CN105367652A - Monoclonal antibody resistant to HPV16L1 protein and preparation method and application thereof - Google Patents
Monoclonal antibody resistant to HPV16L1 protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an antibody or an antigen binding part of human papilloma virus high-risk carcinogenic subtype of 16 type L1protein and/or VLP. The antibody or the antigen binding part comprises a CDR1 area, a CDR2 area and a CDR3 area, shown in the SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively, of a heavy chain variable area, and a CDR1 area, a CDR2 area and a CDR3 area, shown in the SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, of a light chain variable area. Specificity combines the antibody or the antigen binding part of the HPV16L1 protein and/or VLP with other most of HPV subtypes such as HPV6, 11, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 and the like, a cross reaction is avoided, specificity is high, the antibody or the antigen binding part has the characteristics of neutralizing HPV16 pseudovirus and blocking pseudovirus infection, and affinity is high. A double-resistant interlayer ELISA detection reagent kit built through two strains of antibodies can be used for detecting existence of the HPV16L1 protein or research and development of a horizontal reagent kit, treatment of patients and passive immunity on susceptible population, and has good application prospects.
Description
Technical field
The present invention relates to field of immunology, more specifically, the present invention relates to a kind of antibody of specific binding human papillomavirus HPV16L1 albumen, preparation method and the application in vitro in detection kit thereof.
Background technology
Human papillomavirus (HPV) is one group of nonencapsulated small DNA virus, infects human skin and mucous epithelium tissue, can bring out and produces verrucous hyperplasia and even cause innocent and malignant tumour.Good pernicious difference according to bringing out pathology after infection is divided into high-risk HPV and low risk HPV.High-risk HPV infects with the generation of cervical cancer and precancerous lesion closely related, and cancer relevant to mucous membrane in addition and the generation of precancerous lesion are correlated with.HPV16 belongs to high-risk HPV, epidemiology survey and non-clinical statistical data all show HPV16 and cervical cancer and other cancers and present height correlation, the cervical cancer that HPV16 and HPV18 type brings out accounts for more than 70% of all HPV positive cervical cancers, high-risk HPV infects in relevant malignant change the highest with the sickness rate of woman uterus cancer, sickness rate in global range is only second to mammary cancer, is in the second of gynecologic malignant tumor.Prevention HPV infects and the most effective means of cervical cancer inoculates HPV vaccine exactly, the HPV vaccine gone on the market at present, comprise tetravalent vaccine " Gardasil " and the nine valency vaccines " Gardasil9 " of Merck company, and the bivalent vaccine of GSK company " Cervarix ", these three kinds of vaccines all comprise the vaccine composition of HPV16.HPV vaccine can stimulate body to produce protectiveness neutralizing antibody, significant in effective treatment and prevention HPV infection and cervical cancer.
Research finds, the monoclonal antibody with neutralising capacity substantially all identifies conformational epitope, and the monoclonal antibody of most of identification type specific conformation epi-position all has neutralising capacity.Different neutralization monoclonal antibody, by closing virus and the binding site of cell Co receptor, can stop virus infection to enter the generation of born of the same parents, is conducive to explaining that virus infection enters the critical sites of born of the same parents to the research of these monoclonal antibodies in conjunction with epi-position.Current majority is thought, in the HPV of animal infects, serum IgG (mainly and IgG) can with sufficiently high concentration through epithelium of cervix uteri, particularly in squamous, cylindrical epithelium junction, thus is combined with virion and prevents infections and occur.The mensuration of vaccine being brought out to the Neutralization antibody of generation detects the major criterion of preventative vaccine immune protective.Neutralization effect antibody detects in the immunogenicity of HPV vaccine and has important application prospect in assay.Only there is a kind of HPV16 neutralization monoclonal antibody at present---H16.V5 antibody is reported abroad to some extent, and this antibody is successfully applied to the quality control of the effective constituent in HPV16 vaccine production process.
Therefore, research and develop more polymorphic type, for the HPV16 monoclonal antibody of various different epi-position, for better research HPV virus and research and development HPV vaccine, there is vital role, and antibody is applied to diagnosis that HPV infects, prevention and therapy is also very necessary.
Summary of the invention
In order to solve above technical problem, one aspect of the present invention is to provide the antibody of a kind of specific binding human papillomavirus HPV16L1 albumen and/or VLP, includes CDR1, CDR2 and CDR3 region of the variable region of heavy chain shown in SEQIDNO.1-3 and CDR1, CDR2 and CDR3 region of the variable region of light chain shown in SEQIDNO.4-6.
Another aspect of the present invention is to provide the antibody of a kind of specific binding human papillomavirus HPV16L1 albumen and/or VLP, includes the heavy chain variable amino acid sequence as shown in SEQIDNO.7 and the chain variable region amino acid sequence as shown in SEQIDNO.8.
Antibody of the present invention is monoclonal antibody.
As preferably, in an embodiment of the invention, described antibody is the monoclonal antibody that the cell being CGMCCNO.10955 by deposit number produces.Described cell be by immunity after splenocyte and the hybridoma that obtains of myeloma cell fusion, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number is CGMCCNO.10955, and preservation date is on July 6th, 2015.
Antibody described in the present invention is type specific antibody, only can be specific in conjunction with HPV16L1 albumen and/or VLP, and all reactionless to other most HPV types, as HPV6,11,16,18,31,33,35,39,45,51,52,56,59, the types such as 68.
In the present invention, described antibody has Neutralization effect, and HPV16 type pseudovirus capable of blocking infects 293FT cell, with the effect of pseudovirus in having.
As preferably, in an embodiment of the invention, described antibody is unit price or bivalent antibody.
In the present invention, the hybridoma preparation method of the report in Nature256:495 (1975) such as Kohler can be adopted to prepare described monoclonal antibody.First immunogen (adding adjuvant time necessary) immunization mouse or other suitable host animal is used.In the present invention, described immunogen be debaryomyces hansenii cell expressing produce HPV16L1 albumen and/or this HPV16L1 albumen in vitro automatic Composition formed virus-like particle (VLP).The aminoacid sequence of its antigen is as shown in SEQIDNO.11.
The injection system of immunogen or adjuvant is generally subcutaneous multi-point injection or abdominal injection.Adjuvant can utilize freund's adjuvant (Fu Shi thoroughness adjuvant or Fu Shi imperfection adjuvant) or MPL-TDM etc.Animal, after accepting immunity, can produce the lymphocyte of the immunogenic antibody of secretion specific binding in body.Collect object lymphocyte, and with suitable fusogen (as PEG4000) by itself and myeloma cell fusion, thus obtain hybridoma (Goding, MonoclonalAntibodies:PrinciplesandPractice, pp.59-103, AcademicPress, 1996).
The hybridoma of above-mentioned preparation is inoculated in suitable substratum and grows, in described substratum, material that do not merge, parental myeloma cells growth can be suppressed containing one or more.Such as, for the parental myeloma cells lacking enzyme hypoxanthine guanine phosphotransferase (HGPRT or HPRT), add the growth that the materials such as xanthoglobulin, aminopterin-induced syndrome and thymus pyrimidine (HAT substratum) can suppress HGPRT-deficient cells in the medium.
It is high that preferred myeloma cell should have fusion rate, and antibody-secreting ability is stablized, to abilities such as HAT substratum sensitivities.Wherein, myeloma cell first-selected mouse source myelomatosis, as MOP-21 and MC-11 mouse tumor derives strain (THESalkInstituteCellDistributionCenter, SanDiego, Calif.USA), and SP-2/0 or X63-Ag8-653 cell strain (AmericanTypeC μ ltureCollection, Rockville, Md.USA).In addition, human myeloma and people mouse allogenic bone marrow tumor cell strain can also be utilized to prepare people's monoclonal antibody (Kozbor, J.Immunol., 133:3001 (1984); Brodeuretal., MonoclonalAntibodyProductionTechniquesandApplications, pp.51-63, MarcelDekker, Inc., NewYork, 1987).
The substratum of Growth of Hybridoma Cell is for detecting the generation of the monoclonal antibody for specific antigen.Following method can be used to measure the binding specificity of the monoclonal antibody of hybridoma generation: immunoprecipitation or external binding tests, as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA).Such as, the Scatchard analytical method utilizing Munson etc. to describe in Anal.Biochem.107:220 (1980) can measure the avidity of monoclonal antibody.
After determining the specificity of the antibody that hybridoma produces, avidity and reactivity, object cell strain can pass through Goding, MonoclonalAntibodies:PrinciplesandPractice, pp.59-103, AcademicPress, 1996 limiting dilution assays described carry out subcloning.Suitable substratum can be DMEM or RPMI-1640 etc.In addition, hybridoma can also the form of ascitic tumor grow in animal body.
Utilize traditional immunoglobulin purification method, as protein A Sepharose beads, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography etc., the monoclonal antibody that subcloned cells is secreted can be separated from cell culture fluid, ascites or serum, and then obtain described monoclonal antibody.
As preferably, in an embodiment of the invention, the monoclonal antibody obtained described in is 4A3.
Another aspect of the present invention there is provided the antigen-binding portion thereof of a kind of specific binding human papillomavirus HPV16L1 albumen and/or VLP, includes CDR1, CDR2 and CDR3 region of the variable region of heavy chain shown in SEQIDNO.1-3 and CDR1, CDR2 and CDR3 region of the variable region of light chain shown in SEQIDNO.4-6;
Wherein, described antigen-binding portion thereof is selected from Fab, Fab', F (ab')
2, Fd, dAb, complementary determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or double antibody.
The antigen-binding portion thereof of described specific binding HPV16L1 albumen and/or VLP can use and well known to a person skilled in the art that method obtains, and as utilized the method for chemical reagent process, or utilizes the method for protease digestion, as papoid, stomach en-etc.
Another aspect of the present invention there is provided the hybridoma that a kind of deposit number is CGMCCNO.10958, and it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).
Another aspect of the present invention there is provided a kind of test kit, and described test kit comprises antibody or its antigen-binding portion thereof of specific binding human papillomavirus HPV16L1 albumen of the present invention and/or VLP.
As preferably, in an embodiment of the invention, in described test kit, also comprise the second antibody with HPV16L1 albumen and/or VLP specific binding.
More preferably, described second antibody is the monoclonal antibody that the cell being CGMCCNO.10958 by deposit number produces, and preservation date is on July 6th, 2015.
More preferably, described second antibody is 2H8.
As preferably, in an embodiment of the invention, described second antibody adopts the method identical with the antibody of specific binding human papillomavirus HPV16 of the present invention to prepare.
Present invention also offers mentioned reagent box and detect the purposes in the L1 albumen of HPV16 in sample and/or the existence of VLP or level.
Beneficial effect of the present invention:
The antibody of specific binding human papillomavirus HPV16L1 albumen provided by the invention and/or VLP or antigen-binding portion thereof and other hypotypes of most HPV, as HPV6,11,18,31,33,35,39,45,51,52,56,58,59, the type no cross reactions such as 68, specificity is high, in having and HPV16 pseudovirus block its characteristic infected, and affinity is high.Can be used in sample, detecting the existence of HPV16L1 albumen and/or VLP or the research and development of horizontal test kit, and for the treatment of patient with carry out passive immunization to Susceptible population, have a good application prospect.
Biological deposits information:
Deposit number: CGMCCNO.10955
Preservation date: preservation date is on July 6th, 2015
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica
Classification And Nomenclature: hybridoma cell strain
Deposit number: CGMCCNO.10958
Preservation date: preservation date is on July 6th, 2015
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica
Classification And Nomenclature: hybridoma cell strain
Accompanying drawing explanation
Fig. 1 is the electron microscopic observation result of the antigen HPV6L1-VLP of preparation in the embodiment of the present invention 1 (1).
Sequence explanation
SEQIDNO.1-3 is the aminoacid sequence of the variable region of heavy chain CDR1-3 of antibody of the present invention or antigen-binding portion thereof;
SEQIDNO.4-6 is the aminoacid sequence of the variable region of light chain CDR1-3 of antibody of the present invention or antigen-binding portion thereof;
SEQIDNO.7 is the heavy chain variable amino acid sequence of antibody of the present invention;
SEQIDNO.8 is the chain variable region amino acid sequence of antibody of the present invention;
SEQIDNO.9 is the weight chain variable region nucleotide sequence of antibody of the present invention;
SEQIDNO.10 is the light chain variable region nucleotide sequence of antibody of the present invention;
SEQIDNO.11 is the aminoacid sequence of antigen HPV16L1 albumen.
Embodiment
The invention discloses the antibody of a kind of specific binding human papillomavirus HPV16L1 albumen and/or VLP, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has the implication that those skilled in the art understand usually.Further, cell cultures used herein, molecular genetics, nucleic acid chemistry, Immunology Lab operation steps are widely used conventional steps in corresponding field.Meanwhile, in order to understand the present invention better, provide definition and the explanation of relational language below.
The term " antibody " used in the present invention, refers to the immunoglobulin molecules be usually made up of two pairs of polypeptide chains (often pair has " gently " (L) chain and " weight " (H) chain).Light chain of antibody can be categorized as κ and lambda light chain.Heavy chain can be categorized as μ, δ, γ, α or ε, and respectively the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain, variable region and constant region are connected by about 12 or more amino acid whose " J " district, and heavy chain also comprises about 3 or more individual amino acid whose " D " districts.Each heavy chain is by variable region of heavy chain (V
h) and CH (C
h) composition.CH is by 3 structural domain (C
h1, C
h2 and C
h3) form.Each light chain is by variable region of light chain (V
l) and constant region of light chain (C
l) composition.Constant region of light chain is by a domain C
lcomposition.The constant region of antibody can mediated immunity sphaeroprotein and host tissue or the factor, comprises the combination of first component (C1q) of immune various cell (such as, effector cell) and classical complement system.V
hand V
ldistrict also can be subdivided into has denatured region (being called complementary determining region (CDR)), is scattered with the comparatively conservative region being called framework region (FR) therebetween.Each VH and VL is by the following order: 3 CDR and 4 FR that FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arrange from N-terminal to C-terminal form.Variable region (the V that each heavy chain/light chain is right
hand V
l) form antibody binding site respectively.。Term " antibody " does not limit by the method for any specific generation antibody.Such as, it comprises, especially, and recombinant antibodies, monoclonal antibody and polyclonal antibody.Antibody can be the antibody of different isotype, such as, and IgG (such as, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.
The term " antigen-binding portion thereof " used in the present invention, refer to the polypeptide of the fragment comprising full length antibody, the ability of its same antigen keeping specific binding full length antibody to combine, and/or compete the specific binding to antigen with full length antibody, it is also referred to as " Fab ".Usually see, (Paul, W., ed., the 2nd edition, RavenPress, N.Y. (1989), it integrates with in full herein, for all objects with it by reference for FundamentalImmunology, Ch.7.By recombinant DNA technology or by the enzymatic of complete antibody or the Fab of chemical disruption generation antibody.In some cases, Fab comprise Fab, Fab ', F (ab ')
2, Fd, Fv etc.
Wherein, term " Fab fragment " means by V
l, V
h, C
land C
hthe antibody fragment of 1 structural domain composition; Term " F (ab ')
2fragment " mean the antibody fragment of two the Fab fragments comprised by the disulfide bridge connects on hinge area.Term " Fd fragment " means by V
hand C
hthe antibody fragment of 1 structural domain composition; Term " Fv fragment " means by the V of the single armed of antibody
land V
hthe antibody fragment of structural domain composition.
In this article, unless context explicitly points out, otherwise when mentioning term " antibody ", it not only comprises complete antibody, and comprises the Fab of antibody.
The term " monoclonal antibody " used in the present invention refers to, from a segment of an antibody in the antibody molecule of a group very high homology or antibody, also namely except may except the spontaneous mutation of spontaneous appearance, and the identical antibody molecule of a group.Monoclonal antibody has high specific to the single epi-position on antigen.Polyclonal antibody is for monoclonal antibody, and it comprises at least two or more different antibodies usually, and these different antibody identify the different epi-positions on antigen usually.Monoclonal antibody can adopt the hybridoma technology of the reported first such as Kohler to obtain (Nature, 256:495,1975) usually, but recombinant DNA technology also can be adopted to obtain (as see U.S.P4,816,567).
The term " specific binding " used in the present invention refers to, two intermolecular nonrandom association reactions, as antibody and its for antigen between reaction.
The term " neutralizing antibody " used in the present invention refers to, can know or remarkable antibody or the antibody fragment reducing the virulence of target viral or pseudovirus.
The term " epi-position " used in the present invention refers to, the position that on antigen, immunoglobulin (Ig) or antibodies specific combine, can be linear epi-position, also can be the epi-position of conformation.
" monoclonal antibody " and " monoclonal antibody " that use in the present invention has identical implication and is used interchangeably.
In the present invention, amino acid represents with single-letter well known in the art and trigram abbreviation usually, such as: L-Ala can represent with A or Ala.
In the present invention, term " adjuvant " refers to, nonspecific immunity strengthening agent, after mixing with antigen can enhancing body to the immunne response of antigen or change the type of immunne response, include but not limited to that aluminium adjuvant is as aluminium hydroxide, Freund's complete adjuvant, Freund's incomplete adjuvant etc.
In the present invention: term " HPVVLP " refers to, there is the virus-like particle that the HPVL1 albumen of vivoexpression assembles, as HPV16L1-VLP just refers to, the virus-like particle that the HPV16L1 albumen of vivoexpression assembles.
In the present invention, term " HPV pseudovirus " refers to, utilize the characteristic of the nonspecific parcel nucleic acid of HPVVLP, by at cell inner expression HPVL1 and L2 albumen, and the reporter plasmid that the viral DNA of parcel endocellular liberation or external source import, thus the HPV pseudovirus formed, the conventional evaluation being used for the external neutralization of HPV vaccine.Pseudovirus used in the present invention is HPV16 type pseudovirus.
In order to make those skilled in the art understand technical scheme of the present invention better, below in conjunction with specific embodiment, the present invention is described in further detail.
Experiment material and instrument:
1. main agents, test kit and consumptive material
Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA) and PEG4000 are purchased from Sigma company.
DMEM substratum (containing 2%HAT) and foetal calf serum (FBS) purchased from American HyClone company.
The sheep anti-mouse igg Fc purchased from American JacksonImmun company of HRP coupling.
HRP substrate A and B liquid are purchased from Beijing Kwinbon Biotechnology Co., Ltd..
ProteinG affinity column post material is purchased from General Electric (China) Medical Group.
SepharoseCL-4B gel-filtration is from General Electric (China) Medical Group.
10% dimethyl sulfoxide (DMSO) protection liquid: containing 10% dimethyl sulfoxide (DMSO), 20% inactivated fetal bovine serum, 70%RPMI-1640 liquid.
20%FCS-RPMI-1640: containing penicillin 100U/ml, Streptomycin sulphate 100 μ g/ml.
Antibody subtype classification and Detection test kit (PierceRapidELISAMousemAbIsotypingKit) is purchased from Thermoscientific company.
All the other reagent are domestic analytical pure product.
2. key instrument
CO
2incubator is purchased from SANYO company.
Biohazard Safety Equipment is purchased from Baker company.
Inverted fluorescence microscope is purchased from Olympus company.
Flow cytometer is purchased from BD company.
Microplate reader is purchased from Bole's life medical product (Shanghai) Co., Ltd..
IMMAGE800 purchased from American Beckman Coulter Inc..
Ultraviolet spectrophotometer is purchased from LabTech company limited.
3. laboratory animal and cell strain
Laboratory animal is SPF level Balb/c female mice, 8-12 week age, about body weight 28g, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., raises under barrier condition.
SP2/0 cell is purchased from AbMax Biotechnology Co., Ltd..
293FT cell is purchased from Invitrogen company.
HPV16 type pseudovirus, is so kind as to give by U.S. JohnTSchiller.
The preparation of embodiment 1 hybridoma and screening
1. immunogen preparation
The immunogen that the monoclonal antibody of preparation HPV16L1 albumen uses is HPV16L1-VLP albumen, through the amino acid of in-vitro recombination expression as shown in SEQIDNO.11 according to this area ordinary method, it can become VLP by automatic Composition, can virus-like particle as shown in Figure 1 through transmission electron microscope observing, diameter is between 40-60nm, spherical in shape.The Freund's complete adjuvant (CFA) of HPV16L1-VLP albumen and equivalent or freund 's incomplete adjuvant (IFA) are mixed, utilizes ultrasonic fully emulsified, prepare corresponding immunogen.
2. animal immune
Within 0 day, by the immunogen mixed with CFA, through dorsal sc multi-point injection mouse, immunity 3 mouse, only, contained immunogen is 100 μ g to 0.12ml/, and every Balb/c mouse per injection amount is 100 μ g albumen.Within 7th day, carry out the 2nd immunity.Within 11st day, the immunogen for IFA mixing carries out the 3rd immunity, and 0.08ml/ only.Within 14th day, through mouse tail vein blood sampling, separation of serum, utilizes indirect ELISA method shown below to detect antibody titer, and as the result display in table 1, mice serum antibody titer, all at more than 1:50000, all can be used for merging.
Table 1 three mice serum antibody ELISA detected results
3. indirect elisa method
HPV16L1-VLP albumen bag by 96 orifice plates, 100ng/ hole.Negative control (NC) hole adds the PBS solution 100 μ l containing 5% skim-milk, and 4 DEG C are spent the night; Discard liquid in hole, PBS washes plate 3 times, adds the PBS solution containing 5% skim-milk, 200 μ l/ holes, and room temperature closes 1 hour; Discard liquid in hole, PBS washes plate 1 time, and (tail blood is from 1 ︰ 500 to 1 ︰ 50000 gradient dilution to add primary antibodie; Hybridoma Cell Culture supernatant 1 ︰ 1 dilutes; Mouse ascites is ten thousand gradient dilutions from 1:1000 to 1:200; Antibody purification 1 μ g/ml to 0.0005 μ g/ml gradient dilution), incubated at room 1 hour; Discard liquid in hole, PBS washes plate 3 times, adds the sheep anti-mouse igg Fc (1 ︰ 2000 dilutes) of HRP coupling, incubated at room 1 hour; PBS washes plate 5 times, adds substrate A and B liquid, 50 μ l/ holes, room temperature lucifuge reaction 20min; Add 1mol/L sulfuric acid termination reaction, upper microplate reader measures A450 value, with A
450(positive) >2A
450(NC) as judging criterion.
4. the preparation of hybridoma and screening
Get the highest mouse of antibody titer (No. 3 mouse) for merging.Merge and carry out booster immunization by the immunogen mixed with IFA in first 3 days, 0.08ml/ only, gets spleen separating Morr. cell on the 17th day after immunity and carries out cytogamy.First be separated single splenocyte, merge through PEG4000 (500g/L) and rat bone marrow tumour SP2/0 cell in 4:1 ratio, use contains the DMEM substratum of 20%FBS and 2%HAT, 37 DEG C of constant temperature culture 14 days in 96 orifice plates; Collect supernatant, screening positive clone cell, further by the DMEM substratum enlarged culturing containing 20%FBS, collect supernatant, carry out multiple sieve, the positive colony cell expansion sifted out again is cultivated, and collects supernatant, obtains positive colony through freezing and thawing, collects supernatant.Culture supernatant is detected, the cell strain of the screening supernatant positive by indirect elisa method.Result is as shown in table 2.From 700 strain cell strains, screen 550 strain positive cell strains, finally therefrom select and the cell strain of the anti-HPV16L1-VLP protein antibodies of stably excreting can amount to 40 strains.
The cell strain supernatant liquor ELISA detected result of the anti-HPV16L1 protein antibodies of table 2 stably excreting
5. in hybridoma secretion supernatant liquor, antibodies specific detects
Utilize indirect elisa method, coating antigen be respectively HPV6,11,16,18,31,33,35,39,45,51,52,56,58,59, the L1VLP albumen of 68,100ng/ hole.The secretion of this 40 strain of hybridoma is detected at the antibody of cell conditioned medium liquid, result display clone2H8, clone4A3, clone4A6, clone3A4, this six strain of hybridoma of clone3A5 and clone6A6 is only positive to HPV16L1-VLP albumen test, is feminine gender, determines that this 6 strain of hybridoma is type specific antibody to the HPV detected result of other types.
6. ascites preparation
By clone2H8, clone4A3, clone4A6, clone3A4, clone3A5 and clone6A6 six strain of hybridoma prepare ascites, every strain positive colony cell inoculates 2 mouse, ascites is collected after 10-14 days, purify ascites with ProteinG affinity column, obtain antibody purification, upper ultraviolet spectrophotometer measures A280 value, calculating antibody concentration, adjustment antibody concentration is to 2.0mg/ml, and tire by indirect elisa method detection ascites and antibody purification, result as shown in Table 3, 4.Wherein the antibody titer of this two strain of hybridoma of clone2H8 and clone4A3 secretion is the highest, and avidity is best.
Table 3 mouse ascites antibody ELISA detected result
Extent of dilution unit (μ g/ml)
Table 4 purified antibodies ELISA detected result
Extent of dilution unit (μ g/ml)
7. antibody neutralization detects
Use HPV pseudovirus neutralization test method to detect Neutralization effect and the antibody titers thereof of antibody, detect cleaning antibody pseudovirus or the remarkable ability reducing pseudovirus virulence, specific as follows:
1) be laid on by 293FT cell in 96 porocyte culture plates, every porocyte number is 1.5 × 10
4individual/100 μ l, 37 DEG C, 5% concentration C O
2cultivate 6 hours in incubator.
2) pseudovirus and antibody purification are being diluted the medium volume mixture of plate, setting up substratum to substitute control wells (negative control) and the substratum control wells (blank) of antibody simultaneously, plate 4 DEG C will diluted and place 1 hour.
3) the pseudovirus serum mixture (or substratum) drawing 100 μ l from the dilution each hole of plate has been completed in the culture plate corresponding aperture of cell adherent slowly adding in advance, Tissue Culture Plate is placed in 37 DEG C, 5% concentration C O
2cultivate 72 hours in incubator.
4) Tissue Culture Plate is placed in fluorescence microscopy Microscopic observation, infecting the hole that inhibiting rate is greater than 50% is positive hole, the hole being less than 50% is negative hole, record strong positive hole and strong negative hole, all the other hole inner cell trysinizations are also transferred in streaming pipe, flow cytomery fluorocyte is utilized to account for the ratio of total cell, calculate and infect inhibiting rate (infecting inhibiting rate=(the fluorocyte ratio of the fluorocyte ratio/negative control group of l-serologic group) × 100%), infect the dilution inverse of the highest antibody that inhibiting rate is greater than 50% (positive) as in serum and titre.
Result shows, and in 6 strain of hybridoma strains, the antibody of only clone4A3 cell strain secretion has Neutralization effect to HPV16 type pseudovirus, and 4A3 antibody titers (log10) is 4.505, and remaining hybridoma cell strain is all without Neutralization effect.
Table 56 strain monoclonal antibody is to the Neutralization effect of the HPV pseudovirus of 11 types
8. the Secondary Culture of hybridoma and preservation
Above-mentioned hybridoma is proceeded to cultivate, go down to posterity in containing the DMEM substratum of 10% foetal calf serum, after cultivating for 10 generations, hybridoma still can well-grown, stable to go down to posterity, in culture supernatant, antibody titer is at more than 1:10000.Result shows, gained hybridoma cell line can be stablized and goes down to posterity, and can secrete the monoclonal antibody of anti-HPV16 continually and steadily.
After obtaining the hybridoma of the monoclonal antibody needed for producing, a part of hybridoma to be preserved, otherwise in the process of continuous passage, sudden change or chromosomal drift motion may be produced to losing natural characteristics or losing the characteristic producing antibody; In addition in long-term culturing process, do not occur unavoidably to pollute to such an extent as to destroy, being therefore necessary to preserve cell strain.Store method is as follows: remove the old nutrient solution in Tissue Culture Flask, add 10%FCS-1640 liquid, make cell suspension.The centrifugal 10min of 1000rpm/min, removes supernatant.Cell precipitation 10% dimethyl sulfoxide (DMSO) protection liquid makes suspension, makes into 1.0 × 10
7cell/ml.Sampling, expect blue dyeing with platform, living cell counting quantity should more than 95%.Cell divides and is filled in ampulla by final asepsis injector, every bottle of 0.5ml-1.0ml, sealing by fusing ampulla.4 DEG C leave standstill and to be transferred in-70 DEG C of refrigerators 15 hours after 2 hours, finally proceed in liquid nitrogen frozen.
The qualification of embodiment 24A3 antibody
1. antibody obtains
Select BALB/c mouse of growing up, intraperitoneal inoculation pristane, every mouse 0.5ml.7-10 days pneumoretroperitoneum inoculation the 16th generation clone4A3 hybridomas, every mouse 1 × 10
6-2 × 10
6individual.Interval, after 5 days, treats that belly obviously expands, and when touching with hand, skin has tension, gathers ascites with No. 9 syringe needles.
2. the purifying of antibody
By centrifugal for ascites 13000rpm/min 30 minutes, removing cellular constituent and other throw out, collected supernatant.Carry out purifying with ProteinG affinity chromatography and SepharoseCL-4B gel-filtration, finally obtain the monoclonal antibody clone4A3 of HPV16L1 albumen, concentration is at more than 1mg/ml.
3. antibody purity detects
Antibody after purifying is carried out 12%SDS-PAGE electrophoresis, and result shows that purity is more than 95%.
4. antibody class and subgroup identification
Utilize PierceRapidELISAMousemAbIsotypingKit to carry out the detection of Subtypes to 4A3 antibody, use various immunoglobulin (Ig) hypotype (IgG1, IgG
2a, IgG
2b, IgG
3, IgA, IgM, Kappa and Lambda) the Ig hypotype of antibody that produces of the above-mentioned hybridoma of antibody test, the monoclonal antibody that result display clone4A3 cell strain produces all belongs to IgG
1type.
5. light chain of antibody and heavy chain variable region gene sequencing
Extract the mRNA of clone4A3 hybridoma, reverse transcription is cDNA, variable region universal primer is used to carry out high-fidelity PCR amplification, PCR primer fragment is inserted in carrier T and carries out determined dna sequence, the variable region gene sequence of 4A3: heavy chain is as shown in SEQIDNO.9, and light chain is as shown in SEQIDNO.10.The nucleotide sequence of acquisition is translated into aminoacid sequence.The variable region amino acid sequence of 4A3: heavy chain is as shown in SEQIDNO.7, and light chain is as shown in SEQIDNO.8.
6. antibody isotypes specific detection
As antibodies specific detected result display in 5. hybridoma secretion supernatant liquors in embodiment 1,
4A3 antibody is only positive to HPV16L1-VLP albumen test, feminine gender is to other HPV types (6,11,18,31,33,35,39,45,51,52,56,58,59,68) detected result, proves that 4A3 antibody is type monoclonal antibody specific.
7. antibody neutralization qualification
As 7. antibody neutralization detected result displays in embodiment 1,4A3 antibody is the monoclonal antibody with Neutralization effect.
The qualification of embodiment 32H8 antibody
1. antibody obtains
Except inoculation clone2H8 hybridoma, the method identical with embodiment 2-1 is utilized to carry out.
2. the purifying of antibody
Utilize the method identical with embodiment 2-1 to carry out, finally obtain the monoclonal antibody 2H8 of HPV16L1 albumen, concentration is at more than 1mg/ml.
3. antibody purity detects
Utilize the method identical with embodiment 2-1 to carry out, result shows that purity is more than 95%.
4. antibody class and subgroup identification
Utilize the method identical with embodiment 2-1 to carry out, the monoclonal antibody that result display clone2H8 cell strain produces all belongs to IgG
2btype.
5. antibody isotypes specific detection
As antibodies specific detected result display in 5. hybridoma secretion supernatant liquors in embodiment 1,
2H8 antibody is only positive to HPV16L1-VLP albumen test, feminine gender is to other HPV types (6,11,18,31,33,35,39,45,51,52,56,58,59,68) detected result, proves that 2H8 antibody is type monoclonal antibody specific.
6. antibody neutralization qualification
As 7. antibody neutralization detected result displays in embodiment 1,2H8 antibody does not have Neutralization effect.
Embodiment 4HPV16L1 antigen detection kit
The monoclonal antibody 4A3 utilizing embodiment 1 to obtain and 2H8 carries out the development of HPV16 double crush syndrome test kit, in order to detect HPV16L1 antigen levels.
1. the foundation of double antibody sandwich ELISA
1) test kit principle:
4A3 and 2H8 two strain antibody is HPV16 type specific antibody, and 4A3 strain antibody has Neutralization effect.Using 2H8 as coated antibody, using 4A3 as enzyme labelled antibody, set up double crush syndrome detection kit, when measuring samples solution or standard solution being added the pre-coated enzyme plate having 4A3 antibody, add 4A3-HRP ELIAS secondary antibody again, with nitrite ion colour developing, absorption of sample value becomes positive correlation with the content of HPV16L1 albumen in sample in linearity range, compares the content that can draw HPV16L1 albumen in sample with typical curve.Simultaneously also can according to the shade on enzyme plate, by comparing of the HPV16L1 protein standard substance solution colour with series concentration, the concentration range of HPV16L1 albumen in rough judgement sample.
2) the consisting of of test kit:
(1) be coated with the enzyme plate of coated antibody: coated antibody is 2H8 type specificity monoclonal antibody, the hybridoma cell strain clone2H8 strain being CGMCCNO.10958 by preserving number is secreted and is produced.Be buffered liquid with bag and coated antibody is diluted to 5 μ g/ml, be coated on 96 hole enzyme plates, every hole adds 100 μ l, is placed in 4 DEG C of environment and hatches 8 hours.Discard coating buffer, washings washs 3 times, pats dry.Then in enzyme plate, add confining liquid, every hole 100 μ l, 37 DEG C of environment hatch 2 hours.The liquid in hole that inclines pats dry, and preserves after dry with the vacuum-sealing of aluminium film.Wherein, bag used is buffered liquid, and to be pH value be 9.5,0.1mol/L carbonate buffer solution; Confining liquid used be containing final concentration in confining liquid be the bovine serum albumin of 8% (mass percentage), pH value is 8.6, the Veronal sodium-hydrochloride buffer of 0.1mol/L.
(2) enzyme labelled antibody: marker enzyme is horseradish peroxidase, marking method is Over-voltage protection, enzyme labelled antibody is 4A3 type specificity neutralization monoclonal antibody, and the hybridoma cell strain clone4A3 strain being CGMCCNO.10955 by preserving number is secreted and produced, and the working concentration of enzyme labelled antibody is 1 μ g/ml.
(3) standard solution: HPV16L1 protein standard substance solution, 7 bottles, concentration is respectively 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.63ng/ml, 7.813ng/ml, 3.906ng/ml, 0ng/ml.Preparation standard solution is be the phosphate buffered saline buffer of 6.5-6.7,0.1-0.2mol/L containing in the solution of preparation standard substance, final concentration is DMSO, pH of 5-10% (mass percentage).
(4) substrate nitrite ion: be made up of nitrite ion A liquid and nitrite ion B liquid, nitrite ion A liquid is hydrogen peroxide, and nitrite ion B liquid is O-Phenylene Diamine.
(5) stop buffer is 1-2mol/L sulphuric acid soln.
(6) concentrated cleaning solution: pH value is 8.5, containing the final concentration in concentrated cleaning solution be 0.05% (mass percentage) sodiumazide, final concentration in concentrated cleaning solution is the phosphate buffered saline buffer of 2.0% (mass percentage) tween 20,0.3mol/L; 40ml/ bottle, 1 bottle.
(7) the concentrated liquid that redissolves: DMSO, pH of being 5-10% (mass percentage) containing the final concentration in redissolution liquid are the phosphate buffered saline buffer of 6.5-6.7,0.1-0.2mol/L; 200ml/ bottle, 1 bottle.
(8) test kit specification sheets.
2. linearity range and test kit sensitivity checking
HPV16L1-VLP albumen is configured to the diluent of 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.63ng/ml, 7.813ng/ml, 3.906ng/ml, 0ng/ml, to determine the Monitoring lower-cut of test kit to antigen concentration, in order to the sensitivity of detection kit, and revision test 3 times, to record the sensitivity of mean value as test kit of the minimum concentration that result is the positive.The typical curve equation that duplicate detection is 3 times is y=0.0208x-0.0046, y=0.0273x-0.0044 and y=0.0242x-0.0027, R
2be respectively 0.9976,0.9933 and 0.9965, R
2all be greater than 0.99.The sensing range of HPV16L1-VLP antigen is 3.9-62.5ng/ml.The detection sensitivity of HPV16 test kit is 3.9ng/ml.
3. test kit precision checking
1) difference in precision checking-plate
By HPV16L1-VLP albumen doubling dilution to a 5 different antigen concentration, each antigen concentration does 5 multiple holes (n=6), totally 30 samples, by the variation coefficient (Coefficientofvariation in the plate that calculates 30 samples, CV) determine difference in plate, thus Precision Analyze is carried out to this test kit.As shown in Table 5, in detecting for 30 times, in plate, CV (%) value is all less than 15% to result, shows that in this test kit plate, precision is good.
The variation coefficient in table 6HPV16 test kit plate
2) difference between precision checking-plate
By HPV16L1-VLP albumen doubling dilution to a 5 different antigen concentration, each antigen concentration does 5 multiple holes (n=6), totally 30 samples, by difference between variation coefficient determination plate between the plate that calculates 30 samples, thus carry out Precision Analyze to this test kit.As shown in Table 6, in detecting for 30 times, between plate, CV (%) value is all less than 15% to result, shows that between this detection kit plate, precision is good.
The variation coefficient between table 7HPV16 test kit plate
4. test kit accuracy validation
HPV16L1-VLP albumen is diluted to high, medium and low 3 concentration (40ng/ml, 20ng/ml and 10ng/ml), 2 multiple holes done by each sample, and the calculation sample rate of recovery, analyzes the accuracy of the method.Result display recovery of standard addition (95%CI) is between 80.27%--96.93%, and the test kit rate of recovery is good.
5. test kit preservation period test
Test kit preservation condition is 2-8 DEG C, and through the mensuration of 6 months, the maximum absorbance value (zero standard) of test kit, 50% inhibition concentration, HPV16L1 albumen practical measurement value were all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 6 days under 37 DEG C of conditions of preserving by test kit, carry out accelerated aging tests, result shows that this test kit indices meets the requirements completely.Consider that the freezing situation of test kit occurs, test kit is put into-20 DEG C of refrigerator freezings 5 days, measurement result also shows that test kit indices is completely normal.Can show that test kit at least can preserve more than 6 months at 2-8 DEG C from above result.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (12)
1. the antibody of a specific binding human papillomavirus HPV16L1 albumen and/or VLP, it is characterized in that, include CDR1, CDR2 and CDR3 region of the variable region of heavy chain shown in SEQIDNO.1-3 and CDR1, CDR2 and CDR3 region of the variable region of light chain shown in SEQIDNO.4-6.
2. an antibody of specific binding human papillomavirus HPV16L1 albumen and/or VLP, is characterized in that, includes the heavy chain variable amino acid sequence as shown in SEQIDNO.7 and the chain variable region amino acid sequence as shown in SEQIDNO.8.
3. the antigen-binding portion thereof of a specific binding human papillomavirus HPV16L1 albumen and/or VLP, it is characterized in that, include CDR1, CDR2 and CDR3 region of the variable region of heavy chain shown in SEQIDNO.1-3 and CDR1, CDR2 and CDR3 region of the variable region of light chain shown in SEQIDNO.4-6;
Wherein, described antigen-binding portion thereof is selected from Fab, Fab', F (ab')
2, Fd, dAb, complementary determining region fragment, single-chain antibody, humanized antibody, chimeric antibody or double antibody.
4. the antibody as described in claim 1-2, is characterized in that, described antibody includes the weight chain variable region nucleotide sequence as shown in SEQIDNO.9 and the light chain variable region nucleotide sequence as shown in SEQIDNO.10.
5. the antibody as described in claim 1-2, is characterized in that, described antibody be by immunity after splenocyte and the monoclonal antibody that produces of the hybridoma cell line that obtains of myeloma cell fusion.
6. the antibody according to claim 1-2, is characterized in that, described antibody is the monoclonal antibody that the cell being CGMCCNO.10955 by deposit number produces.
7. the antibody according to claim 1-2, is characterized in that, described antibody is the monoclonal antibody with Neutralization effect.
8. the antibody according to claim 1-2, is characterized in that, described antibody is unit price or bivalent antibody.
9. a test kit, is characterized in that, described test kit comprises as the antibody in claim 1-2 as described in any one or antigen-binding portion thereof as claimed in claim 3.
10. test kit according to claim 9, is characterized in that, described test kit also comprises the second antibody with HPV16L1 albumen and/or VLP specific binding.
11. test kits according to claim 10, is characterized in that, described second antibody is the monoclonal antibody that the cell being CGMCCNO.10958 by deposit number produces.
12. purposes of test kit in the existence detecting HPV16L1 albumen and/or VLP in sample or level as claimed in claim 9.
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| CN115925888B (en) * | 2022-11-26 | 2023-06-27 | 山西省中医药研究院（山西省中医院） | Alpaca-derived nano-antibody specifically combined with human papilloma virus HPV and application thereof |
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