CN108624565A - A kind of grand Antibody preparation of monoclonal antibody of anti-hepatitis B surface antigen and screening - Google Patents
A kind of grand Antibody preparation of monoclonal antibody of anti-hepatitis B surface antigen and screening Download PDFInfo
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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Abstract
It is CCTCC No the present invention relates to a kind of monoclonal antibody of combination hepatitis B surface antigen and the deposit number for generating the antibody:The application of the hybridoma cell strain of C2016210 and the preparation method of antibody and antibody in hepatitis B surface antigen detection kit.The present invention provides the antibody that can be combined with hepatitis B surface antigen, it can be combined with the hepatitis B surface antigen of wild type hepatitis B surface antigen or saltant type, with extensive binding ability, false negative testing result can be effectively reduced, improves the accuracy detected to Hepaitis B virus.
Description
Technical field
The present invention relates to the preparation of monoclonal antibody and screening, more particularly to can be with hepatitis B virus surface antigen knot
The monoclonal antibody of conjunction.
Background technology
Hepatitis type B virus (HBV) is a kind of double-stranded DNA virus, belongs to Hepadnaviridae, is the ball of diameter about 42nm
Shape particle.With shell and nucleocapsid two parts, shell thickness 7-8nm, there is S protein, M albumen and L albumen, S protein surface tool
Have and infects related hepatitis B surface antigen (HBsAg) with virus.
Hepatitis type B virus is the most common cause of disease for causing virus hepatitis, can pass through blood, mother and baby, close contact
And sexual transmission, after human infection hepatitis B, easily cause chronic hepatitis B, and then lead to hepatic sclerosis and/or liver cancer.Mesh
Before, there is viral (HBV) the infected of about 400,000,000 chronic hepatitis Bs in the whole world, and Chinese about 100,000,000 carrier, height rank first in the world.According to
Statistics, has 1,000,000~1,500,000 people to die of liver failure, hepatic sclerosis and/or liver caused by acute or chronic HBV infection every year
Cancer.Thus preventing HBV infection becomes world public health problem.
It is widely used using immunological response principle to detect this technology in sample with the presence or absence of analyte
In every field.Being divided in various biological samples (saliva, blood, urine, serum, sweat etc.) can be detected with the principle
Substance is analysed, the health status (early pregnancy, tumour, infectious disease, drugs etc.) of disease and the mankind is detected to achieve the purpose that.This inspection
The cardinal principle of survey technology has the performance specifically bound between being built upon immune molecule, such as antibody resists with antigen, half
Original/antibody, biotin and antibiotic etc..In addition, many such detections can be completed on solid dielectric, such as commonly
It chromatographs in reagent strip, glass or plastic microtiter plates, device for immunochromatography etc..In general, can be on immune specific binding molecule
In conjunction with some solid particles or chemical substance, come qualitative, sxemiquantitative or can be determined by naked eyes or Other Instruments equipment in this way
Amount obtains testing result.This solid particle can be colored colloidal solid (latex or gold particle), this chemicals
Matter can be the substance with chromophoric group, these substances can send out specific wavelength to show inspection under the conditions of other suitable
Survey result.
Since HBV virus variations are very fast, HBsAg amino acid sequences and structure variation can be caused.When HBsAg structure changes
Afterwards, the immune combination of the antibody of the antigen before detection mutation is it is possible that therefore obstacle is established in the immune combination of antigen-antibody
On the basis of HBV detection be susceptible to false negative result.If HBV is not timely detected out, not only Hepatitis B carriers itself
It is impacted, and hepatitis B can be caused with propagation such as blood, mother and baby, father baby, close contact, sexual behaviour.Therefore, should use pair
Wild type and mutant virus have the hbv antibody compared with wide apaptability as raw material.
Monoclonal antibody refers to the antibody only made by a type of cell.Monoclonal antibody is by that can manufacture
Cell after immunocyte and the cancer cell fusion of corresponding antibodies generates, what this fused cell had both constantly been divided with cancer cell
Ability, and the ability that there is immunocyte can generate antibody.It is detected using the immunological method that antigen and antibody specific combines
Hepatitis B surface antigen (HBsAg), it will usually be detected using double antibody sandwich method.One of antibody is fixed on solid-phase media
It is upper to be used as coated antibody, combine solid phase particles or chemical substance as detection antibody on another antibody.Currently, about
The report for a pair of of the monoclonal antibody that can be combined simultaneously with wild type or saltant type HBsAg.Such as US2004/
0219154A1 can simultaneously with wild type and 6 kinds of saltant type hepatitis B surface antigen combined.However, still having many mutation at present
Type HBsAg fails to be identified, therefore, to in combination with wild type and the monoclonal antibody of saltant type HBsAg with very big
Demand.
Invention content
The technical problem to be solved by the present invention is to seek can in conjunction with hepatitis B surface antigen monoclonal antibody, especially together
When in conjunction with wild type and saltant type HBsAg monoclonal antibody.Therefore, in the present invention, by preparing hybridoma cell strain and list
Clonal antibody, the enough monoclonal antibodies combined with wild type or saltant type hepatitis B surface antigen of screening.Specific method is to use
Animal is immunized as antigen, using universal method in HBsAg, then the immunocyte for the animal being immunized and myeloma cell into
Row fusion prepares hybridoma；The monoclonal antibody that hybridoma secrets out of.
First, the present invention provides the hybridization for the monoclonal antibody that one kind can be produced in conjunction with hepatitis B surface antigen (HBsAg)
Tumor cell strain is preserved in Chinese Typical Representative culture center (CCTCC), deposit number CCTCCNo:C2016210, classification life
It is entitled:Hybridoma cell strain 5C7, preservation date are:On December 28th, 2016, preservation address are：Wuhan City, Hubei Province Wuchang District
In Wuhan University.
Preferably, the present invention also provides a kind of monoclonal antibody of combination hepatitis B surface antigen (HBsAg), the monoclonal is anti-
Body is CCTCC No by deposit number:The hybridoma cell strain secretion of C2016210 generates, what which can not only be specific
In conjunction with wild-type HBsAg, additionally it is possible to which, with various mutations type antigen binding, which belongs to IgG1 hypotypes, and light chain is
Kappa.
Preferably, one kind is CCTCC No by deposit number:It is anti-that the hybridoma cell strain of C2016210 generates monoclonal
The method of body, includes the following steps:
(1) screening, which obtains, stablizes hybridoma cell strain:It is taken after blood sampling measures serum titer with outsourcing antigen-immunized animal
Immune animal splenocyte is merged with myeloma cell SP2/0, and HAT is used in combination to screen to obtain stable hybridoma cell strain；
(2) positive cell strain is screened with ELISA method, clones to stablize hybridoma cell strain CCTCC No:C2016210；
(3) hybridoma cell strain is injected into the pretreated F1 mouse peritoneals of atoleine, cultivated, area's ascites,
ProtainA affinity chromatographys obtain monoclonal antibody.
Preferably, which further includes the segment and derivative of the antibody.
Preferably, the present invention also provides be CCTCC No by deposit number:The hybridoma cell strain secretion production of C2016210
Raw monoclonal antibody is in preparing the reagent of condition assessment of virus B hepatitis or hepatitis B relevant disease or kit
Using.
On the other hand, the present invention also provides one kind capable of producing the monoclonal antibody in conjunction with hepatitis B surface antigen (HBsAg)
Hybridoma cell strain, be preserved in Chinese Typical Representative culture center (CCTCC), deposit number is CCTCC No:C2016198,
Classification And Nomenclature is:Hybridoma cell strain 15G6E1, preservation date are:On December 28th, 2016, preservation address are：Wuhan City, Hubei Province
In the Wuhan University of city Wuchang District.
Preferably, the present invention also provides a kind of monoclonal antibody of combination hepatitis B surface antigen (HBsAg), the monoclonal is anti-
Body is CCTCC No by deposit number:The hybridoma cell strain secretion of C2016198 generates, what which can not only be specific
In conjunction with wild-type HBsAg, additionally it is possible to which, with various mutations type antigen binding, which belongs to IgG1 hypotypes, and light chain is
Kappa.
Preferably, it is CCTCC No that the present invention also provides one kind by deposit number:The hybridoma cell strain of C2016198
The method for generating monoclonal antibody, includes the following steps:
(1) screening, which obtains, stablizes hybridoma cell strain:It is taken after blood sampling measures serum titer with outsourcing antigen-immunized animal
Immune animal splenocyte is merged with myeloma cell SP2/0, and HAT is used in combination to screen to obtain stable hybridoma cell strain；
(2) positive cell strain is screened with ELISA method, clones to stablize hybridoma cell strain CCTCC No:C2016198：
(3) hybridoma cell strain is injected into the pretreated Balb/c mouse peritoneals of atoleine, cultivated, area's ascites,
ProtainA affinity chromatographys obtain monoclonal antibody.
Preferably, which further includes the segment and derivative of the antibody.
Preferably, the present invention also provides be CCTCC No by deposit number:The hybridoma cell strain secretion production of C2016198
Raw monoclonal antibody is in preparing the reagent of condition assessment of virus B hepatitis or hepatitis B relevant disease or kit
Using.
In addition, the present invention also provides be respectively CCTCC No by deposit number:C2016198 and CCTCC No:C2016210
Hybridoma cell strain in preparing the reagent of condition assessment of virus B hepatitis or hepatitis B relevant disease or kit
Using.
The present invention also provides be respectively CCTCC No by deposit number:C2016198 and CCTCC No:C2016210's is miscellaneous
The monoclonal antibody that tumor cell strain generates respectively is handed over to prepare the condition assessment of virus B hepatitis or hepatitis B relevant disease
Application in reagent or kit.
Preferably, it is respectively CCTCC No by deposit number:C2016198 and CCTCC No:The hybridoma of C2016210 is thin
The monoclonal antibody that born of the same parents' strain generates respectively can combine wild type and saltant type HBsAg；Wherein, the saltant type HBsAg is same
Wild type relatively in, refer not only to the variation of amino acid sequence, while further including that HBsAg structures caused by amino acid variation change
Become.
Preferably, it is respectively CCTCC No by deposit number:C2016198 and CCTCC No:The hybridoma of C2016210 is thin
The monoclonal antibody that generates respectively of born of the same parents' strain can include in conjunction with the mutational sites saltant type HBsAg：
Mutation at 112,120,129,130,131,132,133,134, G112N, P120S, Q129L, G130K,
T131N、S134F、M133S、Y134N
Mutation is at 116, T116N
Mutation is at 116 and 133, T116N and M133T
Mutation is at 116, embedded 116T
Mutation is at 118, T118A
Mutation is at 126, T126A
Mutation is at 129, Q129R
Mutation is at 129, Q129H
Mutation is at 130, G130R
It is mutated in 130 and 133, G130N and M133T
It is mutated in 133 and 134, M133T and F134L
It is mutated in 133 and 134, M133L and F134L
Mutation is at 134, F134L
Mutation is at 134, Y134N
Mutation is at 143, S143L
Mutation is at 144, D144E
Mutation is at 145, G145A.
Advantageous effect
It, can be with wild type hepatitis B surface antigen or prominent the present invention provides the antibody that can be combined with hepatitis B surface antigen
The hepatitis B surface antigen of modification combines, and has extensive binding ability, can effectively reduce false negative testing result, improves to second
The accuracy of liver hepatitis virus detection.
Description of the drawings
Fig. 1 is that antigen valence measures schematic diagram.
Specific implementation mode
Embodiment 1：Preparing hybridoma cell strain, (deposit number is respectively CCTCC No:C2016198 and CCTCC No:
C2016210)
1.1 immune animals
Animal prepares：The immune animal of hybridoma is used to prepare without specifically limited, can be used mouse, rat, hamster,
Rabbit, BALB/c mouse is as immunity inoculation object.The present embodiment selects 6-8 week old female BAl BIc/c mouse (to be purchased from Shanghai animal
Center), carry out inoculation according to the immunization protocol pre-established.
1.2 antigens prepare
Two kinds of HBsAg antigens (be purchased from outside MP Biomedicals Asia Pacific Pte Ltd) serotype is respectively
Adr and Ay, Adr are the recombinant protein of Bacillus coli expression, and Ay is to purify to obtain in HBV infection person's biological sample)
1.3 immunization protocol
The immunogenicity of specific antigen is affected by various factors, but can enhance immunogenicity with adjuvant.Commonly
Immunologic adjuvant is exactly Freund's adjuvant.It is Freund's complete adjuvant that initial immunity, which uses, and booster immunization uses Freund incomplete
Adjuvant.Typical immunization protocol depends on the repetitious stimulation of antigen, to enhance specific immune response and make its maturation.
HBsAg two kinds of antigens (Adr and Ay) 10-100 μ g are emulsified with Freund's complete adjuvant.Isometric antigen and Freund is complete
Full adjuvant is emulsified into thick emulsion together, and (total amount 0.5-1.0ml) is subcutaneously injected to mouse, rat or hamster multiple spot.
After 10-14 days, by primary immune response, weak antibody response is generated.
First time booster immunization (i.e. second of injections of antigens) uses incomplete Freund's adjuvant.Antigen and Freund are endless
The emulsion of full adjuvant is also subcutaneous multi-point injection.For the quantity of expansion of antigen reactivity B cell, need repeatedly to be reinforced
It is immune.8-10 days after second of booster immunization, animal blood serum is collected, carries out ELISA bioactivities.Serum titer is respectively in Adr
With 1 is all higher than on Ay：After 40000, it is immune that the end before cell fusion can be carried out.It is 3-5 days immune eventually, so that it may to carry out cell fusion.
1.4 cell fusion
1.4.1 the preparation of immunized B cells
The animal being immunized is put to death, spleen or lymph node are taken out under sterile working.Serum free medium cleans, removal
Serum-free DMEM culture solutions are added in extra tissue, grind, and centrifugation obtains antigen reactivity secreting type immunocyte, with no blood
Clear culture medium washing is resuspended for use.
1.4.2 prepared by culture medium and myeloma cell
Using addition 10% fetal calf serum DMEM in high glucose medium culture myeloma cell,
The preparation of feeder cells
It after mouse is put to death, is soaked in 75% alcohol and sterilizes 5min, tear mouse part skin, retain peritonaeum, with nothing
The DMEM serum free mediums (this process cannot puncture internal organ) of bacterium syringe injection 37 DEG C of preheatings of 5ml, soft mouse peritoneal,
Peritoneal fluid is sucked out in suspension abdominal cavity cell.It is resuspended with the culture solution of 15% fetal calf serum DMEM after centrifugation.
It is prepared by splenocyte
The mouse after booster immunization is put to death, sterile to isolate spleen, 75% ethanol wash, then with serum-free DMEM culture solutions
Elution, is placed on sieve and grinds, sterile centrifugation tube will be poured in splenocyte, is resuspended with serum-free DMEM culture solutions after centrifugation, meter
Number.
1.4.3 cell fusion and bed board
Before cell fusion, immunological lymphocyte is mixed according to a certain percentage with myeloma cell.Antigen reactivity point
The ratio for secreting type immunocyte and myeloma cell's mixing is 5：1-10：1.Gently remove supernatant, and handle after cell mixing centrifugation
Cell precipitation is broken up, and is placed in 37 DEG C of water-baths.1 minute inner edge mixing side be added dropwise 1ml50%PEG (molecular weight 1000 to
4000) then proceedes to mix, and the DMEM of 2ml preheatings is added in 2 minutes.It is complete that other 8ml is added in 4 minutes inner edge mixing sides
Culture medium.After centrifugation, supernatant is abandoned, complete medium gently mixing is added, is spread into the 96 hole cells trainings for adding good feeder cells in advance
It supports in plate.Each 100 microlitres of hole.50 5 × HAT of μ l are added after 37 DEG C of 5%CO2 overnight incubations.
Start within the 5th day after fusion, tissue culture plate will change weekly liquid 3 times.It fills into 100-150 microlitres and contains the fresh of 1 × HAT
Complete medium (10%FBS).7-10 days after fusion, observe that most cells culture hole all shows cell growth vigorous (16
Times object lens, under the microscope, cell size takes 1/3 visual field to be advisable 10 times of mesh), taking cells and supernatant, just ELISA is screened.
1.5 enzyme-linked immunosorbent assays (ELISA) screen
The HBsAg antigens of Adr and Ay types are diluted to the solution of 0.5 μ g/ml using 0.01M PBS (pH 7.4) respectively.So
The antigenic solution diluted is added in 96 hole elisa Plates afterwards, per 100 μ l of hole.4 DEG C of coatings are overnight.It then takes out, uses
The ELISA Plate that has been coated with of PBST washing solution (PBS solution be added polysorbas20 0.05%) washing 3 times.Then lock solution is added
(1%BSA is added in PBS solution), per 200 μ l of hole.37 DEG C are closed 30 minutes.After taking-up, confining liquid is abandoned, it is spare.
The cell conditioned medium that can be screened is taken to be added in the elisa plate closed, per 100 μ l of hole, 37 DEG C are reacted 30 points
Clock.PBST washs solution and washs 3 times after taking-up, and the sheep anti-mouse igg secondary antibody of HRP labels is then added, per 100 μ l of hole.37 DEG C anti-
It answers 30 minutes.After taking-up, PBST washs solution and washs 5 times.Then TMB colour developings are added, per 100 μ l of hole, room temperature shading reaction 10
Minute, 100 μ l, 450nm readings of terminate liquid (2M H2SO4) are then added.
1.6 subclones and cell cryopreservation
ELISA is screened the cell in the positive initial apertures of two kinds of hypotype antigens (Adr and Ay), passes through limiting dilution
Method is subcloned.96 porocyte culture plates of subclone need to add feeder cells or conditioned medium in advance to carry
For growing required growth factor.Original positive colony needs that, by multiple subclone, stably excreting antibody could be formed
Cell line.Because of some cell clones, in the lasting incubation of subclone, it may switch to negative and not secrete anti-
Body.During subclone, ELISA method is used to carry out selective mechanisms at any time, to reject the cell of unstable secretory antibody
System.
Once the hybridoma cell line of monoclonal is established, it should just freeze and build library.The cell expansion of monoclonal
Culture, makes cell density maintain 1 × 105-1×106A cell/ml.Before freezing, hybridoma should be at exponential phase.
Cell is collected by centrifugation, with filtration sterilization and is pre-chilled to 4 DEG C of culture medium containing 10%DMSO cell precipitation is resuspended, and adjust thin
Born of the same parents a concentration of 5 × 106A cell/ml.1ml cell suspensions (5 × 10 will be housed6A cell) cryopreservation tube now -70 DEG C freeze number
It, is then transferred into liquid nitrogen stored for extended periods.
The positive cell that 1.7 ELISA are filtered out is subcloned by limiting dilution assay, obtains stable cell line
CCTCC No：C2016198, the antibody which obtains are hybridoma cell strain 15G6E1.In addition, also being stablized
Cell line CCTCC No：C2016210, it is hybridoma cell strain 5C7 which, which obtains antibody,.
The mass production of 2. monoclonal antibody of embodiment
Using hybridoma mass production monoclonal antibody, mainly there are two methods of culture in vivo and in vitro.External training
It is higher to support hybridoma cost, does not use experimental animal.Generally use is that in vivo method prepares monoclonal antibody at present.And handle
The BALB/c mouse that hybridoma is injected into presensitization is intraperitoneal.
The stage for preparing monoclonal antibody ascites in vivo is as follows：
(1) atoleine pre-sensitization mouse：The volume of injecting fluid paraffin is usually 0.5ml.
(2) injection of hybridoma：Between injecting fluid paraffin and hybridoma be spaced in 7-20 days be best
, the quantity for injecting hybridoma is 0.5 × 106-2×106Most preferably.It is usually 7-10 days small after injecting hybridoma
Mouse ascites fluid can be formed.
The collection of ascites：Abdominal cavity is punctured using the enough big syringes of bore, ascites drainage is carried out to obtain abdomen by gravity
Water.Every mouse most multirow is punctured three times to harvest ascites in 7 days, and is put to death after last time is collected.The ascites of collection
1500g centrifuges ascites 10 minutes, abandons precipitation and removes the cell in ascites and grumeleuse.- 20 DEG C or -70 DEG C of freezen protectives.
Purifying, identification and the application on Test paper box of 3. monoclonal antibody of embodiment
The purifying of 3.1 monoclonal antibodies
Monoclonal antibody purification method is essentially the same with purifying immunoglobulin method from ascites.Utilize salt precipitation side
Method, molecular sieve etc..If antibody, which is mouse IgG, also can be used affinity purification method such as：Protein A、Protein G.
Antibody 15G6E1 and 5C7 in the present invention use Protein A to purify.
After ascites and sample-loading buffer mix in equal volume, after on the Protein A columns balanced with sample-loading buffer
Sample.After loading, foreign protein is eluted with 0.01M PBS solutions, stops elution when measuring the OD280 ＜ 0.03 of efflux.Again
With elution purpose antibody, efflux is collected.When collection, hydrochloric acid solution is added dropwise, adjusts antibody-solutions pH to 7-9.It measures
Stop collecting when efflux OD280≤0.1.Dialysis, centrifugation, concentrate solution to need concentration be needed for monoclonal antibody, -20
It DEG C freezes.
The identification of 3.2 monoclonal antibodies
3.2.1 monoclonal antibody subtype identification
Use the hypotype of mouse subtype identification kit (Thermo, Prod#37503) identification antibody 15G6E1 and 5C7, knot
Fruit is as shown in the table.
| Antibody | Hypotype |
| 15G6E1 | IgG1κ |
| 5C7 | IgG1κ |
Monoclonal antibody belongs to IgG1 hypotypes, and light chain is kappa.
In addition, the measurement of antigen valence is as shown in Figure 1.
3.2.2 antibody titer is identified
Two antibody 15G6E1 and 5C7 are measured respectively in HBsAg two antigen As dr and Ay using ELISA method (such as 1.5)
On potency be：
The 3.3 combination situation (application in detection kit) in wild type and saltant type antigen
It applies in kit detection, is mainly used in immunochromatographyassay assay hepatitis B surface antigen HBsAg, detection reagent
Box includes sample pad, label pad, detecting pad and absorption pad, it is also possible to including supporter, sample pad, label pad, detecting pad and suction
Receiving pad, overlap joint is pasted on a support successively, is coated with detection line area along sample flowing upstream on detecting pad, downstream is coated with
Control line area, detection line area are coated with the antibody that can be combined with hepatitis B surface antigen, and control line area is coated with sheep anti-mouse igg,
The detection antibody for being marked with colloidal gold or colored latex particle is coated in label pad.
5C7 (cell line CCTCC No:C2016210 is produced) as capture antibody, 15G6E1 (cell line CCTCC No:
C2016198 is produced) it is used as labelled antibody, control antibodies 1,2,3 and 4 are purchased from Fitzgerald, with wild type and saltant type
Combine situation as follows on antigen：
"+" indicates that testing result is the positive in table, and "-" indicates that testing result is feminine gender.
SEQUENCE LISTING
<110>Ai Bo biological medicines（Hangzhou）Co., Ltd
<120>A kind of grand Antibody preparation of monoclonal antibody of anti-hepatitis B surface antigen and screening
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 226
<212> PRT
<213>Artificial sequence
<400> 1
Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu
15
Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln
30
Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Thr
45
Thr Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His
60
Ser Pro Thr Ser Cys Pro Pro Thr Cys Pro Gly Tyr Arg Trp Met
75
Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys
90
Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro
105
Val Cys Pro Leu Ile Pro Gly Ser Ser Thr Thr Ser Thr Gly Pro
120
Cys Arg Thr Cys Thr Thr Pro Ala Gln Gly Thr Ser Met Tyr Pro
135
Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile
150
Pro Ile Pro Ser Ser Trp Ala Phe Gly Lys Phe Leu Trp Glu Trp
165
Ala Ser Ala Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val
180
Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Val Ile
195
Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Leu Ser
210
Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr
225
Ile
Claims (5)
1. a kind of hybridoma cell strain that can produce the monoclonal antibody in conjunction with hepatitis B surface antigen, deposit number are
CCTCC No:C2016210。
2. a kind of monoclonal antibody of combination hepatitis B surface antigen, which is CCTCC No by deposit number:
The hybridoma cell strain of C2016210 generates.
3. one kind is CCTCC No by deposit number:The method that the hybridoma cell strain of C2016210 generates monoclonal antibody,
Include the following steps:
(1) screening, which obtains, stablizes hybridoma cell strain:It is taken immune after blood sampling measures serum titer with outsourcing antigen-immunized animal
Animal splenocyte is merged with myeloma cell SP2/0, and HAT is used in combination to screen to obtain stable hybridoma cell strain；
(2) positive cell strain is screened with ELISA method, clones to stablize hybridoma cell strain CCTCC No:C2016210；
(3) hybridoma cell strain is injected into the pretreated F1 mouse peritoneals of atoleine, cultivated, take ascites, ProtainA parents
Monoclonal antibody is obtained with chromatography.
4. monoclonal antibody according to claim 2, which is characterized in that the monoclonal antibody further includes the segment of the antibody
And derivative.
5. being CCTCC No by deposit number:The monoclonal antibody that the hybridoma cell strain secretion of C2016210 generates is preparing second
The reagent of the condition assessment of type virus hepatitis or hepatitis B relevant disease or the application in kit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110003325A (en) * | 2019-03-31 | 2019-07-12 | 四川迈克生物新材料技术有限公司 | Hybridoma cell strain and its monoclonal antibody and application generated |
| CN111548412A (en) * | 2020-05-29 | 2020-08-18 | 杭州博岳生物技术有限公司 | Hepatitis B surface antigen monoclonal antibody, preparation method, application and amino acid sequence |
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| CN111548412A (en) * | 2020-05-29 | 2020-08-18 | 杭州博岳生物技术有限公司 | Hepatitis B surface antigen monoclonal antibody, preparation method, application and amino acid sequence |
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