disruption cell (300W/3 second/stop 3 seconds * 30 circulations) in ice-water bath of learning from else's experience, to guarantee clasmatosis, intracellular virus all discharges.
5, viral antigen purifying
5.1 be the viral recovery that guarantees to discharge in the cell, will all discharge rearmounted 18-20 ℃ in clasmatosis, virus and melt 15 minutes, adding formaldehyde saturated aqueous solution (formalin) to the final concentration of formaldehyde in mixed liquor is 0.20v%,
With the stabilized cell crushed material.
5.2 gradient centrifugation
With centrifugal 20 minutes of crushed material 6000rpm/min, get supernatant, the 45000rpm/min high speed centrifugation is 60 minutes again, gets deposition.
Deposition adds the PBS dissolving of the pH7.5 0.05M of 2 times of volume ratios, places above the 10ml 17wt% glycerite, and 60000rpm/min high speed centrifugation 60 minutes is got deposition.
Sediment adds the glycerine of 1 times of volume, obtains Cox B1 viral antigen liquid.
6, adopt step 1-5 to prepare Cox B2, Cox B3, Cox B4, Cox B5, Cox B6 viral antigen liquid respectively.
Each viral antigen liquid that 7, will prepare is respectively got the 1-3 parts by volume and is mixed acquisition CoxB group antigen.
Through detecting, each virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output is all above 2.0mg virus/100g cytopathy venom.
The assembling of embodiment 4 kits
Porous elisa plate and all ingredients preparation in the kit:
1. sample diluting liquid: the 0.01M phosphate buffer (PBS, pH7.2-7.5),＜0.1%NaN
3, 10% calf serum.
2. positive control: contain＜0.1%NaN
3The positive human serum through inactivation of virus.Passed through HBsAg, human immunodeficiency virus (1,2 type) antibody, hepatitis C virus antibody, syphilis helicoid antibody are all negative.
3. negative control: contain＜0.1%NaN
3The negative human serum through inactivation of virus.Passed through HBsAg, human immunodeficiency virus (1,2 type) antibody, hepatitis C virus antibody, syphilis helicoid antibody are all negative.
4.IgG enzyme labelled antibody: the goat anti-human igg antibody of HRPO (HRP) mark.
5. substrate A: 0.1M sodium citrate, 0.01% hydrogen peroxide.
6. substrate B: contain tetramethyl benzidine, dimethyl sulfoxide, SAS.
7. concentrated cleaning solution: (20X) concentrated 0.01MPBS.
8. stop buffer: 2mol/L H
2SO
4
9. be coated with the porous elisa plate of CoxB group antigen layer: seal with confining liquid after the Cox B group antigen for preparing with embodiment 1-3 respectively adopts conventional method to encapsulate elisa plate respectively.
With each one bottle in the sealed reagent bottle that sample diluting liquid, positive control, negative control, IgG enzyme labelled antibody, substrate A, substrate B, concentrated cleaning solution and stop buffer are housed respectively; A shrouding film; After the porous elisa plate porous elisa plate that is coated with CoxB group antigen layer of arbitrary 9 preparation packed, the box body of packing into was in the lump processed CoxB IgG enzyme-linked immunosorbent assay (ELISA) kit.
The condition of storage of kit: 2～8 ℃ of preservations.
The assembling of embodiment 5 kits
Porous elisa plate and all ingredients preparation in the kit:
1. sample diluting liquid: the 0.01M phosphate buffer (PBS, pH7.2-7.5),＜0.1%NaN
3, 10% calf serum.
2. positive control: contain＜0.1%NaN
3The positive human serum through inactivation of virus.Passed through HBsAg, human immunodeficiency virus (1,2 type) antibody, hepatitis C virus antibody, syphilis helicoid antibody are all negative.
3. negative control: contain＜0.1%NaN
3The negative human serum through inactivation of virus.Passed through HBsAg, human immunodeficiency virus (1,2 type) antibody, hepatitis C virus antibody, syphilis helicoid antibody are all negative.
4.IgM enzyme labelled antibody: the mouse-anti people μ chain IgM antibody of HRPO (HRP) mark.
5. substrate A: 0.1M sodium citrate, 0.01% hydrogen peroxide.
6. substrate B: contain tetramethyl benzidine, dimethyl sulfoxide, SAS.
7. concentrated cleaning solution: (20X) concentrated 0.01MPBS.
8. stop buffer: 2mol/L H
2SO
4
9. be coated with the porous elisa plate of CoxB group antigen layer: seal with confining liquid after the Cox B group antigen for preparing with embodiment 1-3 respectively adopts conventional method to encapsulate elisa plate respectively.
With each one bottle in the sealed reagent bottle that sample diluting liquid, positive control, negative control, IgM enzyme labelled antibody, substrate A, substrate B, concentrated cleaning solution and stop buffer are housed respectively; A shrouding film; After the porous elisa plate porous elisa plate that is coated with CoxB group antigen layer of arbitrary 9 preparation packed, the box body of packing into was in the lump processed CoxB IgM enzyme-linked immunosorbent assay (ELISA) kit.
The condition of storage of kit: 2～8 ℃ of preservations.
The detection of embodiment 6 kits
The kit method of application:
Kit can detect serum sample, collects serum by standard method, and is to be measured.As do not measure immediately, serum sample is placed on below-20 ℃ frozen, and avoids multigelation.
1. reagent preparation
1.1 the preparation of solution: get 1 bottle of concentrated cleaning solution in the kit, with for use in the wash bottle (providing for oneself) of packing into after 1: 20 dilution mixing, the dissolving.
1.2 experiment is prepared: sample to be measured is taken out from refrigerator, put after room temperature thaws, sample to be measured is numbered by requirement of experiment.
1.3 after the careful confirmation in sample to be measured numbering back, with test serum with sample diluting liquid (diluting at 1: 100) promptly 10 μ l serum be added in the 1ml sample diluent mixing.
2. the experiment condition that must satisfy
2.1 kit is taken out in 2～8 ℃ of refrigerators, puts room temperature and placed 30 minutes.
2.2 the wavelength that uses ELIASA to measure is 450nm.
2.3 the temperature of adjustment water bath is 37 ℃.
3. the every hole of yin and yang attribute reference substance in the kit adds 100 μ l.
4. operation steps
Application of sample amount (μ l)
Add each 50 μ l/ hole of chromogenic substrate A liquid and B liquid, the mixing that gently shakes, the room temperature lucifuge was placed 10-15 minute, added stop buffer 50 μ l/ holes, slightly vibrated with mixed solution, placed room temperature 5 minutes.
In 15 minutes, detect, read each hole OD value in wavelength 450 nanometers with ELIASA.
5. quality control
The average OD ratio relatively of kit positive reference substance and negative control article should be not less than 3.
6. the result calculates
Calculate the average OD value of each reference substance, sample, and judge the yin and yang attribute of sample according to the ratio of sample and negative control OD.
[kit Cut off value]
The average OD value of Cut off value=negative control X 2.1
Serum sample and ratio negative control OD value >=Cut off value positive (+);
Serum sample and ratio negative control OD value≤Cut off value positive (-).
Embodiment 7 kit specificity and accuracy testings
Get the kit (being coated with the Cox B group antigen of embodiment 1 preparation on the elisa plate) of embodiment 4 and 5 preparations respectively, adopt the method for embodiment 6 to detect sample, adopt neutralization test to contrast simultaneously, observe its specificity and accuracy.
Negative match-rate=100% * [258/ (0+265)]=97.36%
Negative match-rate=100% * [253/ (0+265)]=95.47%
Above-mentioned test findings shows that kit specificity of the present invention is good, and accuracy rate is high, can be used for clinical reference.
The test of embodiment 8 kit specificitys
Get the kit of embodiment 4 and 5 preparations; Adopt the method for embodiment 6 respectively, detect with human immunodeficiency virus (1,2 type), hepatitis B, hepatitis C virus, syphilis, CoxA papova positive sample; All be negative, show that kit specificity of the present invention is good.
Claims (7)
1. CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit; Comprise the porous elisa plate; The reagent bottle of sample diluting liquid, positive control, negative control, IgG enzyme labelled antibody or IgM enzyme labelled antibody, substrate A, substrate B, concentrated cleaning solution and stop buffer is housed respectively, and the inwall of little reacting hole of said porous elisa plate is coated with CoxB group antigen.
2. CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit according to claim 1; It is characterized in that the principal ingredient of the CoxB group antigen that encapsulates on the porous elisa plate is the mixing of Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and the Cox B6 virus of purified and complete inactivation.
3. CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit according to claim 1 is characterized in that, said CoxB group antigen adopts the method that comprises the following steps to prepare:
1, virus amplification: will be selected to cultivate in the Hela cell that arbitrary seed culture of viruses in Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and the Cox B6 virus is seeded to individual layer and obtain the cytopathy venom;
2, virus discharges: the cytopathy venom is repeated freeze thawing more than three times, then ultrasonic disruption cell in ice-water bath;
3, inactivation of virus: will repeat freeze thawing more than three times the cytopathy venom or through the intact deactivation of the cytopathy venom of ultrasonic disruption cell, the cytopathy venom of deactivation need be negative through the inactivation of virus experimental examination;
4, viral antigen purifying:
4.1 will after virus discharge cytopathy venom with inactivation of virus and places 18-20 ℃ to melt appropriate time, add formalin and mix, the final concentration of formaldehyde in mixed liquor is 0.12-0.20v%;
4.2 mixed liquor gradient centrifugation isolated viral:
4.3 adding protection liquid, isolated virus obtains viral antigen liquid;
5, utilize step 1-4 to prepare the viral antigen liquid of Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 respectively, the viral antigen liquid of the Cox B1 that makes, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 is mixed obtaining CoxB group antigen.
4. like the said CoxB IgG/IgM of claim 3 enzyme-linked immunosorbent assay (ELISA) kit, it is characterized in that in the step 4.2, the gradient centrifugation isolated viral specifically comprises the following steps:
4.2.1 mixed liquor low-speed centrifugal at least 20 minutes is got supernatant;
4.2.2 centrifugal at least 60 minutes of supernatant 35000-45000rpm/min gets deposition;
4.2.3 deposition places on the 13-17wt% glycerine water solution after dissolving with PBS, 50000-65000rpm/min is centrifugal to get deposition more than 60 minutes, and deposition is isolated virus.
5. like the said CoxB IgG/IgM of claim 4 enzyme-linked immunosorbent assay (ELISA) kit, it is characterized in that among the step 4.2.1, the centrifugal speed of low-speed centrifugal is 5000-8000rpm/min.
6. like the said CoxB IgG/IgM of claim 4 enzyme-linked immunosorbent assay (ELISA) kit, it is characterized in that among the step 4.2.3, deposition is 1 with the volume ratio of PBS: (2-3).
7. like the said CoxB IgG/IgM of claim 3 enzyme-linked immunosorbent assay (ELISA) kit, it is characterized in that in the step 4.3, said virus is 1 with the volume ratio of protection liquid: (1-2).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108152511A (en) * | 2017-12-20 | 2018-06-12 | 广州瑞辉生物科技股份有限公司 | Coxsack A16 virus antigen polypeptides and its IgM antibody 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 |
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