CN102590504B - 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 the one of enterovirus, after CBV infects, can occur aseptic meningitis, flesh fiber crops low, herpangina, myocarditis and pericarditis, acute hemorrhagic conjunctivitis etc.Coxsackie B group has 6 serotypes.The mensuration of CoxB IgG/IgM can detect whether patient suffers CoxB virus infections recently early, especially the myocarditis of acute stage is carried out to the generaI investigation of CoxB IgG/IgM, diagnosis basis will be provided to cardiomyopathies, the particularly positive of CoxB IgM, by prompting patient likely cardiac muscle be infected in early days, the favourable index of clinical diagnosis and observation can be coordinated simultaneously, the treatment of patient and rehabilitation are had great significance.
The diagnostic method of current infection of Coxsackie B virus mainly contains three kinds, Virus Isolation, Serological testing and Protocols in Molecular Biology.Viral isolation and molecular biology method complicated bothersome, traditional isolation of virus length consuming time, and PCR method to there is testing cost expensive and high to the risk of environmental cross-contamination, process the demand of great amount of samples during cannot viral prevalence being met, fast simultaneously. accurately.Serological progress (express method) has opened up the quick diagnosis technology of this virus, and it has the advantage of high sensitivity and high specific, has played more and more important effect in the diagnosis of virus infections.
Summary of the invention
The object of the invention is to overcome defect of the prior art, a kind of CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit is provided.
Present invention employs following technical proposals and solve above-mentioned technical matters:
A kind of CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit, comprise 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 micro-reacting hole of described 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, described porous elisa plate is 48 hole elisa plates or 96 hole elisa plates.
Sample diluting liquid in kit also can adopt conventional ELISA sample diluting liquid, as the sample diluting liquid containing phosphate buffer, or also can for containing 0.01M phosphate buffer (PBS, pH7.2-7.5) and < 0.1wt%NaN
310wt% calf serum.
Positive control in kit is the positive serum containing CoxB group IgG antibody or CoxB group IgM antibody through inactivation of virus.
Negative control in kit is negative serum.
IgG enzyme labelled antibody in kit is the IgG antibody that horseradish peroxidase (HRP) marks, and IgG antibody can be various types of IgG antibody, as mouse-anti human IgG, goat anti-human igg or rabbit anti-human igg.
IgM enzyme labelled antibody in kit is the μ chain IgM antibody that horseradish peroxidase (HRP) marks.
Substrate A in kit, substrate B are conventional ELISA substrate A and substrate B.
Concentrated cleaning solution in kit can adopt conventional concentrated ELISA cleansing solution, as the phosphate buffer containing tween, or the 0.01MPBS that also can concentrate for 20X.
Stop buffer in kit can adopt conventional ELISA stop buffer.
The principal ingredient of porous elisa plate wrapping the CoxB group antigen of quilt is purifying and the mixing of the Cox B1 of complete inactivation, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 virus.Each viral blending ratio can need adjustment arbitrarily according to detection.Generally, count based on viral total amount, the percentage by weight of each virus is 6-20%.
On porous elisa plate, the method for envelope antigen adopts conventional.
Further, described CoxB group antigen adopts the method comprised the following steps to prepare:
1) virus amplification: the arbitrary seed culture of viruses be selected from Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 virus is seeded in the Hela cell of individual layer and cultivates acquisition cytopathy venom;
2) virus release: cytopathy venom is repeated freeze thawing more than three times, then ultrasonic disruption cell in ice-water bath;
3) inactivation of virus: by the repetition cytopathy venom of freeze thawing more than three times or the cytopathy venom complete inactivation through ultrasonic disruption cell, the cytopathy venom of deactivation need be negative through inactivation of virus experimental examination;
4) viral antigen purifying:
4.1 by be placed in through virus release and the cytopathy venom of inactivation of virus 18-20 DEG C melt appropriate time after, add formalin and mix, the final concentration of formaldehyde in mixed liquor is 0.12-0.20v%;
4.2 mixed liquor gradient centrifugation isolated virals:
4.3 isolated viruses add protection liquid and obtain 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 mixing of the viral antigen liquid of obtained Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 is obtained CoxB group antigen.
In step 1, after the viral seed culture of viruses of Hela cell inoculation CoxB group, routine can be adopted to be suitable for the CMC model of Hela Growth of Cells.Preferably, cited by embodiment, first can put 37 DEG C of absorption 1 hour, after adding cell maintenance medium (RPMI-1640 containing 2wt% calf serum), put 5%CO
2incubator 36 DEG C is cultured to 100% cell generation pathology.
In step 2, ice-water bath temperature is generally 0 DEG C.
In step 3, cytopathy venom complete inactivation can adopt the method for conventional complete inactivation virus, as ultraviolet inactivation.
In step 4, described in step 4.1, formalin can be formalin.
In step 4.1, the time of melting is 15-20min.
In step 4.2, gradient centrifugation isolated viral specifically comprises the following steps:
4.2.1 mixed liquor low-speed centrifugal at least 20 minutes, gets supernatant;
4.2.2 centrifugal at least 60 minutes of supernatant 35000-45000rpm/min, gets precipitation;
4.2.3 after precipitation is dissolved with PBS (phosphate buffer), be placed on 13-17wt% glycerine water solution, centrifugal more than 60 minutes of 50000-65000rpm/min, gets precipitation, and precipitation is isolated virus.
Further, in step 4.2.1, the centrifugal speed of low-speed centrifugal is 5000-8000rpm/min.
In step 4.2.3, PBS used is 0.01-0.05M, pH7.2-7.5.Precipitation is 1 with the volume ratio of PBS: (2-3).
Protect liquid to can be glycerine or dimethyl sulfoxide described in step 4.3, virus can be 1 with the volume ratio of protection liquid: (1-2).
Further, the preparation of described CoxB group antigen also comprises:
Step 5: the multiple mixing in the antigen liquid of the Cox B1 prepared utilizing step 1-4 respectively, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6.
The method of above-mentioned purified virus adopts suitable method process cytopathy venom before viral centrifugal concentrating, especially the process of debita spissitudo formalin, cross linked chain (bridged bond) is formed on the one hand between protein terminal group, slowed down the degradation speed of albumen, the virion in cell is not allowed to be degraded rapidly destruction by other organic solvents, thus play effectively fixing, stable, the effect of protected protein agent, on the other hand, the power that removes slag before the formalin process of debita spissitudo also improves viral centrifugal concentrating, coordinate suitable concentrated centrifugal method again, finally effectively improve the viral purification recovery, enhance 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 is more than 2.0mg/100g.
In kit, the porous elisa plate being coated with CoxB group antigen layer can adopt conventional method by antigen coated to elisa plate after, then obtain by the mode that confining liquid is closed.
In kit except comprising above-mentioned substance, also can comprise reagent, instrument or container required when conventional ELISA tests, as 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 enumerating specific embodiment and set forth the present invention further, should be understood that example is not for limiting the scope of the invention.
The preparation of embodiment 1CoxB group antigen
Reagent:
Cell maintenance medium: 2% calf serum 1640 maintenance medium (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, getting 0.5ml Cox B1 virus seed culture of viruses is seeded in the Hela cell bottle (Hela cell cell culture fluid 36 DEG C cultivation) growing into individual layer, put 37 DEG C of absorption 1 hour, add cell maintenance medium (RPMI-1640 containing 2% calf serum) 10ml again, put 5%CO
2incubator 36 DEG C cultivates about 48-72 hour, observes 100% cell generation pathology, obtains cytopathy venom.
2, after getting 0.5ml freeze thawing, cytopathy venom repeats freeze thawing three times, and object ensures that intracellular virus all discharges.
3, viral complete inactivation: ultra violet lamp cytopathy venom complete inactivation virus.Getting the virus liquid 1ml of deactivation, there is not cytopathy in conventional method subculture three times, shows that namely viral complete inactivation can be used for making antigen.
4, inactivation of virus of learning from else's experience tests the cytopathy venom that is negative ultrasonic disruption cell (300W/3 second/stop 3 seconds × 30 circulations) in ice-water bath, and to ensure clasmatosis, intracellular virus all discharges.
5, viral antigen purifying
5.1 for ensureing the viral recovery of release in cell, to all discharge rearmounted 18-20 DEG C in clasmatosis, virus and melt 15 minutes, adding formaldehyde saturated aqueous solution (formalin) to the final concentration of formaldehyde in mixed liquor is 0.16v%, with stabilized cell crushed material.
5.2 gradient centrifugation
By centrifugal for crushed material 5000rpm/min 20 minutes, get supernatant, then 40000rpm/min high speed centrifugation 60 minutes, get precipitation.
The PBS that precipitation adds the pH7.2 0.01M of 2 times of volume ratios dissolves, and be placed in above 10ml 15wt% glycerite, 50000rpm/min high speed centrifugation 60 minutes, gets precipitation.
Sediment adds the glycerine of 1 times of volume, obtains Cox B1 viral antigen liquid.
6, step 1-5 is adopted to prepare Cox B2, Cox B3, Cox B4, Cox B5, Cox B6 viral antigen liquid respectively.
7, each viral antigen liquid equal-volume of preparation is obtained CoxB group antigen than mixing.
After testing, each virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output, all more than 2.0mg virus/100g cytopathy venom, reaches as high as 2.45mg virus/100g cytopathy venom.
The preparation of embodiment 2CoxB group antigen
Reagent: with embodiment 1
Preparation process:
1, getting 0.5ml Cox B1 virus seed culture of viruses is seeded in the Hela cell bottle (Hela cell cell culture fluid 36 DEG C cultivation) growing into individual layer, put 37 DEG C of absorption 1 hour, add cell maintenance medium (RPMI-1640 containing 2% calf serum) 10ml again, put 5%CO
2incubator 36 DEG C cultivates about 48-72 hour, observes 100% cell generation pathology, obtains cytopathy venom.
2, after getting 0.5ml freeze thawing, cytopathy venom repeats freeze thawing three times, and object ensures that intracellular virus all discharges.
3, viral complete inactivation: ultra violet lamp cytopathy venom complete inactivation virus.Getting the virus liquid 1ml of deactivation, there is not cytopathy in conventional method subculture three times, shows that namely viral complete inactivation can be used for making antigen.
4, inactivation of virus of learning from else's experience tests the cytopathy venom that is negative ultrasonic disruption cell (300W/3 second/stop 3 seconds × 30 circulations) in ice-water bath, and to ensure clasmatosis, intracellular virus all discharges.
5, viral antigen purifying
5.1 for ensureing the viral recovery of release in cell, will clasmatosis, virus all release rearmounted 18-20 DEG C melt 20 minutes, adding formaldehyde saturated aqueous solution (formalin) to the final concentration of formaldehyde in mixed liquor is 0.12v%,
With stabilized cell crushed material.
5.2 gradient centrifugation
By centrifugal for crushed material 8000rpm/min 20 minutes, get supernatant, then 35000rpm/min high speed centrifugation 60 minutes, get precipitation.
Precipitation adds the pH7.4 of 3 times of volume ratios, and the PBS of 0.02M dissolves, and be placed in above 10ml 13wt% glycerite, 65000rpm/min high speed centrifugation 60 minutes, gets precipitation.
Sediment adds the dimethyl sulfoxide of 1 times of volume, obtains Cox B1 viral antigen liquid.
6, step 1-5 is adopted to prepare Cox B2, Cox B3, Cox B4, Cox B5, Cox B6 viral antigen liquid respectively.
7, each viral antigen liquid of preparation is respectively got the mixing of 1-3 parts by volume and obtain CoxB group antigen.
After testing, each virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output is all more than 2.0mg virus/100g cytopathy venom.
Embodiment 3
Reagent: with embodiment 1
Preparation process:
1, getting 0.5ml Cox B1 virus seed culture of viruses is seeded in the Hela cell bottle (Hela cell cell culture fluid 36 DEG C cultivation) growing into individual layer, put 37 DEG C of absorption 1 hour, add cell maintenance medium (RPMI-1640 containing 2% calf serum) 10ml again, put 5%CO
2incubator 36 DEG C cultivates about 48-72 hour, observes 100% cell generation pathology, obtains cytopathy venom.
2, after getting 0.5ml freeze thawing, cytopathy venom repeats freeze thawing three times, and object ensures that intracellular virus all discharges.
3, inactivation of virus: ultra violet lamp cytopathy venom complete inactivation virus.Getting the virus liquid 1ml of deactivation, there is not cytopathy in conventional method subculture three times, shows that namely viral complete inactivation can be used for making antigen.
4, inactivation of virus of learning from else's experience tests the cytopathy venom that is negative ultrasonic disruption cell (300W/3 second/stop 3 seconds × 30 circulations) in ice-water bath, and to ensure clasmatosis, intracellular virus all discharges.
5, viral antigen purifying
5.1 for ensureing the viral recovery of release in cell, will clasmatosis, virus all release rearmounted 18-20 DEG C melt 15 minutes, adding formaldehyde saturated aqueous solution (formalin) to the final concentration of formaldehyde in mixed liquor is 0.20v%,
With stabilized cell crushed material.
5.2 gradient centrifugation
By centrifugal for crushed material 6000rpm/min 20 minutes, get supernatant, then 45000rpm/min high speed centrifugation 60 minutes, get precipitation.
The PBS that precipitation adds the pH7.5 0.05M of 2 times of volume ratios dissolves, and be placed in above 10ml 17wt% glycerite, 60000rpm/min high speed centrifugation 60 minutes, gets precipitation.
Sediment adds the glycerine of 1 times of volume, obtains Cox B1 viral antigen liquid.
6, step 1-5 is adopted to prepare Cox B2, Cox B3, Cox B4, Cox B5, Cox B6 viral antigen liquid respectively.
7, each viral antigen liquid of preparation is respectively got the mixing of 1-3 parts by volume and obtain CoxB group antigen.
After testing, each virus of purifying has typical nucleoprotein ultraviolet absorption curve, and A260/A280 is greater than 1.5, and purification output is all more than 2.0mg virus/100g cytopathy venom.
The assembling of embodiment 4 kit
Porous elisa plate and various preparation of reagents in kit:
1. sample diluting liquid: 0.01M phosphate buffer (PBS, pH7.2-7.5), < 0.1%NaN
3, 10% calf serum.
2. positive control: containing < 0.1%NaN
3the positive human serum through inactivation of virus.Through hepatitis B virus surface antigen, human immunodeficiency virus (1,2 type) antibody, anti-HCV, syphilis helicoid antibody has been feminine gender.
3. negative control: containing < 0.1%NaN
3the negative human serum through inactivation of virus.Through hepatitis B virus surface antigen, human immunodeficiency virus (1,2 type) antibody, anti-HCV, syphilis helicoid antibody has been feminine gender.
4.IgG enzyme labelled antibody: the goat anti-human igg antibody that horseradish peroxidase (HRP) marks.
5. substrate A: 0.1M sodium citrate, 0.01% hydrogen peroxide.
6. substrate B: containing tetramethyl benzidine, dimethyl sulfoxide, sodium acetate solution.
7. concentrated cleaning solution: the 0.01MPBS that (20X) is concentrated.
8. stop buffer: 2mol/L H
2sO
4
9. be coated with the porous elisa plate of CoxB group antigen layer: adopt conventional method to wrap respectively with Cox B group antigen prepared by embodiment 1-3 respectively and closed with confining liquid by after elisa plate.
By each one bottle of 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 arbitrary piece 9 porous elisa plate porous elisa plate being coated with CoxB group antigen layer prepared packs, load a box body in the lump and make CoxB IgG enzyme-linked immunosorbent assay (ELISA) kit.
The condition of storage of kit: 2 ~ 8 DEG C of preservations.
The assembling of embodiment 5 kit
Porous elisa plate and various preparation of reagents in kit:
1. sample diluting liquid: 0.01M phosphate buffer (PBS, pH7.2-7.5), < 0.1%NaN
3, 10% calf serum.
2. positive control: containing < 0.1%NaN
3the positive human serum through inactivation of virus.Through hepatitis B virus surface antigen, human immunodeficiency virus (1,2 type) antibody, anti-HCV, syphilis helicoid antibody has been feminine gender.
3. negative control: containing < 0.1%NaN
3the negative human serum through inactivation of virus.Through hepatitis B virus surface antigen, human immunodeficiency virus (1,2 type) antibody, anti-HCV, syphilis helicoid antibody has been feminine gender.
4.IgM enzyme labelled antibody: the mouse-anti people μ chain IgM antibody that horseradish peroxidase (HRP) marks.
5. substrate A: 0.1M sodium citrate, 0.01% hydrogen peroxide.
6. substrate B: containing tetramethyl benzidine, dimethyl sulfoxide, sodium acetate solution.
7. concentrated cleaning solution: the 0.01MPBS that (20X) is concentrated.
8. stop buffer: 2mol/L H
2sO
4
9. be coated with the porous elisa plate of CoxB group antigen layer: adopt conventional method to wrap respectively with Cox B group antigen prepared by embodiment 1-3 respectively and closed with confining liquid by after elisa plate.
By each one bottle of 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 arbitrary piece 9 porous elisa plate porous elisa plate being coated with CoxB group antigen layer prepared packs, load a box body in the lump and make CoxB IgM enzyme-linked immunosorbent assay (ELISA) kit.
The condition of storage of kit: 2 ~ 8 DEG C of preservations.
The detection of embodiment 6 kit
Kit using method:
Kit can detect serum sample, collects serum by standard method, to be measured.If do not measured immediately, serum sample be placed on less than-20 DEG C frozen, and avoid multigelation.
1. preparation of reagents
The preparation of 1.1 solution: get the concentrated cleaning solution 1 bottle in kit, stand-by to load in wash bottle (providing for oneself) after 1: 20 dilution mixing, dissolving.
1.2 Preparatory work of experiments: taken out from refrigerator by sample to be measured, after putting thaw at RT, empirically require sample to be measured to number.
1.3 by after careful confirmation after sample numbering to be measured, is added in 1ml sample diluent, mixing by test serum sample diluting liquid (1: 100 dilution) i.e. 10 μ l serum.
2. the experiment condition that must meet
Kit takes out by 2.1 in 2 ~ 8 DEG C of refrigerators, puts room temperature and places 30 minutes.
2.2 wavelength using microplate reader to measure are 450nm.
The temperature of 2.3 adjustment water baths is 37 DEG C.
3. the every hole of yin and yang attribute reference substance in kit adds 100 μ l.
4. operation steps
Application of sample amount (μ l)
Add chromogenic substrate A liquid and each 50 μ l/ holes of B liquid, mixing of gently shaking, room temperature lucifuge places 10-15 minute, adds stop buffer 50 μ l/ hole, and slight oscillatory, with mixed solution, places room temperature 5 minutes.
Detected by microplate reader in 15 minutes, read each hole OD value in wavelength 450 nanometers.
5. quality control
The average relative OD ratio of kit positive reference substance and negative controls should be not less than 3.
6. result calculates
Calculate the mean 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]
Cut off value=negative control mean OD value X 2.1
Serum sample be positive (+) with the ratio >=Cut off value of negative control OD value;
Serum sample be positive (-) with the ratio≤Cut off value of negative control OD value.
Embodiment 7 kit specificity and accuracy testing
The kit (elisa plate being coated with Cox B group antigen prepared by embodiment 1) of Example 4 and 5 preparation, adopts the method for embodiment 6 to detect sample, adopts neutralization test to compare simultaneously, observe its specificity and accuracy respectively.
Negative match-rate=100% × [258/ (0+265)]=97.36%
Negative match-rate=100% × [253/ (0+265)]=95.47%
Above-mentioned test findings shows, kit specificity of the present invention is good, and accuracy rate is high, can be used for clinical reference.
Embodiment 8 kit specific test
Kit prepared by Example 4 and 5, adopt the method for embodiment 6 respectively, detect by 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 excellent.
Claims (4)
1. the CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit preparation method of CoxB group antigen, described CoxB IgG/IgM enzyme-linked immunosorbent assay (ELISA) kit comprises porous elisa plate, respectively sample diluting liquid is housed, positive control, negative control, IgG enzyme labelled antibody or IgM enzyme labelled antibody, substrate A, substrate B, the reagent bottle of concentrated cleaning solution and stop buffer, the inwall of micro-reacting hole of described porous elisa plate is coated with CoxB group antigen, the principal ingredient of described CoxB group antigen is purified and the Cox B1 of complete inactivation, Cox B2, Cox B3, Cox B4, the mixing of Cox B5 and Cox B6 virus, described CoxB group antigen adopts the method comprised the following steps to prepare:
1) virus amplification: the arbitrary seed culture of viruses be selected from Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 virus is seeded in the Hela cell of individual layer and cultivates acquisition cytopathy venom;
2) virus release: cytopathy venom is repeated freeze thawing more than three times, then ultrasonic disruption cell in ice-water bath;
3) inactivation of virus: by the repetition cytopathy venom of freeze thawing more than three times or the cytopathy venom complete inactivation through ultrasonic disruption cell, the cytopathy venom of deactivation need be negative through inactivation of virus experimental examination;
4) viral antigen purifying:
4.1 by be placed in through virus release and the cytopathy venom of inactivation of virus 18-20 DEG C melt appropriate time after, add formalin and mix, the final concentration of formaldehyde in mixed liquor is 0.12-0.20v%;
4.2 mixed liquor gradient centrifugation isolated virals: gradient centrifugation isolated viral specifically comprises the following steps:
4.2.1 mixed liquor low-speed centrifugal at least 20 minutes, gets supernatant;
4.2.2 centrifugal at least 60 minutes of supernatant 35000-45000rpm/min, gets precipitation;
4.2.3 precipitate after dissolving with PBS, be placed on 13-17wt% glycerine water solution, centrifugal more than 60 minutes of 50000-65000rpm/min, gets precipitation, and precipitation is isolated virus;
4.3 isolated viruses add protection liquid and obtain viral antigen liquid;
5) step 1 is utilized)-4) prepare the viral antigen liquid of Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 respectively, the mixing of the viral antigen liquid of obtained Cox B1, Cox B2, Cox B3, Cox B4, Cox B5 and Cox B6 is obtained CoxB group antigen.
2. preparation method as claimed in claim 1, it is characterized in that, in step 4.2.1, the centrifugal speed of low-speed centrifugal is 5000-8000rpm/min.
3. preparation method as claimed in claim 1, is characterized in that, in step 4.2.3, precipitation is 1:2 ~ 1:3 with the volume ratio of PBS.
4. preparation method as claimed in claim 1, is characterized in that, in step 4.3, described virus is 1:1 ~ 1:2 with the volume ratio of protection liquid.
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| 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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