CN1284860C - Single clone antibody of antimutagen hepatitis B virus surface antigen - Google Patents
Single clone antibody of antimutagen hepatitis B virus surface antigen Download PDFInfo
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- CN1284860C CN1284860C CN 200510012334 CN200510012334A CN1284860C CN 1284860 C CN1284860 C CN 1284860C CN 200510012334 CN200510012334 CN 200510012334 CN 200510012334 A CN200510012334 A CN 200510012334A CN 1284860 C CN1284860 C CN 1284860C
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Abstract
The present invention relates to a monoclonal antibody of anti-mutagen hepatitis B virus surface antigens, which is secreted by a hybridoma cell strain CGMCC HBSP2. Hepatitis B viruses comprising fourteen mutagen strains and wild strains can be simultaneously detected according to the monoclonal antibody of anti-mutagen hepatitis B virus surface antigens prepared by the monoclonal antibody; HBsAB in a conventional detecting reagent kit of hepatitis B surface antigens can be replaced for making a diagnostic reagent kit of the hepatitis B surface antigens. By adopting the antibody, missed diagnosis due to diagnosis by a conventional reagent can be avoided for diagnosing variant hepatitis B virus carriers and non-variant hepatitis B virus carriers or hepatitis B patients; consequently, the patients are treated in time. Particularly, when blood donors are screened out, variant virus carriers who can not be detected by the conventional reagent kit can be detected for avoiding using blood of the hepatitis B patients as healthy blood transported to blood recipients for ensuring the quality of a blood source.
Description
Technical field
The invention belongs to hepatitis B virus antibody, be specifically related to a kind of monoclonal antibody that resists the multi-functional hepatitis B virus surface antigen of wild and sudden change.
Background technology
Hepatitis B is a global health problem.The whole world has the chronic carrier of 300,000,000 5 thousand ten thousand HBV approximately, and the patient who dies from hepatitis B every year has 200 ten thousand, makes it to become the whole world the 9th and causes dead disease greatly.The chronic carrier of China HBV accounts for 1/3 of the whole world, is the highest country of HBV virus carrying rate in the world.Estimation crowd HBsAg positive rate is 10.3%.Asymptomatic HBsAg carrier more than 100,000,000 is arranged, and existing disease patient has surpassed 3,000 ten thousand people, and sickness rate is about 30.41%.Hepatitis B is serious threat China people's health not only, also brings serious economical load for society.
The mutation rate of HBV is higher, there are some researches show, the mutation rate of HBV reaches more than 30%.The different genes district of HBV all can undergo mutation, but the variation of main harm maximum occurs in S district and the variation of preceding S district: S gene " a " determinant G 145R variation, can produce the immunologic escape strain, and antigenicity changes, reduce with the avidity of anti--HBs, cause conventional hepatitis b vaccine immune invalid.Use the lamivudine therapy chronic viral hepatitis B, the incidence of YMDD variant is 14～32% after 1 year, 2 years be 38%, 3 year to be 49%, 4 year to be 66% (Chinese hepatopathy magazine 2004 the 12nd the 7th phase of volume the 425th page " lamivudine clinical application expert common recognition in 2004 ").
Will treat huge hepatitis B morbidity crowd like this, at first must find hepatitis B patient, this just need diagnose, and it serves to show the importance that detects hepatitis B reagent.At present both at home and abroad hepatitis diagnostics has a variety ofly, if any the simple hepatitis B surface antigen that detects, also has and detects hepatitis B surface antigen simultaneously, also has DNA to hepatitis B virus to detect.The detection method that adopts has multiple, and the immunodetection based on antigen antibody reaction is arranged, and also has and utilizes biochip to detect, and concrete detection technique is also different, is multifarious.
But these diagnostic reagents have a fatal defective, can not detect for the hepatitis B virus that sudden change has taken place exactly, and especially the hepatitis B virus of variation has taken place surface antigen HBsAg, can't diagnose with conventional reagent, will cause like this and fail to pinpoint a disease in diagnosis.Make hepatitis B virus carriers or hepatitis B patient can not get making a definite diagnosis, thereby also just can not obtain treatment.Even more serious is when the blood donor is carried out examination, if adopt conventional test kit to carry out, just the blood of hepatitis B patient may be defeated by the blood recipient as healthy blood, thereby cause very serious cross infection.
At this situation, development can detect the test kit of the hepatitis B virus that comprises mutant strain and wild strain, just has very important significance.
Summary of the invention
The monoclonal antibody that the technical problem that the present invention mainly solves provides a kind of anti-mutation hepatitis B virus surface antigen with and preparation method thereof, this is a kind of hepatitis B surface antibody that can detect sudden change hepatitis B virus surface antigen and wild hepatitis B virus surface antigen simultaneously.Adopt this hepatitis B surface antibody can prepare the detection kit that can detect the new hepatitis B virus surface antigen that comprises mutant strain and wild strain simultaneously.
Technical solution of the present invention is: a kind of monoclonal antibody of anti-mutation hepatitis B virus surface antigen, with blood source Hepatitis B virus vaccine routine immunization BALB/C mice, detect hepatitis B surface antibody with the ELISA method and confirm the immunity success, obtain anti-hbs monoclonal antibodies by hybridoma cell technology.
The present invention is by the monoclonal antibody of hybridoma cell strain HBSP2 excretory anti-mutation hepatitis B virus surface antigen.This microorganism (strain) has been stored in CGMCC, deposit number on November 23rd, 2004: 1252.
The preparation method of a kind of hybridoma cell strain CGMCC HBSP2, this method may further comprise the steps:
(1) preparation immunogen: haematogenous Hepatitis B virus vaccine HBsAg albumen is dropped on the nitrocellulose filter;
(2) immunity: surgical procedures is imbedded nitrocellulose membrane and is treated in the mice immunized spleen routinely, at interval two week the back booster immunizations, abdominal injection recombination mutation HBsAg albumen and freund 's incomplete adjuvant mixed solution, immunity for the third time plays each interval 3 thoughtful 1 month;
(3) screening: with enzyme-linked immunosorbent assay (ELISA) screening immune mouse serum;
(4) cytogamy, screening and cloning: select the highest mouse extracting spleen cell of antiserum titre and the external fusion of myeloma cell, through the hybridoma cell strain CGMCC HBSP2 of three subclone acquisition stably excreting anti-mutation HBsAg protein monoclonal antibodies.
Above-mentioned MONOCLONAL ANTIBODIES SPECIFIC FOR method by hybridoma cell strain CGMCC HBSP2 excretory anti-mutation hepatitis B virus surface antigen may further comprise the steps:
(1) extraction and purification of monoclonal antibody: with the hybridoma cell strain CGMCC HBSP2 (deposit number: 1252) after the external enlarged culturing, regularly collect supernatant liquor of the stably excreting anti-mutation HBsAg protein monoclonal antibody that obtains;
(2) ascites preparation: the cell strain after the enlarged culturing is squeezed into the BALB/C mice abdominal cavity to produce a large amount of ascites, handle mouse peritoneal, hybridoma is injected mouse peritoneal, treat to use when mouse peritoneal is enough big syringe sucking-off ascites, salting-out process purifying with lipopenicillinase alkane; In the ascites preparation, the hybridoma number of the cell strain after the enlarged culturing being squeezed into the mouse abdominal cavity is 1 * 10
7Individual, collect ascites after 7-10 days, behind extraction, purifying, can obtain anti-mutation HbsAg protein monoclonal antibody.
The present invention is by hybridoma cell strain CGMCC HBSP2 (deposit number: 1252) excretory anti-mutation hepatitis B virus surface antigen monoclonal antibody, the application in preparation hepatitis B surface antigen diagnostic kit.Can replace the HbsAb in the conventional hepatitis B surface antigen detection kit.
Monoclonal antibody according to the anti-mutation hepatitis B virus surface antigen of the present invention preparation, can detect the hepatitis B virus that comprises 14 kinds of mutant strains and wild strain simultaneously, can replace the HBsAb in the conventional hepatitis B surface antigen detection kit, make the hepatitis B surface antigen diagnostic kit.That causes when adopting this antibody can avoid with the diagnosis of conventional reagent fails to pinpoint a disease in diagnosis, and variation and unmanifest hepatitis B virus carriers or hepatitis B patient is made a definite diagnosis, thereby obtained in time to treat.Particularly when the blood donor is carried out examination, can detect the variant viral carrier who adopts conventional test kit can not detect, avoid the blood of hepatitis B patient is defeated by the blood recipient as healthy blood, guarantee the blood source quality.
Embodiment
Embodiment one: hybridoma technology prepares the monoclonal antibody of anti-hepatitis B virus surface antigen (HBsAg)
One, material
The BALB/C mouse inbred lines, female, 8 ages in week.SP2/0 myeloma cell strain, RPMI1640 substratum, newborn calf serum, xanthoglobulin-methotrexate-thymus pyrimidine (HAT), xanthoglobulin-thymus pyrimidine (HT), polyoxyethylene glycol (PEG4000), (above provide) by Huamei Bio-Engrg Co..The sheep anti mouse immunoglobulin (Ig) of horseradish peroxidase (HRP) mark (Wuhan doctor's moral biological products company).
Two, method
1. immunogen preparing 10 μ g (5 μ l, 2 μ g/ μ l) antigen drops on the nitrocellulose filter (NC) of 2mm * 2mm, dries.
2. immune spleen is implanted into immunity, mouse Patients Under Ketamine Anesthesia, the spleen district epilation of the fixing back of four limbs, alcohol disinfecting, the 5mm otch is made under the midaxillary line rib edge in a left side, goes into abdomen and involves the spleen lower edge, cuts the spleen coating, insert the NC film of adsorption antigen, hemostasis by compression, otch holostrome one pin is sewed up.
The immunization protocol first immunisation adopts spleen to be implanted into; Interval booster immunizations after 2 weeks, abdominal injection antigen and freund 's incomplete adjuvant mixed solution (1: 1) 1ml; The 3rd immunity plays each interval 3 thoughtful 1 month.
3.ELISA screening adds 37 ℃ of bags of 10 μ g/ml surface antigen solution by 2h with phosphoric acid buffer (pH5.8) the treat enzyme target 4h that contains 0.2% glutaraldehyde, contains 37 ℃ of sealings of PBS (pH7.4) 2h of 10%NBS, PBS/T flushing 3 times, 4 ℃ of maintenances.Every bag of time spent is added dilute serum or Hybridoma Cell Culture supernatant 100 μ l by the hole, hatches 30min for 37 ℃, and PBS/T flushing 5 times adds 1: 5000 HRP-goat anti-mouse igg, hatches 30min for 37 ℃, adds substrate-p-diaminodiphenyl (TMB), substrate-hydrogen peroxide (H at last
2O
2) color development at room temperature 10-15min, with the negative contrast of SP2/0 myeloma cell's culture supernatant.
4. cytogamy, screening and cloning were merged preceding 3 days, to the no adjuvant antigen 1 00 μ g of passages through which vital energy circulates injection under the highest mouse socket of the eye of serum titer, according to a conventional method, getting mouse boosting cell and SP2/0 cell (5: 1) merges, HAT selects to cultivate, with above-mentioned ELISA method screening, 3 limited dilution cloningizations of positive aperture.
5. after the hybridoma enlarged culturing of the extraction and purification of monoclonal antibody through the clone, collected 1 supernatant liquor every 3 days, the centrifugal 10min of 1000r/min, supernatant liquor behind sulfate of ammoniac precipitation classification purifying-20 ℃ frozen.
6. the ascites preparation is injected 0.5ml liquid lipopenicillinase alkane, the hybridoma of having cultivated with every injection of method after 10 days 10 at every BALB/C mice intraperitoneal
6Individual, mouse web portion obviously swells after 10 days, and hand has touched fluctuation, gathers ascites with No. 16 syringe needles.The centrifugal 30min of 1500r/min collects supernatant, and-20 ℃ frozen.
7.mAb preliminary evaluation
7.1 titration detects the hybridoma culture supernatant and induces the ascites antibody immunology with indirect elisa method and tires.Envelope antigen is a recombination mutation HBsAg albumen, and the enzyme labelling thing is a sheep anti mouse HRP ELIAS secondary antibody, and enzyme substrates is TMB-H
2O
2
7.2McAb type identification is pressed Luo Shi (Roche) company mouse immuning ball protein subclass and is measured the test kit description operation.
7.3 the monoclonal antibody specific assay protein immunization marking (Western Blot) analysis is at first carried out polyacrylamide gel (SDS-PAGE) electrophoresis, electricity goes on the pvdf membrane again, after one, two resistive connections close, and diaminobenzidine (DAB) colour developing.
Embodiment two: anti--HBsAg monoclonal antibody detects the variation surface antigen
1. express variation surface antigen construction of recombinant plasmid and expression
Survey a pair of primer of design at HBV S gene two, design 21 pairs of site-specific mutant primers according to the mutational site of the S gene of bibliographical information, by PCR method rite-directed mutagenesis HBV S gene.The S gene clone that will make a variation is to carrier for expression of eukaryon PCR3.1, and order-checking is used for experiment after confirming correctly.Make up 21 kinds of recombinant plasmids of expressing variation HBsAg altogether.
With 21 kinds of recombinant plasmid transfection COS-7 cells of expressing variation HBsAg, collect supernatant.
2. anti--HBsAg monoclonal antibody E1C detects with the reactivity of variation surface antigen
With purifying anti--HBs monoclonal antibody E1C, adds 21 kinds respectively and contains the cells and supernatant that becomes hepatitis B surface antigen by the ELISA reacting hole with suitable extent of dilution bag after the sealing, 37 ℃, 1 hour; The washing back adds resisting-HBs of HRP mark, 37 ℃, 45 minutes more; The washing back adds TMB developer, colorimetric.According to the ratio calculation S/N value of color, judged result (seeing Table).
3. domestic HBsAg test kit commonly used detects the variation surface antigen
Select the HBsAg detection kit of domestic 8 manufacturer production to detect the cells and supernatant of expressing the variation surface antigen respectively.Detection method is undertaken by the test kit specification sheets, and HBsAg detection kit commonly used detects the ability (seeing Table two) of variation HBsAg in the comparator.
4. the relatively sensitivity of monoclonal antibody E1C and two kinds of domestic reagent box detection R145R HBsAg.
With cell expressing contain times dilution of G145R HBsAg supernatant from 1: 5 to 1: 640, detected the reactivity (seeing Table three) of series of diluted samples by the back with monoclonal antibody E1C bag.
The qualification result of embodiment 1 and embodiment 2 is as follows:
1. the foundation of hybridoma cell line is tired with clone's immune mouse blood ELISA and is all reached 1: 10
4More than, HAT culture medium culturing after extracting spleen cell and the SP2/0 cytogamy, the cell clone growth is seen in the part hole about 1 week, the back cell growth of 2 weeks surpasses hole floorage 1/3, being fused into power is 75%, the specific antibody positive rate is 28.3%, keeps the best hybridoma cell strain E1C of a strain after 3 time cloningizations.
2. the evaluation of anti-mutation HBsAg monoclonal antibody
Detecting in E1C ascitic type monoclonal antibody and the cells and supernatant monoclonal antibody 2.1 tire with indirect elisa method tires and is respectively 1: 6.4 * 10
4, 1: 160.
Monoclonal antibody E
1CELISA measures OD behind the gradient dilution
450Be worth as follows:
| E 1C | |||||
| Hybridoma Cell Culture supernatant ascites | 1∶10 2.698 1∶1000 2.872 | 1∶100 1.734 1∶10000 1.814 | 1: 1,000 1.053 1: 10 10,000 | 1: 10,000 0.477 1: 100 10,000 | Control 0.046 1: 1,000 10,000 |
Annotate: positive criteria: detect hole OD value/control group OD value＞2.1
2.2 immunoglobulin (Ig) (Ig) classification and hypotype are measured through identifying that gained cell strain E1C excretory immunoglobulin (Ig) is IgG2a, the light chain type is κ.
2.3 specificity Western-Blot identifies that E1C can combine with sudden change HBsAg albumen generation specificity.
Table one: anti-hepatitis b HBsAg monoclonal antibody detects the result of variation HBsAg
| Sample | E1C |
| HBsAg adr | 100 |
| HBsAg (ayw) HBsAg adw G145R L98V Y100S C124R T123N M133T Y134N P142S G145K D144A M133I Q129R D144E A166V P127S T118K M125T number positive Hela cell | 100 100 63.0 41.82 34.38 1.95 1.95 57.25 63.55 50.74 93.54 104.44 29.48 69.52 94.95 114.32 129.51 68.23 32.81 15/17 0.34 |
Annotate: detected result is 100% with the OD value of wild HBsAg, and the OD value percentage ratio by comparison of all the other samples is detected result.
Table two: domestic 9 kinds of HBsAg detection kit detect the result of variation HBsAg
| Sample | First | Second | Third | Fourth | Penta | Oneself | Heptan | Hot | The ninth of the ten Heavenly Stems |
| HBsAg(adr) G145R L98V Y100S C124R T123N M133T Y134N P142S G145K D144A M133I Q129R D144E A166V P127S | 100 10.35 75.35 89.68 0.59 0.58 6.54 48.89 47.28 83.48 118.54 19.56 68.65 69.41 83.14 93.08 | 100 0.68 76.68 4.12 0.92 0.84 3.49 48.23 1.95 92.01 112.96 11.56 53.42 78.52 214.63 85.44 | 100 0.06 24.13 0.56 1.71 1.37 1.37 4.88 3.53 91.84 82.22 0.41 0.12 86.55 183.2 91.55 | 100 4.42 77.07 4.11 1.49 2.09 7.16 61.28 14.76 95.43 92.87 7.64 70.44 70.43 94.22 70.45 | 100 9.19 47.31 13.73 0.88 1.83 6.7 23.39 15.84 82.47 97.14 17.18 5.45 85.04 93.53 91.51 | 100 2.22 31.78 6.18 1.88 3.81 4.14 0.98 4.05 70.55 84.13 10.26 49.44 53.57 92.2 62.68 | 100 2.28 21.97 37.04 2.48 4.83 8.91 2.28 1.82 48.87 93.32 14.75 53.14 40.67 107.19 78.54 | 100 3.51 51.07 9.55 2.04 4.91 6.29 14.56 25.58 79.92 96.27 17.67 46.18 66.49 105.37 84.28 | 100 2.24 47.64 6.91 0.98 2.49 59.13 58.04 2.54 67.14 57.27 10.53 3.68 72.83 132.0 85.15 |
| T118K M125T detects number positive COS-7 cell | 64.25 42.53 14/17 0.25 | 38.46 2.62 11/17 0.36 | 21.38 36.54 8/17 0.12 | 68.79 3.51 12/17 0.13 | 48.54 3.98 14/17 0.09 | 39.58 48.71 11/17 0.28 | 64.74 37.05 12/17 0.41 | 48.82 4.65 13/17 0.48 | 80.47 1.58 11/17 0.21 |
Annotate: detected result is 100% with the OD value of wild HBsAg, and the OD value percentage ratio by comparison of all the other samples is detected result.
Table three: the relatively sensitivity of monoclonal antibody E1C and two kinds of reagent detection G145R HBsAg
| 1∶5 | 1∶10 | 1∶20 | 1∶40 | 1∶80 | 1∶160 | 1∶320 | 1∶640 | |
| Second first E1C negative control | 0.161 0.612 2.568 0.036 | 0.081 0.304 2.041 0.012 | 0.063 0.236 1.642 0.042 | 0.051 0.108 1.027 0.036 | 0.052 0.074 0.638 0.014 | 0.048 0.051 0.312 0.022 | 0.057 0.057 0.148 0.031 | 0.042 0.027 0.061 0.014 |
Annotate: the OD value representation of result after with colorimetric
Embodiment three: the preparation of hepatitis B surface antigen detection kit
Replace HBsAb in the conventional hepatitis B surface antigen detection kit with the anti-mutation HBsAg monoclonal antibody of the present invention preparation, the same in all the other reagent and material and the conventional hepatitis B surface antigen detection kit.The detection method also hepatitis B surface antigen detection kit with routine is the same.
Claims (3)
1, a kind of monoclonal antibody by hybridoma cell strain HBSP2 excretory anti-mutation hepatitis B virus surface antigen; This hybridoma cell strain is stored in CGMCC, deposit number: 1252.
2, a kind of MONOCLONAL ANTIBODIES SPECIFIC FOR method by hybridoma cell strain CGMCC HBSP2 excretory anti-mutation hepatitis B virus surface antigen is characterized in that may further comprise the steps:
(1) extraction and purification of monoclonal antibody: with the hybridoma cell strain CGMCC HBSP2 (deposit number: 1252), after the external enlarged culturing, regularly collect supernatant liquor of the stably excreting anti-mutation HbsAg protein monoclonal antibody that obtains;
(2) ascites preparation: the cell strain after the enlarged culturing is squeezed into the BALB/C mice abdominal cavity to produce a large amount of ascites, handle mouse peritoneal, hybridoma is injected mouse peritoneal, treat to use when mouse peritoneal is enough big syringe sucking-off ascites, salting-out process purifying with lipopenicillinase alkane; In the ascites preparation, the hybridoma number of the cell strain after the enlarged culturing being squeezed into the mouse abdominal cavity is 1 * 10
7Individual, collect ascites after 7-10 days, behind extraction, purifying, can obtain anti-mutation HBsAg protein monoclonal antibody.
3, a kind of by hybridoma cell strain CGMCC HBSP2 (deposit number: 1252) excretory anti-mutation hepatitis B virus surface antigen monoclonal antibody, the application in preparation hepatitis B surface antigen diagnostic kit.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101871944B (en) * | 2010-06-02 | 2013-07-10 | 中国农业科学院茶叶研究所 | Qualitative detection method of tea geometrid nuclear polyhedrosis virus |
| CN102863527B (en) * | 2012-05-25 | 2014-03-19 | 中国疾病预防控制中心病毒病预防控制所 | Humanized hepatitis b virus resistance surface antigen gene engineering antibodies and preparation method and application thereof |
| CN105132376B (en) * | 2015-06-26 | 2018-02-09 | 南京红十字血液中心 | One can the how individual epitopes of specific recognition HBsAg monoclonal antibody and its application |
| CN105602906B (en) * | 2015-12-22 | 2019-06-18 | 菲鹏生物股份有限公司 | Hybridoma, monoclonal antibody and the application of anti-mutation Hepatitis B virus surface antigen monoclonal antibody can be secreted |
| CN108624565A (en) * | 2017-03-17 | 2018-10-09 | 艾博生物医药(杭州)有限公司 | A kind of grand Antibody preparation of monoclonal antibody of anti-hepatitis B surface antigen and screening |
| CN108624564A (en) * | 2017-03-17 | 2018-10-09 | 艾博生物医药(杭州)有限公司 | The preparation and screening of the grand antibody of monoclonal antibody of anti-hepatitis B surface antigen |
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