CN1693454A - Hybridized tumour cell strain of anti hpatitis B virus surface antigen and its monoclonal antibody - Google Patents
Hybridized tumour cell strain of anti hpatitis B virus surface antigen and its monoclonal antibody Download PDFInfo
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Abstract
A hybridoma cell strain (CCTCC-C200505) is disclosed, which can secrete the monoclonal antibody of HBV surface antigen. An EL1SA method created by enveloping the monoclonal antibody can detect wild-type HBsAg and 13 variant HBsAgs.
Description
Technical field
The present invention relates to biotechnological formulation, relate to a kind of secretion anti-hepatitis B virus surface former anti-(Hepatitis B surface antigen, the hybridoma cell strain of monoclonal antibody HBsAg) and secreted monoclonal antibody thereof particularly.
Technical background:
HBsAg is the important symbol of diagnosis of hepatitis b virus infection.Mostly at present domestic and international HBsAg detection kit is to utilize the ELISA detection method of anti--HBsAg monoclonal antibody or polyclonal antibody foundation.The antibody difference that all ingredients box uses, the ability difference of detection variation hepatitis B virus.Present result of study shows that the ability of domestic HBsAg detection kit detection variant is not good, and major cause is the monoclonal antibody that does not have to discern variant.Therefore be necessary to develop and detect monoclonal antibody wild and variation HBsAg, set up detection method, with the loss of remarkable reduction HBsAg variant.
The reason of HBsAg variation is very complicated.Think the formation of anti-HBV immunoglobulin (Ig) behind the hepatitis b vaccine immune at present, and the high titre immunoglobulin prevention, the persistent infection of the pressure of medicine and virus suddenlys change relevant with HBV.Use high titre immunoglobulin also can cause HBV S transgenation, the liver transplantation patient uses 30% the people of still having an appointment behind the high titre immunoglobulin to infect HBV.
Promote Hepatitis B virus vaccine and inoculated against since HBV infects since the eighties in last century, the prevalence rate that HBV infects significantly reduces, and human beings'health has been played great role.But along with the prolongation that vaccine is used the time limit, the immune evasion strain that can escape the S transgenation of Hepatitis B virus vaccine gets more and more.Report that at present China's Hepatitis B virus vaccine and HBIG mother and baby block the loser, S genovariation rate is about 20%-40%; Carrier's aberration rate is 31% behind the simple hepatitis b vaccine immune of China.The surface antigen variant still has pathogenic and transmission capacity, may become the potential contagium, and the existing immunizing power of possible breakthrough crowd forms new infection and becomes new public health problem.Therefore, the prevention and the diagnosis of research S transgenation strain, to the infection of control HBV, the examination of carrying out HBV infection popular monitoring and blood donor is significant.
The prevention of HBV S transgenation strain and diagnosis are also significant to blood donor's examination.China mainly is by serological method to blood donor's examination at present.The appearance of hepatitis B variant has proposed problem for the examination of blood products.The antigenicity of variant and wild strain HBsAg has very big change; Behind the site amino acid variations such as surface antigen " a " determinant 145, antigenicity can be sent out the bigger change of dirt, about 60% variation surface antigen weakens with antigen reactivity at the monoclonal antibody of wild-type HBsAg or disappears, can not detect with domestic and foreign current surface antigen diagnostic kit, loss does not wait from 12%-90%.The easy omission of patient that variant infects, the blood products that variant pollutes also be difficult for finding, the prevention and control of HBV are brought difficulty.
The method that is used to detect the HBV sudden change at present mainly contains direct order-checking, limiting dilution PCR, colony screening and special gene sequence solid state polymerization enzyme chain reaction (SS-SPPCR).Special gene sequence solid state polymerization enzyme chain reaction (SS-SPPCR) is to detect the polyinfection sensitivity HBV point mutation and differentiation street strain and variant and special novel method.The specificity of these methods detection transgenations is fine, but the expense costliness needs many large-scale instrument and equipments and those skilled in the art, complicated operation, and detection time is long, is difficult for promoting the use of in China.
The common method that detects HBsAg is ELISA, and determining method susceptibility and specific key are the monoclonal antibodies that suitable anti--HBsAg is arranged.There are many research units to prepare the related monoclonal antibody that can be applied to clinical detection at present, but mainly are to be used to detect wild strain, can better discern the monoclonal antibody of variant and not appear in the newspapers as yet so far.Therefore, it is significant that screening can be discerned the monoclonal antibody of variant.
Summary of the invention
The objective of the invention is in order to solve the deficiency that the above-mentioned background technology exists, propose to prepare hybridoma cell strain and the excretory monoclonal antibody thereof of a kind of resisting-HBsAg, make the reagent of its preparation can when detecting the hepatitis B virus wild strain, can also detect multiple hepatitis B virus variation strain.
Hybridoma cell strain of the present invention obtains by immunity, fusion and screening, belongs to mouse source hybridoma D12E9F11, and its CCTCC preserving number is 200505, and it can secrete the monoclonal antibody of anti-HBsAg.
The secreted monoclonal anti physical efficiency of this hybridoma cell strain is carried out specific association reaction with wild-type and mutant HBsAg simultaneously.
The feature of hybridoma of the present invention:
Clone title (Cell line Name): mouse source hybridoma D12E9F11
Plant name (Species): Mouse
Derived tissues (Tissue Derived): murine myeloma cell SP2/0
Morphology (Morphology): Lymphoblast
Nutrient solution (Medium and Supplement) goes down to posterity: RPMI1640 90%; FCS10%
Growth conditions (Growth Condition): 37 ℃, CO
25%
Frozen matrix (Freeze Medium): FCS 90%, and DMSO 10%
Secretory product (antibody) feature:
Antibody classification: 1gGl
Antibody titer: supernatant 〉=1 * 10
3ELISA
Ascites 〉=1 * 10
7ELISA
Affinity of antibody: be used for the wild-type HBsAg detection sensitivity and can reach 0.5ng/ml, surpass the standard (standard is 1.0ng/ml) of China's food and sanitary inspection office promulgation, the sensitivity that detects common variation Gly145Arg HBsAg can reach 1.0ng/ml.
Viability (Viability): 〉=90%
Purposes of the present invention:
Direct purposes: foundation can detect hepatitis B virus mutant strain and the wild strain clinical detection experimental technique in interior hepatitis B virus surface antigen simultaneously.Comprise ELISA, the colloid gold immune dialysis, the solid phase of multiple antigen of hepatitis B virus and liquid phase analysis, and panimmunity pathology detection technology etc.The foundation of these methods can break through the restriction of conventional hepatitis B virus surface antigen detection reagent on multiple variant detects to a great extent.
Indirect purposes: the experimental technique and the reagent that adopt this cell strain secretory antibody to be developed can be widely used in the basis of hepatitis B, clinical and epidemiological study, in particular for the screening to hepatitis B virus variant the infected in the blood donation personnel.
The present invention is to promoting the control and the research of China's viral hepatitis, and clinical, the epidemiology development of further propelling hepatitis B and the propagation of prevention post-transfusion hepatitis are had and significant values.
Embodiment
One, the foundation of the cell strain of monoclonal antibody of anti--HBsAg
1, immunogenic preparation
Immunogen is isolating HBsAg from the HBV infected person anteserum.
2, immune animal:
Get HBsAg 20ug and be diluted in the 100ul physiological saline, add the aluminum hydroxide adjuvant of equivalent, behind the mixing, at BALB/c mouse (6 age in week) the subcutaneous multi-point injection of nape portion.At interval after 2 and 4 weeks, get with amount antigen and add Freund supplementary immunization twice, immunity finishes the back and gathers a little mouse blood, detects HBsAb and tires.Merged preceding 2 days, with same dose antigen abdominal injection supplementary immunization.
3, immunizing potency detects
Use the HBsAg of Ke Hua biotech firm pre-bag in Shanghai to be detected the detection of tiring of immunity back mice serum by 96 hole polystyrene plates.
Mouse serum is with containing 10% bovine serum PBS with 10
2-10
6Doubly dilution adds 96 orifice plates, 0.1ml/ hole, 37 ℃, 60 minutes.Get rid of liquid in the hole, button is done, and fills with each hole with washings, leaves standstill 15 seconds, gets rid of liquid in the hole, pats dry.Repeat 5 times.Every hole adds l: the sheep anti-mouse igg 50ul of HRP marks of 4000 dilutions in each hole, 37 ℃, 45 minutes.Repeat washing.Add 50ul and contain 0.1% (W/V) O-Phenylene Diamine 0.1%, 50ul hydrogen peroxide PH5.0 citric acid phosphoric acid damping fluid, the vibration mixing is put 37 ℃, 15 minutes.After 1M sulfuric acid color development stopping, the vibration mixing is in the 492nm wavelength readings.With the positive school valency of judging immune mouse serum in the measured value and ratio 〉=2.1 of control value.
3, cytogamy is built strain
Behind the art time booster immunization, the aseptic mouse spleen of getting is collected splenocyte, washes 3 times with serum-free medium, and counting is also measured cell viability.The myeloma cell of results logarithmic phase makes viable count, and cell viability should be not less than 95%.With 1 * 10
8Splenocyte and 2 * 10
7The myeloma cell is mixing in the 50ml conical centrifuge tube, and is centrifugal, and abandoning supernatant adds the 0.7ml molecular weight and be 4000 PEG as fusogen, makes its fusion, selects culture medium culturing with HAT, and hybridoma can be grown, and treats that cell grows to 10
5During/ml, the same immunizing potency assay method screening detects emiocytosis antibody situation;
4, limiting dilution assay is cultivated the cloning of positive colony
Clone with in the curved suction pipe piping and druming Hybridoma Cell Culture plate mesopore is suspended in the complete culture solution.Counting cells is adjusted cell concn to 100/ml, 50/ml, 10/ml.Respectively with the hybridoma suspension inoculation of three kinds of density in 96 well culture plates that contain feeder cell, every hole adds 0.1ml, makes every hole on average contain 10,5,1 cells.At 37 ℃, 5%CO
2And leave standstill cultivation under the saturated humidity condition.Can half amount change liquid after 1 week.Cultivate and got culture supernatant in 10-14 days and carry out the screening of the same immunizing potency assay method and detect emiocytosis antibody situation.The cell of getting in 20 best holes of the cultivation of cloning for the first time back antibody-secreting level carries out the cloning cultivation second time.Method is the same.Till clone's secreting specificity antibody of 100%.Filter out the best cell of a strain antibody character,, be made into 10 with the nutrient solution of 10%DMSO with the further enlarged culturing of positive colony
6/ ml cell, frozen.The strain stably excreting antibody cell strain of building together.Called after: D12E9F11.
Two, MONOCLONAL ANTIBODIES SPECIFIC FOR
Preparation contains the ascites of monoclonal antibody specific: select the female multiparity mouse of BALB/C for use, 1 week before the inoculation hybridoma is earlier to mouse peritoneal injection 0.5ml pristane.Collect well-grown hybridoma, centrifuge washing 1 time is resuspended in the serum-free medium, adjusts cell density, every mouse peritoneal injection 10
6Individual hybridoma.Inoculating cell 7-12 days, visible mouse web portion obviously expanded.The sterilization skin of abdomen connects syringe needle No. 8 with the 5ml syringe, thrusts the abdominal cavity, collects ascites.The centrifugal 15min of 3000rpm discards the upper strata grease, draws faint yellow ascites, packing, and-70 ℃ of preservations are standby.
Three, Purification of Monoclonal Antibodies
Get the mouse ascites of 1ml preparation, add the saturated ammonium sulphate solution of equivalent, make to reach 50% saturation ratio, stirring at room 2 hours, standing over night, 12000rpm, 4 ℃ centrifugal 30 minutes, supernatant discarded.Throw out is dissolved with PBS, adds the saturated sulfuric acid amine aqueous solution of 0.8 volume, make to reach 40% saturation ratio, stirring at room 2 hours, standing over night, 12000rpm, 4 ℃ centrifugal 30 minutes, supernatant discarded.Throw out is dissolved with PBS, adds the saturated ammonium sulphate solution of 0.7 volume, make to reach 35% saturation ratio, stirring at room 2 hours, standing over night, 12000rpm, 4 ℃ centrifugal 30 minutes, supernatant discarded.Throw out dissolves with PBS, puts into dialysis tubing, and dialysis tubing is put into the container that fills 1 liter of PBS, dialyses 24 hours, changes PBS therebetween twice.Measure the concentration of antibody purification, and observe antibody purification by the PAGE protein electrophoresis, 4 ℃ of preservations are standby.
Four, the immunology character analysis of monoclonal antibody
1, specific assay
With D12E9F11 monoclonal antibody bag by after, use the HBV cAg, and non-HBV antigen is antigen to be checked, be the detection antigen measurings with anti--HBsAg of HRP mark is how anti-.The result shows except that reacting with the HBV surface antigen, not have specific reaction with HBV cAg and contrast antigen.
2, monoclonal antibody immunoglobulins and subclass are measured
The 30ml cells and supernatant adds 4 ℃ of solid ammonium sulfates by 25.8%, and 2 hours, 3000 left the heart 30 minutes, and solution dissolves with the neutral PBS of 0.1ml.Do immune double diffusion with the anti-mouse 1g of rabbit blood grouping serum (Sigma), observe precipitation line after 24 hours.Monoclonal antibody is IgG1 as a result.
3, the analysis in monoclonal antibody identification HBsAg site
The same with HBsAg bag by 96 orifice plates, 50ul HBsAb positive patients serum, the monoclonal antibody of 50ulHRP mark, 37 ℃ 1 hour, the same colour developing liquid that adds in washing back is surveyed the 492nm absorption value.With odd contradictive hydroperitoneum the monoclonal antibody of same HRP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculates inhibiting rate with known monoclonal antibody.Inhibiting rate is (1-measures OD/ negative control OD) * 100.〉=50% inhibiting rate is positive.27 parts of surface antibody positive serums have all shown inhibiting rate in various degree (seeing Table 1) to three groups of antibody as a result, show that thus the antibody after the natural infection can suppress the reaction of monoclonal antibody and HBsAg, so the site of pointing out this monoclonal antibody to discern is present on the native antigen also.
Table 1. competition inhibition method is measured the antibody result
| Inhibiting rate (%) | Positive serum (part) | Negative serum (part) |
| ????0-9 ????10-19 ????20-29 ????30-39 ????40-49 ????50-59 ????≥60 | ????1 ????2 ????3 ????2 ????6 ????7 ????6 | ????7 ????5 ????7 ????5 ????3 ????0 ????0 |
Five, the applied research of monoclonal antibody
Monoclonal antibody detect the variation surface antigen ability
Application ELISA discerns the antigenic ability of common HBV S genovariation to this strain monoclonal antibody and detects, and contrasts with 8 kinds of homemade HBSAg detection kit commonly used.Detected result sees Table 3.This monoclonal antibody (D12E9F11) can detect 13 kinds in 15 kinds of variations, and 8 kinds of HBsAg detection kit commonly used are respectively the detection of variant: Wuhan is long upright 7 kinds, 8 kinds, ten thousand safe 7 kinds, 6 kinds in section China, 8 kinds of Rong Sheng, 6 kinds in moon gulf, magnificent 7 kinds are newly created in the Xiamen, like 7 kinds on grace ground.Monoclonal antibody D12E9F11 detects the more conventional test kit of ratio significantly for high to variation HBsAg's, and the ELISA colored intensity also is significantly higher than test kit.
Express recombinant plasmid wild and variation HBsAg in table 2. experiment
| Sample | Serotype | The mutational site | Sudden change betides the position of MHR | Country | Sample source |
| ????1056Sp ????91-4696 ????AP3.1 ????Arg145dr ????Arg145dw ????Arg145yw ????BA2.4 ????BA3.2 ????BA3.4 ????C126 ????Gly?dr ????Gly?dw ????Gly?yw ????HK188 ????M5 | ????ayw ????adw ????adw ????adr ????adw ????ayw ????ayw ????ayw ????ayw ????adr ????adr ????adw ????ayw ????adr ????ayw | P120S/S143L D99N/122NT123/G145R D144A G145R G145R G145R Y100C/P120T T123N/C124R T123N I126T/S143T standard sequence standard sequence standard sequence L98V/Q101R Y100S/T118V/R122K/M133I/ Y134N/P142S/S143L/G145K | ????2、4 ????1，2，4 ????4 ????4 ????4 ????4 ????1，2 ????2 ????2 ????3，4 ? ? ? ????1 ????1-4 | Spain Indonesia Britain China Italy Germany Pakistani China Spain Spain Hong-Kong Saudi Arabia | HBIG treated HBsAg carrier positive control positive control positive control Marrow donation person renal transplant patient after HBIG treated liver transfer operation after the HBIG treatment liver transfer operation after the liver transfer operation of HBIG treatment artificial mutation immuning failure immuning failure after the liver transfer operation of intravenous drug users immunity inoculation |
| ????SA4 ????SA6 ????SA7 | ????adw ????adw ????ayw | ????M133T/Y161F ????Q129R/G130N/A166V ????M133T | ????3，5 ????3，5 ????3 | South Africa South Africa | Acute hepatitis B chronic hepatopathy chronic hepatopathy |
Table 3: monoclonal antibody D12E9F11 and HBsAg detection kit commonly used detect comparison wild and variation HBsAg.
| Sample | Long upright | Newly create in the Xiamen | Ten thousand Thailands | Section China | Rong Sheng | Moon gulf | Magnificent | Like grace ground | D12E9F11 |
| 1056sp | ????+ | ????+ | + | ??- | ??+ | ???- | ????- | ????- | ????+ |
| 91-4696 AP3.1 Arg145dr Arg145dw Arg145yw BA2.4 BA3.2 BA3.4 C126 Gly Dr Gly Dw Gly Yw HK188 M5 SA4 SA6 SA7 coated antibody detects antibody | ++------+++++--++ how anti-monoclonal antibody is | ++------++ ++ ++ +++-+++how anti-monoclonal antibody is | ++------+++++--++ how anti-monoclonal antibody is | ++---+--++ ++ ++ +++--+-how anti-monoclonal antibody is | ++--+---+++++--+-how anti-monoclonal antibody is | ++------+++++--++ how anti-monoclonal antibody is | ++--+---+++++--++ how anti-monoclonal antibody is | ++------+++++--++ how anti-monoclonal antibody is | ++++++--++ ++ ++ +++++++how anti-monoclonal antibody is |
Annotate: with S/N value judged result, S/N is positive greater than 2.1, and is negative less than 2.1.
Table 4: the monoclonal antibody D12E9F11 detection of tiring:
| Sample | Extent of dilution | |||||||
| Cells and supernatant ascites | ??10 -1??2.690 | 10 -21.916 | ?10 -3?0.643 | 10 -40.277 ≥2.900 | 10 -50.097 ≥2.900 | ?10 -6?0.042 ?2.435 | 10 -70.040 0.299 | Negative control 0.024 0.029 |
Six, brief summary
Conventional MONOCLONAL ANTIBODIES SPECIFIC FOR technology is adopted in this patent invention, uses that isolating HBsAg is the immunogen immune mouse from the HBV infected person anteserum, after cytogamy, screening, successful screens the monoclonal antibody that the HBV variant can be better discerned in a strain.The epi-position of identification is present on the self-faced antigen.The ELISA method that is established with this kind monoclonal antibody bag, not only can detect wild-type HBsAg, can also detect 13 kinds of variation HBsAg in 15 kinds of variations except that T123M/C124R and T123N, the ability that detects variant obviously is better than domestic like product.The minimum concentration that this monoclonal antibody detects wild HBsAg reaches 0.5ng/mL, meets the standard that country formulates.Domestic still do not have report that the hybridoma cell strain that can secrete the monoclonal antibody that detects variant is arranged at present.Cell strain with this kind character is the initiative of our unit.
The method that the ELISA that uses cell strain excretory antibody of the present invention to set up detects surface antigen can replace the relatively poor existing HBsAg diagnostic reagent of present domestic HBV immune evasion variant detectivity.Being used for other experimental techniques that the HBV variant infects clinical detection and research,, good application prospects is arranged also as immunofluorescence detection, immune colloidal gold technique and radioimmunity detection etc.Present technique is in case enforcement can be widely used in the basis of hepatitis B, clinical and epidemiological study, in particular for the screening to hepatitis B virus variant the infected in the blood donation personnel.To promoting the control and the research of China's viral hepatitis, clinical, the epidemiology development of further propelling hepatitis B and the propagation of prevention post-transfusion hepatitis are had and significant values.
Claims (2)
1, a kind of hybridoma cell strain, CCTCC preserving number are CCTCC-C200505, and it can secrete the monoclonal antibody of hepatitis B vaccine surface antigen.
2, a kind of by the described hybridoma cell strain excretory of claim 1 monoclonal antibody, this antibodies specific ground hepatitis B vaccine surface antigen.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102906111A (en) * | 2009-12-24 | 2013-01-30 | 株式会社绿十字 | Human antibodies specifically binding to the hepatitis B virus surface antigen |
| CN104845938A (en) * | 2015-04-17 | 2015-08-19 | 北京科兴中维生物技术有限公司 | Hepatitis b virus surface antigen monoclonal antibody and application thereof |
| CN105602906A (en) * | 2015-12-22 | 2016-05-25 | 菲鹏生物股份有限公司 | Hybridoma cell capable of secreting monoclonal antibody resisting mutated HBV (hepatitis B virus) surface antigen, monoclonal antibody and application |
| 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 |
Family Cites Families (3)
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| CN1044384C (en) * | 1994-08-25 | 1999-07-28 | 中国药品生物制品检定所 | Building of anti-type-C hepatitis virus antigen monoclonal antibody cell strain and monoclonal antibody thereof |
| CN1089802C (en) * | 1998-07-31 | 2002-08-28 | 中国药品生物制品检定所 | Monoclone antibody cell strain and its monoclone antibody with hepatitis C virus resisting non-structure zone 4 antigen |
| CN1264573C (en) * | 2003-09-26 | 2006-07-19 | 陕西九州科技股份有限公司 | Method for preparing targeting preparation of human monoclonal antibody crosslinking interferon for anti hepatitis b virus |
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| CN102906111A (en) * | 2009-12-24 | 2013-01-30 | 株式会社绿十字 | Human antibodies specifically binding to the hepatitis B virus surface antigen |
| CN102906111B (en) * | 2009-12-24 | 2014-11-05 | 株式会社绿十字 | Human antibodies specifically binding to the hepatitis B virus surface antigen |
| CN104845938A (en) * | 2015-04-17 | 2015-08-19 | 北京科兴中维生物技术有限公司 | Hepatitis b virus surface antigen monoclonal antibody and application thereof |
| CN105602906A (en) * | 2015-12-22 | 2016-05-25 | 菲鹏生物股份有限公司 | Hybridoma cell capable of secreting monoclonal antibody resisting mutated HBV (hepatitis B virus) surface antigen, monoclonal antibody and 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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