CN105367652B - A kind of monoclonal antibody, preparation method and the application of anti-HPV16 L1 albumen - Google Patents
A kind of monoclonal antibody, preparation method and the application of anti-HPV16 L1 albumen Download PDFInfo
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Abstract
The present invention provides a kind of high-risk carcinogenic hypotypes of human papilloma virus --- the antibody or antigen-binding portion thereof of 16 type L1 albumen and/or VLP, include heavy chain variable region shown in SEQ ID NO.1-3 the region CDR1, CDR2 and CDR3 and SEQ ID NO.4-6 shown in light chain variable region the region CDR1, CDR2 and CDR3.The antibody or antigen-binding portion thereof and other major part HPV hypotypes of specific binding HPV16L1 albumen and/or VLP provided by the invention, the type no cross reaction of such as HPV6,11,18,31,33,35,39,45,51,52,56,58,59,68, it is specific high, with the characteristic for neutralizing HPV16 pseudovirus and it being blocked to infect, and compatibility is high.The double crush syndrome detection kit established using two strain antibodies can be used for the presence of HPV16L1 Protein Detection or the research and development of horizontal kit, and carry out passive immunity for the treatment of patient and to Susceptible population, have a good application prospect.
Description
Technical field
The present invention relates to field of immunology, more particularly it relates to a kind of specific binding human papilloma virus
Antibody, preparation method and its application in detection kit in vitro of HPV16L1 albumen.
Background technique
Human papilloma virus (HPV) is one group of nonencapsulated small DNA virus, infects application on human skin and mucous epithelium tissue, can
It induces and generates verrucous hyperplasia or even initiation innocent and malignant tumour.It is divided into high-risk-type according to the good pernicious difference for inducing lesion after infection
HPV and low risk HPV.High-risk HPV infection is closely related with the generation of cervical carcinoma and precancerous lesion, additionally related to mucous membrane
Cancer and precancerous lesion generation it is related.HPV16 belongs to high-risk HPV, and epidemiological survey and non-clinical statistical data are shown
HPV16 and cervical carcinoma and the presentation of other cancers are highly relevant, and the cervical carcinoma that HPV16 and HPV18 type induces accounts for all HPV positives palace
70% or more of neck cancer, high-risk HPV infect the disease incidence highest in relevant malignant change with woman uterus cancer, global range
Interior disease incidence is only second to breast cancer, is in the second of gynecologic malignant tumor.Prevent HPV infection and the most effective side of cervical carcinoma
Method is exactly to be inoculated with HPV vaccine, the HPV vaccine listed at present, tetravalent vaccine " Gardasil " and nine valences including Merck company
Vaccine " Gardasil9 " and the bivalent vaccine " Cervarix " of GSK company, these three vaccines all include HPV16 vaccine at
Point.HPV vaccine can stimulate body to generate protectiveness neutralizing antibody, have effectively treating and preventing in HPV infection and cervical carcinoma
It is significant.
The study found that the monoclonal antibody with neutralising capacity all identifies comformational epitope, and most of identification type specificities substantially
The monoclonal antibody of comformational epitope all has neutralising capacity.The knot that different neutralization monoclonal antibodies pass through closing virus and cell Co receptor
Coincidence point, can prevent virus infection from entering the generation of born of the same parents, be conducive to explain that virus infection enters to the research of these monoclonal antibody combination epitopes
The critical sites of born of the same parents.Majority is thought at present, and in the HPV infection of animal, serum IgG (mainly neutralization IgG) can be with sufficiently high
Concentration pass through epithelium of cervix uteri, especially in squamous, columnar epithelium junction, thus in conjunction with virion and the hair that prevents infections
It is raw.It is the major criterion for detecting preventative vaccine immune protective to the measurement that vaccine induces the Neutralization antibody generated.In
It is with important application prospects in the immunogenicity detection of HPV vaccine and assay with active antibodies.It is only a kind of at present
HPV16 neutralization monoclonal antibody --- H16.V5 antibody is reported that the antibody is successfully applied to HPV16 vaccine production process in foreign countries
In effective component quality control.
Therefore, research and develop more polymorphic type, for the HPV16 monoclonal antibody of various different epitopes, for preferably study HPV viruse with
And research and development HPV vaccine plays a significant role, and antibody is applied to the diagnosis of HPV infection, is prevented and treated to be also very necessary.
Summary of the invention
In order to solve the above technical problems, it is an aspect of the invention to provide a kind of specific binding human papilloma virus
The antibody of HPV16L1 albumen and/or VLP, include heavy chain variable region shown in SEQ ID NO.1-3 CDR1, CDR2 and
The region CDR1, CDR2 and CDR3 of light chain variable region shown in the region CDR3 and SEQ ID NO.4-6.
Another aspect of the present invention is to provide a kind of specific binding human papilloma virus HPV16L1 albumen and/or VLP
Antibody, comprising just like heavy chain variable amino acid sequence shown in SEQ ID NO.7 and the light chain as shown in SEQ ID NO.8
Variable region amino acid sequence.
Antibody of the present invention is monoclonal antibody.
Preferably, in an embodiment of the invention, it is CGMCC that the antibody, which is by deposit number,
The monoclonal antibody that the cell of NO.10955 generates.The cell be by after immune splenocyte and myeloma cell's fusion
Obtained hybridoma is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC),
Deposit number is CGMCC NO.10955, and the deposit date is on July 6th, 2015.
Heretofore described antibody is type specific antibody, is only capable of the combination HPV16L1 albumen and/or VLP of specificity,
And it is reactionless to other most HPV types, such as HPV6,11,16,18,31,33,35,39,45,51,52,56,59,68
Equal types.
In the present invention, the antibody has neutralization activity, and HPV16 type pseudovirus can be blocked to infect 293FT cell, had
Neutralize the effect of pseudovirus.
Preferably, in an embodiment of the invention, the antibody is unit price or bivalent antibody.
In the present invention, the hybridoma preparation side that can be reported in Nature 256:495 (1975) using Kohler etc.
Method prepares the monoclonal antibody.First with immunogene (adding adjuvant when necessary) inoculation mouse or other suitable
Host animal.In the present invention, the immunogene is the HPV16L1 albumen and/or be somebody's turn to do that the expression of Hansenula yeast cell generates
HPV16L1 albumen assembles the virus-like particle (VLP) of formation automatically in vitro.The amino acid sequence of its antigen such as SEQ ID
Shown in NO.11.
The injection system of immunogene or adjuvant is usually subcutaneous multi-point injection or intraperitoneal injection.Adjuvant can use Freund assistant
Agent (Freund completeness adjuvant or Freund imperfection adjuvant) or MPL-TDM etc..Animal can generate in vivo after receiving to be immunized
The lymphocyte of the antibody of secretion specific binding immunogene.Purpose lymphocyte is collected, and (such as with suitable fusion agent
PEG4000) it is merged with myeloma cell, thus obtain hybridoma (Goding, Monoclonal Antibodies:
Principles and Practice, pp.59-103, Academic Press, 1996).
The hybridoma of above-mentioned preparation is inoculated into suitable culture medium and is grown, contains one in the culture medium
Kind or a variety of substances for being able to suppress parental myeloma cells growth that is not merging.For example, for lacking enzyme hypoxanthine guanine
The parental myeloma cells of phosphotransferase (HGPRT or HPRT) add hypoxanthine, aminopterin-induced syndrome and thymus gland in the medium
The substances such as pyrimidine (HAT culture medium) will can inhibit the growth of HGPRT- deficient cells.
Preferred myeloma cell should have fusion rate high, and antibody-secreting ability is stablized, sensitive to HAT culture medium to wait energy
Power.Wherein, myeloma cell's first choice source of mouse myeloma, such as derivative strain (the THE Salk of MOP-21 and MC-11 mouse tumor
Institute Cell Distribution Center, San Diego, Calif.USA) and SP-2/0 or X63-Ag8-
653 cell strains (American Type C μ lture Collection, Rockville, Md.USA).Furthermore it is also possible to utilize
Human myeloma and people's mouse allogenic bone marrow tumor cell strain prepare people's monoclonal antibody (Kozbor, J.Immunol., 133:3001 (1984)；
Brodeur et al., Monoclonal Antibody Production Techniques and Applications,
Pp.51-63, Marcel Dekker, Inc., New York, 1987).
The culture medium of Growth of Hybridoma Cell is used to detect the generation of the monoclonal antibody for specific antigen.Following side can be used
Method measures the binding specificity of the monoclonal antibody of hybridoma generation: immunoprecipitation or it is external combine test, as radio-immunity tries
Test (RIA), enzyme-linked immunosorbent assay (ELISA).For example, using Munson etc. in Anal.Biochem.107:220 (1980)
Described in Scatchard analytic approach can measure the affinity of monoclonal antibody.
After determining the specificity of antibody of hybridoma generation, affinity and reactivity, aim cell strain can pass through
Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103, Academic
The limiting dilution assay of Press, 1996 descriptions carry out subcloning.Suitable culture medium can be DMEM or RPMI-1640 etc..Separately
Outside, hybridoma can be grown in animal body in the form of ascites tumor.
Using traditional immunoglobulin purification method, such as protein A agarose gel, hydroxyapatite chromatography, gel electricity
Swimming, dialysis or affinity chromatography etc. can isolate the monoclonal antibody that subcloned cells are secreted from cell culture fluid, ascites or serum
Come, and then obtains the monoclonal antibody.
Preferably, in an embodiment of the invention, the obtained monoclonal antibody is 4A3.
Another aspect of the present invention there is provided a kind of specific binding human papilloma virus HPV16L1 albumen and/or
The antigen-binding portion thereof of VLP, include heavy chain variable region shown in SEQ ID NO.1-3 the region CDR1, CDR2 and CDR3 and
The region CDR1, CDR2 and CDR3 of light chain variable region shown in SEQ ID NO.4-6；
Wherein, the antigen-binding portion is selected from Fab, Fab', F (ab')2, it is Fd, dAb, complementary determining region segment, single-stranded
Antibody, humanized antibody, chimeric antibody or double antibody.
The antigen-binding portion thereof of the specific binding HPV16L1 albumen and/or VLP can be used those skilled in the art public
The method known obtains, such as using the method for chemical reagent processing, or using the method for protease digestion, such as papain, stomach
Protease etc..
Another aspect of the present invention is the hybridoma of CGMCC NO.10958 there is provided a kind of deposit number,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).
For another aspect of the present invention there is provided a kind of kit, the kit includes specific binding of the invention
The antibody or its antigen-binding portion thereof of human papilloma virus HPV16L1 albumen and/or VLP.
Preferably, in an embodiment of the invention, in the kit also comprising with HPV16L1 albumen and/
Or the secondary antibody of VLP specific binding.
It is highly preferred that the secondary antibody is that the monoclonal generated by the cell that deposit number is CGMCC NO.10958 resists
Body, the deposit date is on July 6th, 2015.
It is highly preferred that the secondary antibody is 2H8.
Preferably, in an embodiment of the invention, the secondary antibody uses and specificity of the present invention
In conjunction with the identical method preparation of antibody of human papilloma virus HPV16.
The present invention also provides mentioned reagent boxes in test sample presence or the water of the L1 albumen of HPV16 and/or VLP
Purposes in flat.
Beneficial effects of the present invention:
Antibody or the antigen binding of specific binding human papilloma virus HPV16L1 albumen and/or VLP provided by the invention
Part and other hypotypes of most HPV, such as the type of HPV6,11,18,31,33,35,39,45,51,52,56,58,59,68
No cross reaction, specificity is high, has the characteristic for neutralizing HPV16 pseudovirus and it being blocked to infect, and compatibility is high.It can be used for sample
The presence of HPV16L1 albumen and/or VLP or the research and development of horizontal kit are detected in product, and for the treatment of patient and to easy
Touching group carries out passive immunity, has a good application prospect.
Biological deposits information:
Deposit number: CGMCC NO.10955
Preservation date: the deposit date is on July 6th, 2015
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address: north
No. 3 Institute of Microorganism, Academia Sinica, institute of the Chaoyang District Jing Shi North Star West Road 1
Classification naming: hybridoma cell strain
Deposit number: CGMCC NO.10958
Preservation date: the deposit date is on July 6th, 2015
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address: north
No. 3 Institute of Microorganism, Academia Sinica, institute of the Chaoyang District Jing Shi North Star West Road 1
Classification naming: hybridoma cell strain
Detailed description of the invention
The Electronic Speculum that Fig. 1 is the antigen HPV 6L1-VLP prepared in the embodiment of the present invention 1 (1) observes result.
Sequence explanation
SEQ ID NO.1-3 is the amino acid sequence of the heavy chain variable region CDR1-3 of antibody of the present invention or antigen-binding portion thereof
Column；
SEQ ID NO.4-6 is the amino acid sequence of the light chain variable region CDR1-3 of antibody of the present invention or antigen-binding portion thereof
Column；
SEQ ID NO.7 is the heavy chain variable amino acid sequence of antibody of the present invention；
SEQ ID NO.8 is the chain variable region amino acid sequence of antibody of the present invention；
SEQ ID NO.9 is the weight chain variable region nucleotide sequence of antibody of the present invention；
SEQ ID NO.10 is the light chain variable region nucleotide sequence of antibody of the present invention；
SEQ ID NO.11 is the amino acid sequence of antigen HPV16L1 albumen.
Specific embodiment
The invention discloses a kind of specific binding human papilloma virus HPV16L1 albumen and/or the antibody of VLP, abilities
Field technique personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replaces
Change and change apparent to those skilled in the art, they are considered as being included in the present invention.Side of the invention
Method and application be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, spirit and
To method described herein and application is modified or appropriate changes and combinations in range, carry out implementation and application the technology of the present invention.
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The normally understood meaning of personnel institute.Also, cell culture used herein, molecular genetics, nucleic acid chemistry, immunological experiment
Room operating procedure is widely used conventional steps in corresponding field.Meanwhile for a better understanding of the present invention, it is provided below
The definition and explanation of relational language.
Term used in the present invention " antibody ", refer to usually by two pairs of polypeptide chains (it is each pair of have " light " (L) chain and
One " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can be classified as μ, δ,
γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.In light chain and heavy chain, it can be changed
Area is connected with constant region by area " J " of about 12 or more amino acid, and heavy chain also includes about 3 or more amino acid
The area " D ".Each heavy chain is by heavy chain variable region (VH) and heavy chain constant region (CH) composition.Heavy chain constant region is by 3 structural domain (CH1、
CH2 and CH3) it forms.Each light chain is by light chain variable region (VL) and constant region of light chain (CL) composition.Constant region of light chain is by a structure
Domain CLComposition.The constant region of antibody can mediated immunity globulin and host tissue or the factor, the various cells including immune system
The combination of the first component (C1q) of (for example, effector cell) and classical complement system.VHAnd VLArea, which can be also subdivided into, has height
The region (referred to as complementary determining region (CDR)) of denaturation is interspersed with the more conservative region for being known as framework region (FR).Each VH and
VL is by the following order: 3 CDR that FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are arranged from amino terminal to carboxyl terminal
It is formed with 4 FR.Variable region (the V of each heavy chain/light chain pairHAnd VL) it is respectively formed paratope.Term " antibody " not by
Any specific method limitation for generating antibody.Such as comprising, particularly, recombinant antibodies, monoclonal antibody and Anti-TNF-α
Body.Antibody can be the antibody of different isotypes, for example, IgG (for example, IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1,
IgA2, IgD, IgE or IgM antibody.
Term used in the present invention " antigen-binding portion thereof " refers to the polypeptide of the segment comprising full length antibody, keeps
The ability for the same antigen that specific binding full length antibody is combined, and/or tied with specificity of the full length antibody competition to antigen
It closes, also referred to as " antigen-binding fragment ".Usually referring to, Fundamental Immunology, Ch.7 (Paul, W., ed.,
Second edition, Raven Press, N.Y. (1989), is incorporation by reference, for all purposes in its entirety.It can pass through
Recombinant DNA technology or the antigen-binding fragment that antibody is generated by the enzymatic or chemical disruption of complete antibody.In some cases,
Antigen-binding fragment includes Fab, Fab ', F (ab ')2, Fd, Fv etc..
Wherein, term " Fab segment " means by VL、VH、CLAnd CHThe antibody fragment of 1 structural domain composition；Term " F (ab ')2
Segment " means the antibody fragment of two Fab segments comprising connecting by the disulphide bridges on hinge area.Term " Fd segment " means
By VHAnd CHThe antibody fragment of 1 structural domain composition；Term " Fv segment " means by the V of the single armed of antibodyLAnd VHStructural domain composition
Antibody fragment.
It herein, unless clearly indicated by the context, not only include complete anti-otherwise when referring to term " antibody "
Body, and the antigen-binding fragment including antibody.
Term used in the present invention " monoclonal antibody " refers to, one in the antibody molecule from a group very high homology
One segment of antibody or antibody, namely in addition to the natural mutation of possible spontaneous appearance, the identical antibody molecule of a group.It is single
It is anti-that there is high specific to the single epitope on antigen.Polyclonal antibody is usually wrapped for monoclonal antibody
Different epitopes containing at least two kinds of or more different antibodies, on these the generally recognized antigens of different antibody.Monoclonal antibody
The hybridoma technology that Kohler etc. is reported for the first time usually can be used and obtain (Nature, 256:495,1975), but weight can also be used
Group DNA technique obtains (such as referring to U.S.P 4,816,567).
Term used in the present invention " specific binding " refers to, two intermolecular nonrandom association reactions, such as antibody
Reaction between its targeted antigen.
Term used in the present invention " neutralizing antibody " refers to, can understand or significantly reduce the poison of target viral or pseudovirus
The antibody or antibody fragment of power.
Term used in the present invention " epitope " refers to, the position that immunoglobulin or antibody specificity combine on antigen,
It can be linear epitope, be also possible to the epitope of conformation.
" monoclonal antibody " and " monoclonal antibody " used in the present invention have the same meaning and are used interchangeably.
In the present invention, amino acid is usually indicated with single-letter well known in the art and trigram abbreviation, such as: alanine
It can be indicated with A or Ala.
In the present invention, term " adjuvant " refers to, nonspecific immunity strengthening agent can enhance body fight after mixing with antigen
The type of former immune response or change immune response, including but not limited to aluminium adjuvant such as aluminium hydroxide, Freund's complete adjuvant, not
Family name's Freund's incomplete adjuvant etc..
In the present invention: term " HPV VLP " refers to there is virus-like made of the HPV L1 albumen assembling of vivoexpression
Grain, refers to if HPV 16L1-VLP, virus-like particle made of the HPV16L1 albumen assembling of vivoexpression.
In the present invention, term " HPV pseudovirus " refers to, using the characteristic of the nonspecific package nucleic acid of HPV VLP, passes through
HPV L1 and L2 albumen is expressed in the cell, and wraps up the viral DNA of endocellular liberation or the reporter plasmid of external source importing, thus
The HPV pseudovirus of formation, the evaluation conventionally used for the external neutralization of HPV vaccine.Pseudovirus used in the present invention is HPV
16 type pseudovirus.
It is right combined with specific embodiments below in order to make those skilled in the art more fully understand technical solution of the present invention
The present invention is described in further detail.
Experimental material and instrument:
1. main agents, kit and consumptive material
Freund's complete adjuvant (CFA), incomplete Freund's adjuvant (IFA) and PEG4000 are purchased from Sigma company.
DMEM culture medium (containing 2%HAT) and fetal calf serum (FBS) are purchased from U.S. HyClone company.
The sheep anti-mouse igg Fc of HRP coupling is purchased from U.S. Jackson Immun company.
HRP substrate A and B liquid are purchased from Beijing Kwinbon Biotechnology Co., Ltd..
Protein G affinity column column material is purchased from General Electric (China) Medical Group.
Sepharose CL-4B gel filtration is from General Electric (China) Medical Group.
10% dimethyl sulfoxide protects liquid: containing 10% dimethyl sulfoxide, 20% inactivated fetal bovine serum, 70%RPMI -1640
Liquid.
20%FCS -1640 culture medium: 100U/ml containing penicillin, 100 μ g/ml of streptomysin.
Antibody subtype classification and Detection kit (Pierce Rapid ELISA Mouse mAb Isotyping Kit) purchase
From Thermo scientific company.
Remaining reagent is domestic analysis net product.
2. key instrument
CO2Incubator 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..
IMMAGE 800 is purchased from Beckman Coulter Inc., the U.S..
Ultraviolet specrophotometer is purchased from LabTech Co., Ltd.
3. experimental animal and cell strain
Experimental animal is SPF grades of Balb/c female mices, and 8-12 week old, weight 28g or so, purchased from Beijing, dimension tonneau China is real
Zoo technical Co., Ltd is tested, is raised under the conditions of barrier.
2/0 cell of SP is purchased from AbMax Biotechnology Co., Ltd..
293FT cell is purchased from Invitrogen company.
HPV16 type pseudovirus is given by U.S. John T Schiller.
The preparation and screening of 1 hybridoma of embodiment
1. prepared by immunogene
Preparing immunogene used in the monoclonal antibody of HPV16L1 albumen is HPV16L1-VLP albumen, is according to ability
Domain conventional method can be assembled into VLP through in-vitro recombination expression amino acid as shown in SEQ ID NO.11 automatically, through transmission electricity
The visible virus-like particle shown in FIG. 1 of sem observation, diameter are spherical in shape between 40-60nm.By HPV16L1-VLP albumen with etc.
The Freund's complete adjuvant (CFA) or freund 's incomplete adjuvant (IFA) of amount mix, fully emulsified using ultrasound, and preparation is exempted from accordingly
Epidemic focus.
2. animal immune
With the immunogene mixed with CFA, through dorsal sc multi-point injection mouse, be immunized 3 mouse within 0 day, 0.12ml/ only,
Contained immunogene is 100 μ g, and every Balb/c mouse per injection amount is 100 μ g albumen.It carries out within 7th day being immunized for the 2nd time.11st
It carries out the 3rd time for the immunogene of IFA mixing and is immunized, and 0.08ml/ is only.It takes a blood sample through mouse tail vein within 14th day, separates serum,
Antibody titer is detected using indirect ELISA method as shown below, as in table 1 the results show that mice serum antibody titer is equal
In 1:50000 or more, it is used equally for merging.
1 three mice serum antibody ELISA testing results of table
3. indirect elisa method
HPV16L1-VLP albumen is coated with 96 orifice plates, the hole 100ng/.The negative control hole (NC) is added containing 5% skimmed milk power
100 μ l of PBS solution, 4 DEG C overnight；Liquid in hole is discarded, PBS board-washing 3 times, the PBS solution for containing 5% skimmed milk power, 200 μ are added
The hole l/, room temperature are closed 1 hour；Discard liquid in hole, PBS board-washing 1 time, primary antibody is added, and (tail blood is from 1 ︰, 500 to 1 ︰, 50000 gradient
Dilution；1 ︰ 1 of Hybridoma Cell Culture supernatant dilution；Mouse ascites ten thousand gradient dilution from 1:1000 to 1:200；1 μ g/ of antibody purification
Ml is to 0.0005 μ g/ml gradient dilution), it is incubated at room temperature 1 hour；Liquid in hole is discarded, PBS board-washing 3 times, HRP coupling is added
Sheep anti-mouse igg Fc (1 ︰ 2000 dilution) is incubated at room temperature 1 hour；PBS board-washing 5 times, substrate A and B liquid, 50 holes μ l/, room temperature is added
It is protected from light 20min；1mol/L sulfuric acid is added and terminates reaction, upper microplate reader measures A450 value, with A450(positive) > 2A450(NC)
As judgment criteria.
4. the preparation and screening of hybridoma
Take the highest mouse of antibody titer (No. 3 mouse) for merging.Fusion first 3 days with the immunogene mixed with IFA into
Row booster immunization, 0.08ml/ only, take spleen separating Morr. cell to carry out cell fusion on the 17th day after immune.Single spleen is separated first
Cell is merged through PEG4000 (500g/L) with 2/0 cell of rat bone marrow tumour SP in 4:1 ratio, with containing 20%FBS's and 2%HAT
DMEM culture medium, 37 DEG C constant temperature incubation 14 days in 96 orifice plates；Collect supernatant, screening positive clone cell, further with containing
The DMEM culture medium of 20%FBS expands culture, collects supernatant, carries out secondary screening, and the positive colony cell expansion culture that secondary screening goes out is received
Collect supernatant, frozen and recovered and obtain positive colony, collects supernatant.Culture supernatant, screening supernatant sun are detected by indirect elisa method
The cell strain of property.The results are shown in Table 2.550 plants of positive cell strains are screened from 700 plants of cell strains, finally therefrom select energy
The cell strain of enough anti-HPV16L1-VLP protein antibodies of stably excreting is 40 plants total.
The cell strain supernatant ELISA testing result of the anti-HPV16L1 protein antibodies of 2 stably excreting of table
5. hybridoma secretes antibody specificity detection in supernatant
Using indirect elisa method, coating antigen be respectively HPV6,11,16,18,31,33,35,39,45,51,52,56,58,
59,68 L1VLP albumen, the hole 100ng/.40 strain of hybridoma secretion is detected in the antibody of cell supernatant, is tied
Fruit shows this six strain of hybridoma of clone2H8, clone4A3, clone4A6, clone3A4, clone3A5 and clone6A6
It is only the positive to the reaction of HPV16L1-VLP albumen, the HPV testing result to other types is feminine gender, determines 6 plants of hybridomas
Cell is type specific antibody.
6. prepared by ascites
Six plants of hybridomas of clone2H8, clone4A3, clone4A6, clone3A4, clone3A5 and clone6A6 are thin
Born of the same parents prepare ascites, and 2 mouse of every plant of positive colony cell inoculation collect ascites after 10-14 days, pure with Protein G affinity column
Change ascites, obtain antibody purification, upper ultraviolet specrophotometer measures A280 value, and calculating antibody concentration, adjustment antibody concentration is extremely
2.0mg/ml detects ascites and antibody purification potency by indirect elisa method, as a result as shown in Table 3, 4.Wherein clone2H8 and
The antibody titer highest of this two strain of hybridoma of clone4A3 secretion, affinity are best.
3 mouse ascites antibody ELISA testing result of table
Dilution unit (μ g/ml)
4 purified antibodies ELISA testing result of table
Dilution unit (μ g/ml)
7. antibody neutralization detects
Using the neutralization activity and its antibody titer of HPV pseudovirus neutralization test method detection antibody, cleaning antibody is detected
Pseudovirus or the ability for significantly reducing pseudovirus virulence, specific as follows:
1) 293FT cell is laid in 96 porocyte culture plates, every hole cell number is 1.5 × 104A/100 μ l, 37 DEG C,
5% concentration C O2It is cultivated 6 hours in incubator.
2) by pseudovirus and antibody purification in the dilution medium volume mixture of plate, while the control of culture medium substitution antibody is set up
Hole (negative control) and culture medium control wells (blank control) will dilute 4 DEG C of plate and place 1 hour.
3) from dilution each hole of plate in draw 100 μ l pseudovirus serum mixture (or culture medium) it is adherent be slowly added into advance
It has completed in the culture plate corresponding aperture of cell, tissue culture plate is placed in 37 DEG C, 5% concentration C O2It is cultivated 72 hours in incubator.
4) tissue culture plate is placed in fluorescence microscopy under the microscope, hole of the infection inhibiting rate greater than 50% is positive hole, small
It is negative hole in 50% hole, records strong positive Kong Yuqiang negative hole, remaining hole inner cell pancreatin digests and is transferred to streaming
Guan Zhong accounts for the ratio of total cell using flow cytomery fluorecyte, calculates infection inhibiting rate (infection inhibiting rate=(l-
Fluorecyte ratio/negative control group fluorecyte ratio of serum group) × 100%), infection inhibiting rate is greater than 50% (sun
Property) highest antibody dilution dilution factor of the inverse as serum.
The results show that only the antibody of clone4A3 cell strain secretion has HPV16 type pseudovirus in 6 strain of hybridoma strains
There is neutralization activity, 4A3 antibody titer (log10) is 4.505, remaining hybridoma cell strain is without neutralization activity.
Neutralization activity of the 56 plants of monoclonal antibodies of table to the HPV pseudovirus of 11 types
8. secondary culture and the preservation of hybridoma
Above-mentioned hybridoma is continued to culture, passage, training in the DMEM culture medium containing 10% fetal calf serum
Support to after 10 generations, hybridoma still is able to well-grown, stablizes passage, in culture supernatant antibody titer 1:10000 with
On.The result shows that gained hybridoma cell line can stablize passage, the monoclonal antibody of anti-HPV16 can be continually and steadily secreted.
After the hybridoma for obtaining the monoclonal antibody needed for generating, a part of hybridoma is saved, is otherwise existed
During continuous passage, it is possible to create mutation or drifting about for chromosome generate the spy of antibody down to forfeiture inherent characteristic or loss
Property；In addition in long-term incubation, pollution does not occur inevitably so that destroying, it is therefore necessary to protect to cell strain
It deposits.Store method is as follows: removing the old culture solution in Tissue Culture Flask, 1640 liquid of 10%FCS-is added, cell is made to suspend.
1000rpm/min is centrifuged 10min, removes supernatant.Cell precipitation protects liquid that suspension is made with 10% dimethyl sulfoxide, make into 1.0 ×
107Cell/ml.Sampling, expects blue dyeing with platform, living cell counting quantity should be 95% or more.It finally will be thin with asepsis injector
Born of the same parents dispense into ampulla, every bottle of 0.5ml-1.0ml, seal ampulla.4 DEG C stand 2 hours after to be transferred in -70 DEG C of refrigerators 15 small
When, it is finally transferred in liquid nitrogen and freezes.
The identification of 2 4A3 antibody of embodiment
1. antibody obtains
Selection adult BALB/c mouse, intraperitoneal inoculation norphytane, every mouse 0.5ml.Intraperitoneal inoculation the 16th after 7-10 days
For clone4A3 hybridoma, every mouse 1 × 106-2×106It is a.It obviously expands, touches to abdomen after 5 days in interval
When, skin has tension, acquires ascites with No. 9 syringe needles.
2. the purifying of antibody
Ascites 13000rpm/min is centrifuged 30 minutes, cell component and other sediments is removed, collects supernatant.With
Protein G affinity chromatography and Sepharose CL-4B gel filtration are purified, and the Dan Ke of HPV16L1 albumen is finally obtained
Grand antibody clone4A3, concentration is in 1mg/ml or more.
3. antibody purity detects
Antibody after purification is subjected to 12%SDS-PAGE electrophoresis, the results showed that purity is 95% or more.
4. antibody class and subgroup identification
Subtypes are carried out to 4A3 antibody using Pierce Rapid ELISA Mouse mAb Isotyping Kit
Detection, uses various immunoglobulin hypotype (IgG1, IgG2a、IgG2b、IgG3, IgA, IgM, Kappa and Lambda) antibody
The Ig hypotype for the antibody that above-mentioned hybridoma generates is detected, the monoclonal antibody that clone4A3 cell strain generates as the result is shown is equal
Belong to IgG1Type.
5. antibody light chain and heavy chain variable region gene sequencing
MRNA, the reverse transcription cDNA for extracting clone4A3 hybridoma carry out high guarantor using variable region universal primer
PCR product segment is inserted into carrier T and carries out determined dna sequence by true PCR amplification, the variable region gene sequence of 4A3: heavy chain
As shown in SEQ ID NO.9, light chain is as shown in SEQ ID NO.10.The nucleotide sequence of acquisition is translated into amino acid sequence.
The variable region amino acid sequence of 4A3: heavy chain is as shown in SEQ ID NO.7, and light chain is as shown in SEQ ID NO.8.
6. antibody isotypes specific detection
If antibody specificity testing result is shown in 5. hybridomas secretion supernatant in embodiment 1,
4A3 antibody is only the positive to the reaction of HPV16L1-VLP albumen, to other HPV types (6,11,18,31,33,35,
39,45,51,52,56,58,59,68) testing result be feminine gender, it was demonstrated that 4A3 antibody is type monoclonal antibody specific.
7. antibody neutralization is identified
If 7. antibody neutralization testing results are shown in embodiment 1,4A3 antibody is anti-for the monoclonal with neutralization activity
Body.
The identification of 3 2H8 antibody of embodiment
1. antibody obtains
Other than being inoculated with clone2H8 hybridoma, carried out using with method identical in embodiment 2-1.
2. the purifying of antibody
It is carried out using with method identical in embodiment 2-1, finally obtains the monoclonal antibody 2H8 of HPV16L1 albumen, it is dense
Degree is in 1mg/ml or more.
3. antibody purity detects
It is carried out using with method identical in embodiment 2-1, the results showed that purity is 95% or more.
4. antibody class and subgroup identification
It is carried out using with method identical in embodiment 2-1, the monoclonal that clone2H8 cell strain generates as the result is shown is anti-
Body belongs to IgG2bType.
5. antibody isotypes specific detection
If antibody specificity testing result is shown in 5. hybridomas secretion supernatant in embodiment 1,
2H8 antibody is only the positive to the reaction of HPV16L1-VLP albumen, to other HPV types (6,11,18,31,33,35,
39,45,51,52,56,58,59,68) testing result be feminine gender, it was demonstrated that 2H8 antibody is type monoclonal antibody specific.
6. antibody neutralization is identified
If 7. antibody neutralization testing results are shown in embodiment 1,2H8 antibody does not have neutralization activity.
4 HPV16L1 antigen detection kit of embodiment
The monoclonal antibody 4A3 and 2H8 obtained using embodiment 1 carries out grinding for HPV16 double crush syndrome kit
System, to detect HPV16L1 antigen levels.
1. the foundation of double antibody sandwich ELISA
1) kit principle:
4A3 and two strain antibody of 2H8 are HPV16 type specific antibody, and 4A3 strain antibody has neutralization activity.With 2H8
As coated antibody, using 4A3 as enzyme labelled antibody, establish double crush syndrome detection kit, when by measuring samples solution or
The pre-coated ELISA Plate for having 4A3 antibody is added in standard solution, adds 4A3-HRP ELIAS secondary antibody, is developed the color with developing solution,
Sample absorption value and the content of HPV16L1 albumen in sample are positively correlated in the linear range, can be obtained compared with standard curve
Out in sample HPV16L1 albumen content.Simultaneously can also according to the shade on ELISA Plate, by with series of concentrations
The comparison of HPV16L1 protein standard substance solution colour, the concentration range of HPV16L1 albumen in rough judgement sample.
2) composition of kit are as follows:
(1) be coated with the ELISA Plate of coated antibody: coated antibody is 2H8 type specificity monoclonal antibody, is CGMCC by deposit number
2H8 plants of secretions of hybridoma cell strain clone of NO.10958 generate.Coated antibody is diluted to 5 μ g/ml with coating buffer,
It is coated in 96 hole elisa Plates, 100 μ l are added in every hole, are placed in 4 DEG C of environment and are incubated for 8 hours.Discard coating buffer, cleaning solution washing 3
It is secondary, it pats dry.Then confining liquid, every 100 μ l of hole are added in ELISA Plate, 37 DEG C of environment are incubated for 2 hours.The liquid in hole that inclines is clapped
It is dry, it is saved after dry with aluminium film vacuum sealing.Wherein, it is that 9.5,0.1mol/L carbonate is slow that coating buffer used, which is pH value,
Fliud flushing；Confining liquid used is that the bovine serum albumin(BSA) containing 8% (mass percentage) final concentration of in confining liquid, pH value are
8.6, barbital sodium-hydrochloride buffer of 0.1mol/L.
(2) enzyme labelled antibody: marker enzyme is horseradish peroxidase, and labeling method is Over-voltage protection, enzyme labelled antibody 4A3
Type specificity neutralization monoclonal antibody is generated, enzyme by 4A3 plants of secretions of hybridoma cell strain clone that deposit number is CGMCC NO.10955
The working concentration of labeling antibody is 1 μ g/ml.
(3) standard solution: HPV16L1 protein standard substance solution, 7 bottles, concentration be respectively 125ng/ml, 62.5ng/ml,
31.25ng/ml,15.63ng/ml,7.813ng/ml,3.906ng/ml,0ng/ml.Preparing standard solution is containing preparing
The phosphorus of the DMSO, pH 6.5-6.7,0.1-0.2mol/L of final concentration of 5-10% (mass percentage) in the solution of standard items
Phthalate buffer.
(4) substrate developing solution: being made of developing solution A liquid and developing solution B liquid, and developing solution A liquid is hydrogen peroxide, developing solution B
Liquid is o-phenylenediamine.
(5) terminate liquid is 1-2mol/L sulfuric acid solution.
(6) concentrated cleaning solution: pH value 8.5, containing final concentration of 0.05% in concentrated cleaning solution, (quality percentage contains
Amount) sodium azide, final concentration of 2.0% (mass percentage) Tween-20 in concentrated cleaning solution, 0.3mol/L phosphoric acid
Salt buffer；40ml/ bottles, 1 bottle.
(7) liquid is redissolved in concentration: DMSO, pH containing the final concentration of 5-10% (mass percentage) in redissolution liquid are
The phosphate buffer of 6.5-6.7,0.1-0.2mol/L；200ml/ bottles, 1 bottle.
(8) kit specification.
2. the range of linearity and kit sensitivity verifying
By HPV16L1-VLP albumen be configured to 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.63ng/ml,
The dilution of 7.813ng/ml, 3.906ng/ml, 0ng/ml, to determine kit to the Monitoring lower-cut of antigen concentration, to examine
The sensitivity of test agent box, and repeat to test 3 times, to measure average value that result is positive minimum concentration as kit
Sensitivity.The calibration curve equation for repeating to detect 3 times is y=0.0208x-0.0046, y=0.0273x-0.0044 and y=
0.0242x-0.0027, R2Respectively 0.9976,0.9933 and 0.9965, R2It is all larger than 0.99.The inspection of HPV16L1-VLP antigen
Survey range is 3.9-62.5ng/ml.The detection sensitivity of HPV16 kit is 3.9ng/ml.
3. kit precision is verified
1) difference in precision verifying-plate
By HPV16L1-VLP albumen doubling dilution to 5 different antigen concentrations, each antigen concentration is 5 multiple holes (n=
6), totally 30 samples pass through the coefficient of variation (Coefficient of variation, CV) in the plate for calculating 30 samples and determine
Difference in plate, to carry out Precision Analyze to the kit.As a result as shown in Table 5, in 30 detections, CV (%) value is equal in plate
Less than 15%, show that precision is good in the kit plate.
The coefficient of variation in 6 HPV16 kit plate of table
2) difference between precision verifying-plate
By HPV16L1-VLP albumen doubling dilution to 5 different antigen concentrations, each antigen concentration is 5 multiple holes (n=
6), totally 30 samples, the coefficient of variation determines difference between plate between the plate by calculating 30 samples, to carry out essence to the kit
Density analysis.As a result as shown in Table 6, in 30 detections, CV (%) value is respectively less than 15% between plate, shows between the detection kit plate
Precision is good.
The coefficient of variation between 7 HPV16 kit plate of table
4. kit accuracy validation
HPV16L1-VLP albumen is diluted to high, medium and low 3 concentration (40ng/ml, 20ng/ml and 10ng/ml), each
Sample makees 2 multiple holes, calculates sample recovery rate, analyzes the accuracy of this method.Recovery of standard addition (95%CI) exists as the result is shown
Between 80.27%--96.93%, the kit rate of recovery is good.
5. kit storage life is tested
Kit preservation condition is 2-8 DEG C, by measurement in 6 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, HPV16L1 albumen actual measured value are within normal range (NR).Consider in transport and use process, meeting
There is improper preservation condition to occur, kit is placed 6 days under conditions of 37 DEG C of preservations, carries out accelerated aging tests, as a result
Show that the kit indices comply fully with requirement.In view of kit freezing happens, kit is put into -20 DEG C of ice
Case freezes 5 days, and measurement result also indicates that kit indices are completely normal.It can show that kit can be in 2- from result above
8 DEG C can at least save 6 months or more.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (11)
1. the antibody of a kind of specific binding human papilloma virus HPV16L1 albumen and/or VLP, which is characterized in that include
Light chain shown in the region CDR1, CDR2 and CDR3 of heavy chain variable region shown in SEQ ID NO.1-3 and SEQ ID NO.4-6 can
Become the region CDR1, CDR2 and CDR3 in area.
2. it is a kind of specific binding human papilloma virus HPV16L1 albumen and/or VLP antibody, which is characterized in that comprising just like
Heavy chain variable amino acid sequence shown in SEQ ID NO.7 and the chain variable region amino acid sequence as shown in SEQ ID NO.8
Column.
3. the antigen-binding portion thereof of a kind of specific binding human papilloma virus HPV16L1 albumen and/or VLP, which is characterized in that
Include heavy chain variable region shown in SEQ ID NO.1-3 the region CDR1, CDR2 and CDR3 and SEQ ID NO.4-6 shown in
The region CDR1, CDR2 and CDR3 of light chain variable region；
Wherein, the antigen-binding portion is selected from Fab, Fab', F (ab')2, Fd, dAb, complementary determining region segment, single-chain antibody,
Humanized antibody, chimeric antibody or double antibody.
4. such as antibody claimed in claims 1-2, which is characterized in that the antibody includes just like weight shown in SEQ ID NO.9
Chain variable region nucleotide sequence and the light chain variable region nucleotide sequence as shown in SEQ ID NO.10.
5. such as antibody claimed in claims 1-2, which is characterized in that the antibody is by the splenocyte and marrow after being immunized
The monoclonal antibody that the hybridoma cell line that oncocyte merges generates.
6. antibody according to claim 1 to 2, which is characterized in that it is CGMCC that the antibody, which is by deposit number,
The monoclonal antibody that the cell of NO.10955 generates.
7. antibody according to claim 1 to 2, which is characterized in that the antibody is that have the monoclonal of neutralization activity anti-
Body.
8. antibody according to claim 1 to 2, which is characterized in that the antibody is unit price or bivalent antibody.
9. a kind of kit, which is characterized in that the kit include antibody as described in any one of claim 1-2 or
Antigen-binding portion thereof as claimed in claim 3.
10. kit according to claim 9, which is characterized in that the kit also include with HPV16L1 albumen and/
Or the secondary antibody of VLP specific binding.
11. kit according to claim 10, which is characterized in that it is CGMCC that the secondary antibody, which is by deposit number,
The monoclonal antibody that the cell of NO.10958 generates.
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| CN108586607B (en) * | 2018-04-13 | 2020-09-11 | 郑州大学 | Preparation method and application of anti-HPV16 L1 protein monoclonal antibody |
| CN109180810B (en) * | 2018-09-27 | 2021-05-07 | 国药中生生物技术研究院有限公司 | Antibody specifically binding norovirus GI.1 genotype VP1 protein or VLP, and preparation method and application thereof |
| CN110646612A (en) * | 2019-09-30 | 2020-01-03 | 源道隆（苏州）医学科技有限公司 | Reagent and kit for detecting serum HPV antibody |
| CN114195886B (en) * | 2021-11-12 | 2023-05-23 | 郑州大学 | anti-HPV 39L1 protein monoclonal antibody, preparation and application thereof |
| CN115112885B (en) * | 2022-06-18 | 2023-07-28 | 杭州爱光健康科技有限公司 | HPV detection kit and preparation method and application thereof |
| 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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| CN101186636A (en) * | 2007-12-06 | 2008-05-28 | 三峡大学 | Human papillomavirus coat protein L1 short peptide and its application |
| CN101914139A (en) * | 2010-07-16 | 2010-12-15 | 四川大学 | Human papillomavirus (HPV) capsid protein L1 polypeptide and its preparation and application |
| CN103483447A (en) * | 2012-06-08 | 2014-01-01 | 厦门大学 | Broad spectrum monoclonal antibodies or antigen binding fragments thereof of anti-HPV L1 protein, and applications thereof |
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| CN101186636A (en) * | 2007-12-06 | 2008-05-28 | 三峡大学 | Human papillomavirus coat protein L1 short peptide and its application |
| CN101914139A (en) * | 2010-07-16 | 2010-12-15 | 四川大学 | Human papillomavirus (HPV) capsid protein L1 polypeptide and its preparation and application |
| CN103483447A (en) * | 2012-06-08 | 2014-01-01 | 厦门大学 | Broad spectrum monoclonal antibodies or antigen binding fragments thereof of anti-HPV L1 protein, and applications thereof |
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