CN103130890B - Human papillomavirus (HPV) 16E7 monoclonal antibody and relevant hybridoma cell strain and application thereof - Google Patents
Human papillomavirus (HPV) 16E7 monoclonal antibody and relevant hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention provides a human papillomavirus (HPV) 16E7 monoclonal antibody. The HPV 16E7 monoclonal antibody combines protein specificity shown in SEQ ID NO:2, and the monoclonal antibody is obtained by animal immunity, wherein a fusion protein formed by the protein shown in the SEQ ID NO:2 and a known label and the fusion protein is used as antigen fro the animal immunity. The monoclonal antibody is obtained by two hybridoma cell lines with preservation numbers of CGMCC519 or CGMCC5200. The invention further provides application technology of relevant bybridoma cell strains and the antibody for detecting a biomarker, especially for detecting a HPV carcinogenic protein which is specifically expressive to tumor cells. The HPV 16E7 monoclonal antibody is capable of specifically detecting HPV16E7, can be used as a reagent of high sensitivity and high specificity, and is used for establishing of cancer detection method such as developing body external kit products. The antibody is capable of specifically combining cervical cancer cells transformed by HPV virus, can be used for researching of treatment for cancers, and developing target treating medicines. The HPV 16E7 monoclonal antibody is high in detection agent specificity, flexible in reaction, low in cost, and suitable for large-scaled popularization.
Description
Technical field
The present invention relates to genetically engineered and immunological technique field, more specifically, relate to monoclonal antibody technique field, refer to a kind of HPV16E7 monoclonal antibody, relevant hybridization tumor cell strain and application especially.
Background technology
Cervical cancer is the common cancer of female reproductive system, occupies female malignant second.Large quantity research finds, human papillomavirus HPV (human papilloma virus) is the arch-criminal causing cervical cancer, can also cause other tumour multiple, comprise reproductive tract, mammary gland, digestive tube and respiratory cancer.The propagation of HPV in recent years in population of China is growing on and on, and the research work of the cancerous precaution of the Forbidden City neck, treatment is extremely important.
1949, first Sttauss observed human papillomavirus (HPV) particle under Electronic Speculum in wart body leach liquor.Zur Hansen in 1976 propose HPV may be spread through sex intercourse carcinogenic factor after, HPV infects the heat subject becoming tumour virus etiological study with the research of Cervical Cancer.HPV be a kind of have species specificity addicted to epithelium virus, belong to the small DNA virus of double-strand closed loop, comprise about 8000 base pairs.Comprising 8 early stage open reading frames (E1-E8), 2 late period single open reading frame and 1 non-coding Chang Kong district.In early days in open reading frame, E6 and E7 gene pairs Growth of Cells stimulates the most important, and E6, E7 albumen of E6, E7 coding causes cervical epithelial cells immortalization.And late period reading frame L1 and L2 gene to encode respectively the main of HPV and secondary capsid protein, be assembled into the capsid of HPV.
The cause-effect relationship that the E6 of high-risk hypotype HPV, E7 albumen causes normal epithelium cell to change into cancer cell is proved by science.Therefore, need the hybridoma that a kind of HPV16E7 monoclonal antibody and secretion HPV16E7 monoclonal antibody are provided, this hybridoma energy stably excreting HPV16E7 monoclonal antibody, for studying the function of HPV16E7 further, the research for cancer therapy and cancer detection lays the foundation.
Summary of the invention
Main purpose of the present invention is exactly with not enough for above Problems existing, a kind of HPV16E7 monoclonal antibody, related manufacturing processes, hybridoma cell strain and application are provided, this HPV16E7 monoclonal anti physical efficiency specific detection HPV16E7, can be used for the infection of the high-risk oncovirus hypotype of HPV16, and the specific detection of cervical cancer early diagnosis.Specificity is high, is quick on the draw, and cost is low, is suitable for large-scale promotion application.
In order to solve above-mentioned purpose, in a first aspect of the present invention, providing a kind of HPV16E7 monoclonal antibody, being characterized in, described HPV16E7 monoclonal antibody is the monoclonal antibody to the albumen energy specific binding shown in SEQ ID NO:2.
" specificity " described in the present invention refers to the recognition capability of monoclonal antibody to corresponding antigens or approximate antigenic substance.Specificity is high, just strong to the recognition capability of antigen.Therefore, above-mentioned HPV16E7 monoclonal antibody can specific recognition and in conjunction with the albumen shown in SEQ ID NO:2.Albumen shown in SEQ ID NO:2 can directly synthesize, and also can be prepared by gene engineering method.In a specific embodiment of the present invention, the albumen shown in SEQ ID NO:2 can be produced by SEQ ID NO:1 the 1 to 291 nucleotide coding.
Preparing monoclonal antibody of the present invention antigen used is obtained by engineered method, utilizes the polynucleotide sequence SEQ ID NO:1 expressing in prokaryotic organism and contain coding antigen of the present invention.Those skilled in the art knows the carrier being applicable to express antigen of the present invention usually.Suitable carrier can be selected according to selected suitable promotor and goal gene sequence to be expressed.Any suitable host cell expression antigen of the present invention can be used.The example of suitable host comprises: prokaryotic cell prokaryocyte is as intestinal bacteria, genus bacillus, streptomycete etc.Transduction, to transform or the method for transfection is known in the art, include, but not limited to virus infection, calcium chloride transfection method, lipofection, electroporation or microprojectile bombardment methods etc.In order to activating promoters, select needed for transformant or amplification gene, can cultivate in the conventional nutrient culture suitably modified transduceed, the host cell of transfection or conversion.The culture condition such as the temperature used in cultivation, pH value are all generally determined by the host cell of selected expression specified protein, and these conditions are all well known to those skilled in the art.In order to obtain recombinant protein in a large number, can inducible promoter be adopted, and utilize the expression of inductor induced gene.In fact, " inductor " can be any material inducing genetic expression in host, can be chemical substance or environmental stimulus.
Preferably, described monoclonal antibody is that the fusion rotein adopting the albumen shown in SEQ ID NO:2 and known label to be formed obtains as antigen-immunized animal.
More preferably, described known label is, but is not limited to, histidine-tagged (His label), glutathione S-transferase (GST label), Trx label, S-tag label, HA label, HSV label, Myc label or VSV-G label.In a specific embodiment of the present invention, described known label is GST label, forms GST-HPV16E7 fusion rotein with the albumen shown in SEQ ID NO:2.
More preferably, described monoclonal antibody adopts described fusion rotein to obtain as mice immunized with antigen.
Further, described monoclonal antibody has specificity to HPV16E7, does not have specificity to HPV16L2.
Preferably, the hybridoma cell strain that described monoclonal antibody is CGMCC5199 or CGMCC5200 by preserving number produces.
In a second aspect of the present invention, provide two kinds of hybridoma cell strains, be characterized in, described hybridoma cell strain is for generation of above-mentioned HPV16E7 monoclonal antibody, and the preserving number of described hybridoma cell strain is CGMCC5199 or CGMCC5200.
Above-mentioned hybridoma cell strain has been deposited in one of International Depository Authority, and " (China General Microbiological Culture Collection Center; CGMCC; address: great Tun road, Chaoyang District, city of BeiJing, China; Institute of Microorganism, Academia Sinica; postcode: 100101); preservation date: on September 8th, 2011, Classification And Nomenclature is mouse hybridoma cell (Mouse hybridoma) to China Committee for Culture Collection of Microorganisms's common micro-organisms " center ".
The fusion rotein that the preparation method of above-mentioned HPV16E7 monoclonal antibody adopts the albumen shown in SEQ ID NO:2 and known label to be formed is prepared as antigen.
Preferably, described known label is, but is not limited to, histidine-tagged (His label), glutathione S-transferase (GST label), Trx label, S-tag label, HA label, HSV label, Myc label or VSV-G label.In a specific embodiment of the present invention, described known label is GST label, forms GST-HPV16E7 fusion rotein with the albumen shown in SEQ ID NO:2.
Preferably, described fusion protein immunization mouse, get splenocyte and the myeloma cell fusion of mouse, filter out the hybridoma can secreted and the albumen shown in SEQ ID NO:2 be had to the monoclonal antibody of specific reaction, from the culture supernatant of described hybridoma or from the animal ascites after the described hybridoma of injection, obtain described monoclonal antibody.
More preferably, the preserving number of described hybridoma cell strain is CGMCC5199 or CGMCC5200.
In of the present invention one preferred specific embodiment, using GST-HPV16E7 fusion rotein as mice immunized with antigen, the myeloma cell of the splenocyte and syngeneic animal of getting mouse is merged.Screening can produce the hybridoma of object antibody, carries out colonized culture, and sets up hybrid cell strain.Aforesaid method is only exemplary, and such as can use the same method other Mammalss of immunity, using its splenocyte as immunocyte.The myeloma cell that is suitable for can also be selected for merging, such as, be used for from the myeloma cell of rat, mouse or hamster.The fusion of immunocyte and myeloma cell can conventionally be carried out.
The hybridoma that screening produces object antibody carries out mono-clonal.The hybridoma cell strain of the generation obtained monoclonal antibody of the present invention, can in ordinary culture medium Secondary Culture or preserve for a long time in liquid nitrogen.When collecting monoclonal antibody of the present invention from hybridoma, antibody can be obtained from hybridoma vitro culture supernatant liquor, or hybridoma is injected suitable Mammals and obtains antibody from animal ascites.A kind of front method is suitable for obtaining highly purified antibody, and a kind of rear method is suitable for obtaining antibody in a large number.By the antibody that aforesaid method obtains, can purify by ordinary method, such as, saltout, gel-filtration, the method such as affinity chromatography.
In a third aspect of the present invention, provide the application of above-mentioned HPV16E7 monoclonal antibody in the diagnostic tool of cervical cancer or other human cancer caused by preparation detection and/or auxiliary diagnosis human papilloma virus infection.
In a fourth aspect of the present invention, provide a kind of immunoassay, be characterized in, described immunoassay adopts above-mentioned HPV16E7 monoclonal antibody.
Preferably, described immunoassay is ELISA immunoassay, immunochormatography, immunocytochemical stain assay method or immunohistochemical staining assay method.
In a fifth aspect of the present invention, provide a kind of test kit, be characterized in, described test kit comprises above-mentioned HPV16E7 monoclonal antibody.
For a person skilled in the art, according to content of the present invention, utilizing said monoclonal antibody, prepare corresponding testing product, such as Kit and/or Rapid detection test strip etc., is apparent.Therefore, be also the present invention's content required for protection.
Beneficial effect of the present invention is:
1, HPV16E7 monoclonal antibody of the present invention has specific monoclonal antibody to the albumen shown in SEQ ID NO:2, can specifically in conjunction with the albumen shown in SEQ ID NO:2, thus can be used for the cervical cancer cell of HPV infection, its cell expressing HPV16E7 albumen, HPV16E7 monoclonal antibody can provide specific detection, and specificity is high.
2, HPV16E7 monoclonal antibody of the present invention has specificity to HPV16E7, specificity is not had to HPV16L2, thus the normal human cells that can carry out caused by HPV infects changes into the specific detection of intraepithelial neoplasia cells (CIN) or cancer cells, make up the deficiency that there is no HPV method for detecting specificity at present, utilize HPV16E7 monoclonal antibody to carry out detection specificity high, be quick on the draw.
3, immunoassay of the present invention and test kit can be used for the species specificity detection of HPV positive tumor cell, make up the deficiency that there is no cervical cancer method for detecting specificity at present.HPV16E7 monoclonal antibody can biomarker in specific binding tumour cell, HPV16E7 or HPV18E7 albumen.There is provided specificity high, be quick on the draw, the low diagnostic techniques of cost, is suitable for high throughput testing and large-scale promotion application.
Accompanying drawing explanation
Fig. 1 is the ELISA detected result that fusion mouse 6 Post-immunisation serum are tired.
Fig. 2 is the SDS-PAGE qualification result of the HPV16E7 monoclonal antibody after purifying, and wherein, E1-E4 is the numbering of the monoclonal antibody of different batches wash-out.
Fig. 3 and Fig. 4 be different hybridoma produce HPV16E7 monoclonal antibody respectively with HPV16E7L2 and HPV18E7 in conjunction with detected result.
Fig. 5, Fig. 6 and Fig. 7 are HPV16E7 monoclonal antibody 8C10 antibody and 8E5 antibody carry out immunocytochemical stain experiment respectively results to HPV positive cervical cancer cell SiHa, and wherein Fig. 5 is Negative control mice IgG.
Fig. 8 and Fig. 9 is that HPV16E7 monoclonal antibody 1A5 carries out the result of immunohistochemical experiment to Cervix Squamous Cell cancer pathological section, and wherein Fig. 8 is negative control.
Embodiment
The present inventor, through extensive and deep research, obtains a kind of HPV16E7 monoclonal antibody, and this HPV16E7 monoclonal antibody has excellent specific binding effect to HPV16E7.Complete the present invention on this basis.
In order to more clearly understand technology contents of the present invention, be described with reference to the accompanying drawings especially exemplified by following examples.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.
1, experiment material and method
1.1 reagent and medicine
Fu Shi completely, Freund's incomplete adjuvant (Freund ' s Adjuvant complete or incomplete); PEG (Polyethyleenglycol4000*PEG*Macrogolum 4,000 lot#BCBCO873), purchased from Fluka company; Horseradish peroxidase-labeled sheep anti-mouse igg (Goat anti-mouse IgG-HRP lot#15-035-164), purchased from Jackson company; DMEM (Dulbecco ' s Modified Eagle Medium, lot#SH3002.01B), purchased from HyClone company; Foetal calf serum, purchased from Hyclone company; TMB (Tetramethylbenzidine), available from Sigma; Mouse monoclonal antibody subclass parting kit (Mouse Monoclonal Antiboay IsotypingReagents, 058K4836); IHC test kit (Dako, EnVision+System-HRP Labelled PolymerAnti-mouse, K4000); Other related reagent is the pure or analytical pure of top grade.
1.2 instruments and consumptive material
Superpurgative working table (Boxun is purchased from Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); CO
2constant incubator (German Thermo); Ultralow Temperature Freezer (Forma-86C Germany Thermo); YDS-50B-125 type liquid nitrogen biological container (Cengdu Jinfeng Liquid Nitrogen Container Co. Ltd.); Simple microscope (XDS-1A); Electronic balance (plum Teller-Tuo benefit instrument Shanghai company limited); 85-1 constant temperature blender with magnetic force (Jie Ruier Electrical Appliances Co., Ltd of Community of Jin Tan County city); Table-type high-speed refrigerated centrifuge (German eppendorf); HHS type electric-heated thermostatic water bath (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); MULTISKAN MK3 microplate reader (Thermo company); Ultrapure water preparation device (MILLPORE company of the U.S.); GZX-9240MBE digital display air dry oven (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.); WellWASK4 MK2 washes trigger (purchased from Thermo company); 96 holes, 24 porocyte culture plates (NUNC company); 96 hole enzyme plates (NUNC company).Single track, multichannel pipettor (German Eppendorf company); Tissue Culture Flask, Crossbred Cattle Boydii blood cell counting plate etc.
1.3 cell strains, tissue sample and animal
SP2/0 myeloma cell, clone SiHa:SP2/0 is given by Beijing Tumour Hospital teacher Shou Chengchao laboratory, and clone SiHa is purchased from Shanghai Inst. of Life Science, CAS cellular resources center.
Tissue slice: tumor biopsy is bought in Ai Lina bio tech ltd, Xi'an.
4 ~ 6 week age female BAl BIc/c mouse: buy in Yangzhou University's Experimental Animal Center.
1.4 main solution systems
Bag is buffered liquid (carbonate solution): get 0.2mol/L Na
2cO
38mL, with 0.2mol/L NaHCO
317mL mixes, then adds 75mL distilled water, and adjust pH is to 9.6.
Dilution buffer (PBS, without calcium and magnesium):
Lavation buffer solution (PBS-Tween 20,0.5 ‰): Tween-200.5mL, join in 1L dilution buffer (PBS), stirring and evenly mixing, between adjust pH to 7.0 ~ 7.2.
20 × substrate uses liquid A: get TMB 21mg, be fully dissolved in 5mL ethanol.
100 × substrate uses liquid B: get urea hydrogen peroxide 16.5mg, be dissolved in 1mL distilled water.
Stop buffer (2mol/L H
2sO
4): distilled water 178.3mL, drips the vitriol oil (98%) 21.7mL.
Incomplete DMEM substratum: be purchased from Hyclone company.
Complete DMEM substratum: be purchased from Hyclone company.
HAT substratum: DMEM substratum 99mL completely, adds 500 × HAT stock solution 1mL, prepare under aseptic condition.
The preparation of 1.5 monoclonal antibodies
1.5.1 prepared by antigen:
Prepare prokaryotic expression carrier and express GST-HPV16E7L2 (aminoacid sequence see shown in SEQ ID NO:3) as immunizing antigen, using HPV16E7L2-His (aminoacid sequence see shown in SEQ ID NO:4) as detectable antigens.The detection protokaryon protein sequence that all the other relate to is GST-HPV16E7 (aminoacid sequence see shown in SEQ ID NO:5), GST-HPV16L2 (aminoacid sequence see shown in SEQ ID NO:6), GST-HPV18E7 (aminoacid sequence see shown in SEQID NO:7).
1.5.2 mouse immune program
Be immunogen with GST-HPV16E7L2, immune programme for children is in table 1.
Table 1 mouse immune program
Note: after exempting from three, blood sampling survey in 7 days is tired
1.5.3 cytogamy and cultivation
1.5.3.1 the preparation of feeder layer cells (merge and get Turnover of Mouse Peritoneal Macrophages the day before yesterday)
Get above ICR mouse in 8 week age, de-cervical vertebra is lethal, 75% alcohol-pickled 5min; 10ml substratum is added in 15ml centrifuge tube; Tear mouse skin with autoclaving scissors, tweezers and expose peritonaeum, draw RPMI-DMEM 5ml with 5ml sterilizing syringe; Peritonaeum mentioned by tweezers, and substratum is injected abdominal cavity, gently abdomen massage 1min, extracts substratum; The cell of extraction is spread in 96 orifice plates.
1.5.3.2 cytogamy
Myeloma cell SP2/0 and splenocyte are merged, merge ratio splenocyte: SP2/0=5: 1, fused cell is sub-packed in 96 well culture plates, is placed in 37 DEG C, 5%CO
2in constant incubator, selectivity is cultivated.Liquid is entirely changed 3 times with HAT substratum after merging; When hybridoma covers with a field of microscope, carry out ELISA detection.
1.5.3.3 the screening of positive hybridoma
During detection, employing indirect ELISA screens: antigen selects HPV16E7L2-His fusion rotein, 2ug/ml wrapper sheet, 4 DEG C are spent the night, after PBST washing pats dry, add 5% skim-milk to close, 37 DEG C of effect 2h or 4 DEG C spend the night, after PBST washing pats dry, add hybridoma supematant and the positive (P), negative (N) and blank (blank directly adds ELIAS secondary antibody) are set, add anti-37 DEG C of reaction 30 ~ 45min, the PBST washings of sheep anti mouse-HRP two after 37 DEG C of reaction 1h, PBST washings pat dry again to pat dry rear TMB and develop the color and 2M H
2sO
4stop.
ELISA the selection result with OD value be greater than 0.6 for going out positive hole, and to recheck.After twice continuous detecting is the positive, this positive porocyte is increased, in time frozen and subclone.
1.5.3.4 the subclone in positive hole, enlarged culturing and frozen
For the hole that 2 screenings are all the positive, adopt limiting dilution assay to carry out subclone, get one piece of 96 porocyte culture plate, in every hole, add 150ul HAT nutrient solution; To needing the positive hole of subclone to blow and beat suspension gently, drawing 100ul cell suspension and being added in 96 porocyte plates, doubling dilution from the first hole.Kong Zhongyue is chosen in cell counting to be had the hole of 100 cells and joins in the loading slot containing 6ml HAT nutrient solution, is added in the cell plate of paving feeder layer, adds first three columns by 100ul/ hole; Add HAT substratum 3ml again, 100ul/ hole is added in the cell plate of paving feeder layer, three row in adding; Add HAT substratum 5ml again, 100ul/ hole is added in the cell plate of paving feeder layer, six row after adding.When the positive rate calculating every block plate reaches 100%, stable cell line can be obtained.Transfer in Tissue Culture Plate, enlarged culturing is also frozen in a large number.
1.5.3.5 a large amount of productions (ascites preparation) of monoclonal antibody
Mass propgation hybridoma (8C10), with 1 × 10
6cells/ amount abdominal injection only uses the BALB/C female mice in the 8-10 of whiteruss sensitization all ages in advance, ascites is gathered after about 10 days, detected the ascites positive by ELISA with HPV16E7L2-His bag and tired, by mouse ascites 2ml, utilizing Binding buffer to dilute 10 times.Prepare Protein G, 2ml beads is added in post, utilizes 200ml Binding buffer to balance simultaneously.After balance, add in post lightly by sample, flow rate control is at per minute 0.5ml/min.After post crossed by sample, carry out Wash Step, utilize 10ml Binding buffer and 10ml Washing buffer to clean, the Washing buffer after last 1ml cleaning carries out OD260,280 determination of protein concentration, to prove cleaning performance.Before carrying out wash-out, first in EP pipe, add 100ul Neutralizing Buffer, carry out wash-out, add Elution buffer, each 1ml, collects 6ml altogether, afterwards, then uses the excessive wash-out of 10ml Elution buffer.After recycling 20ml Binding buffer (plus NaN3 0.05%) balances, be stored in Binding buffer-NaN3.
1.5.4 the Function Identification of monoclonal antibody
1.5.4.1 the specificity of monoclonal antibody and cross reaction qualification
Detect monoclonal antibody to the reaction of several albumen of HPV16E7L2-His, HPV18E7-His, GST-HPV16E7, GST-HPV16L2, GST-HPV18E7.ELISA method is adopted to identify: antigen selects HPV16E7L2-His, GST-HPV16E7, GST-HPV16L2, GST-HPV18E7 and HPV18E7-His fusion rotein, the equal 2ug/ml wrapper sheet of antigen, 4 DEG C are spent the night, after PBST washing pats dry, add 5% skim-milk to close, 37 DEG C of effect 2h or 4 DEG C spend the night, after PBST washing pats dry, add positive colony supernatant and negative SP2/0 (N) and blank (blank directly adds ELIAS secondary antibody) are set, 37 DEG C of reaction 1h, the anti-37 DEG C of reaction 30 ~ 45min of sheep anti mouse-HRP two are added again after PBST washing pats dry, PBST washing pats dry rear TMB and develops the color and 2M H
2sO
4stop.
1.5.4.2 the hypotype qualification of monoclonal antibody
Adopt the hypotype identification kit of Sigma company, article No. is: 058K4836.Testing sequence presses shop instruction operation.
1.5.4.3 monoclonal antibody specificity qualification ELISA:
With GST-HPV16L2, GST-HPV18E7 and GST-HPV16E7 identifies, 4ug/ml spends the night bag quilt, and 5% skim-milk 37 DEG C closes 2 hours, carries out specificity identification to 13 strain HPV16 monoclonal antibodies, 37 DEG C act on 1 hour, PBST machine-washes 4 times, the anti-37 DEG C of effects of 1: 8000 dilution sheep anti mouse two 45 minutes, and PBST machine-washes TMB colour developing after 4 times, stop, reading.
1.5.4.4 monoclonal antibody specificity qualification Western blot:
1.5.4.4.1 the qualification of monoclonal antibody
Be with loading, 100V voltage, transferring film 45min with the albumen 1ug/ of HPV16-E7L2 and HPV18-E7 prokaryotic expression, 5% skim-milk room temperature closes 2 hours, adds positive hybridoma supernatant 4 DEG C and spends the night, add sheep anti mouse-HRP after washing 4 times, and room temperature reaction 1h, DAB develop the color.
1.5.4.4.2 the qualification of monoclonal antibody
Be with loading, 100V voltage, transferring film 45min with the albumen 1ug/ of HPV16-L2 and HPV16-E7 prokaryotic expression, 5% skim-milk room temperature closes 2 hours, adds monoclonal antibody 4 DEG C and spends the night, add sheep anti mouse-HRP after washing 4 times, and room temperature reaction 1h, DAB develop the color.
1.5.4.5 monoclonal antibody detects in conjunction with tumour cell: immunocytochemical stain (ICC)
Cervical cancer cell lines Siha cell is selected to carry out immunocytochemical stain test to monoclonal antibody.Specific experiment method is as follows: 1. Siha cell divides in 96 orifice plates, adherent growth 2 days; 2. 4% paraformaldehyde fixing rear 0.1%Triton, 3%H2O2 processes 10min respectively; 3. 5% bovine serum 37 DEG C is closed and is added antibody (5 μ g/ml) after 2 hours, and 37 DEG C are reacted 2 hours; 4. PBS adds anti-1: 2000 dilution of sheep anti mouse two after washing 3 times, and 37 DEG C are reacted 1 hour; 5. after PBS washes 3 times, DAB colour developing, hatches 10min, takes pictures for 37 DEG C.
1.5.4.6 monoclonal antibody is in conjunction with the detection of tumor biopsy: immunohistochemical methods (IHC)
The pathological section of kinds cancer is selected to detect monoclonal antibody.Section puts into dimethylbenzene 15 minutes, then put into successively dehydrated alcohol, 95%, 90%, 80%, the alcohol of 70% 5 minutes.Antigen retrieval buffers is put in section, more than 90 DEG C 15 minutes.To be cooled to room temperature, use 1%Trion process 10 minutes, use 3% hydrogen peroxide process 10 minutes.Room temperature closes 2 hours.Then add antibody (5 μ g/ml), 4 DEG C are spent the night.Wash 3 times gently, each 3 minutes, add EnVision+System-HRP Labelled Polymer Anti-mouse, room temperature 30min.Wash 5 times gently, each 3 minutes, add DAB room temperature 2-5 minute, washing.Haematoxylin redyeing 2 minutes, washing.70%, 80%, 90%, 95% each 2 minutes, dehydrated alcohol 4 minutes are put in dehydration successively, are putting into dimethylbenzene 5 minutes, mounting, microscopic examination.
2 results
2.1 merge mouse positive serum tires
Shown in Figure 1, after 6 immunity, the ELISA of serum titer detects and reaches 1: 25, more than 000.
2.2 merge rear screening
Merged 8 blocks of 96-porocyte plates altogether, fusion rate about 65%, positive rate be 9.78% (be positive hole with OD >=0.6), wherein OD >=1.0 has 12 strains, within second day, carries out ELSIA reinspection, picks out the positive holes of 17 strains and carries out subclone.
Screen after 2.3 subclones
Subcloned cells plate changes liquid entirely through twice, ELISA detected result: 17 strains have 5 strains to turn out cloudy in positive hole, and all the other 12 strains are still positive, and positive rate is 70.6%.
2.4 monoclonal antibody anti-HPV16E7 are to the cross reactivity of HPV-18E7 albumen
The emiocytosis liquid of monoclonal antibody strain is detected by ELISA method.HPV16E7L2-His and HPV18E7-His two kinds of albumen all use 2ug/ml wrapper sheet.Found that: the emiocytosis liquid of monoclonal antibody strain 4G1,6F2,3G5,3A5,1A5,4C8,4G5 is to HPV18 type albumen (E7) no cross reaction.The emiocytosis liquid of monoclonal antibody strain 8E5,8C10,8G9 has stronger cross reaction to HPV18E7-His albumen.The emiocytosis liquid of monoclonal antibody strain 8B12,1F6 has more weak cross reaction.Result is summed up in table 2.Wherein Eclectics's oncocyte of monoclonal antibody strain 1A5 (cell strain ATTO-11-1) to be namely preserving number be CGMCC5199, Eclectics's oncocyte of monoclonal antibody strain 8C10 (cell strain ATTO-11-2) to be namely preserving number be CGMCC5200, be deposited in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", preservation date: on September 8th, 2011.
Table 2ELISA detects monoclonal antibody to HPV18E7 cross reaction
The Subtype qualification of 2.5 clones
Ask for an interview table 3, the result of the antibody of 12 strain clone secretions being carried out to Subtype is: 1F6,4G5,8B12,8G9 antibody is IgG1 type, and 3A5,3G5,8C10,8E5 antibody is IgG2b type, and 1A5,6F2,4G1 antibody is IgG2a type, and 4C8 antibody is IgG3 type.
The hypotype qualification of table 3 monoclonal antibody
| Antibody | Hypotype |
| 8E5 | IgG2b |
| 8C10 | IgG2b |
| 8G9 | IgG1 |
| 8B12 | IgG1 |
| 1F6 | IgG1 |
| 1A5 | IgG2a |
| 3A5 | IgG2b |
| 3G5 | IgG2b |
| 4C8 | IgG3 |
| 4G1 | IgG2a |
| 4G5 | IgG1 |
| 6F2 | IgG2a |
2.6 ELISA qualifications
Identify the result obtained through ELISA, see the following form: 4G1 and 4G5 antibody is for HPV16-L2, and wherein 4G5 and E7 antibody has cross reaction; 1A5,3A5,3G5,6F2,8C10 and 8G9 antibody is all for HPV16-E7.
2.7 SDS-PAGE qualifications
After purifying, monoclonal antibody (8C10 antibody) is through SDS-PAGE qualification result, obtains the monoclonal antibody 8C10 antibody of purity more than 95%, sees Fig. 2.
2.8 Western blot identify
Collect 12 strain clone supernatants to be Western blot and to detect, wherein SP2/0 cell conditioned medium does negative control, GST antibody does positive control, refer to Fig. 3 and 4, result is: 12 strain monoclonal antibodies all have combination with HPV16E7L2, wherein be combined with HPV16E7L2 protein-specific and with 7 strain 1A5,3A5,3G5,4G1,4G5,6F2, the 8G9 that have of HPV18E7 albumen no cross reaction, 4G1 and HPV16L2 has combination, and all the other antibody all belong to HPV16E7.
2.9 immunocytochemical stains (ICC) are identified
Utilize mIgG (negative control), monoclonal antibody 8C10,8E5 carry out immunocytochemical stain qualification to the Intrauterine device bleeding cultivated.Result shows that the cervical cancer tumer line dyeing negative control mIgG that monoclonal antibody 8C10 and 8E5 can make HPV16 infect specifically then fails to make cell dyeing, and in the cell of stained positive, HPV16E7 is arranged in tenuigenin and nucleus, sees Fig. 5, Fig. 6 and Fig. 7.
3.0 tumor biopsy immunohistochemical stainings test (IHC)
HPV16E7 monoclonal antibody 1A5 is utilized to carry out immunohistochemical methods detection to kinds cancer pathological section.Result shows that the paraffin section sample of monoclonal antibody 1A5 to Cervix Squamous Cell cancer, endometrioid adenocarcinoma, ovary mucinous adenocarcinoma, breast ductal cancer patient has positive staining (presenting brown colouring in fig .9) in cytoplasm.Confirm these samples by HPV virus infection by HC2 bis-generation hybrid capture technology before this.As shown in Figure 8, with the sample not using this monoclonal antibody to hatch as negative control.And the sample using monoclonal antibody 1A5 to hatch presents positive staining result, progressive one confirms that the HPV16E7 of this monoclonal antibody and sample is specific binding.The place of arrow indication is specific stain, sees Fig. 9.
In sum, HPV16E7 monoclonal anti physical efficiency specific detection HPV16E7 of the present invention, can be used for HPV and causes cancerous tumor cell, comprise the specific detection of precancerous lesion or cervical cancer cell.
In this description, the present invention is described with reference to its specific embodiment.But, still can make various amendment and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Claims (9)
1. a HPV16E7 monoclonal antibody, is characterized in that, described HPV16E7 monoclonal antibody is the monoclonal antibody to the albumen energy specific binding shown in SEQ IDNO:2;
The hybridoma cell strain that described monoclonal antibody is CGMCC5199 or CGMCC5200 by preserving number produces.
2. HPV16E7 monoclonal antibody according to claim 1, is characterized in that, described monoclonal antibody is that the fusion rotein adopting the albumen shown in SEQ ID NO:2 and known label to be formed obtains as antigen-immunized animal.
3. HPV16E7 monoclonal antibody according to claim 2, is characterized in that, described monoclonal antibody adopts described fusion rotein to obtain as mice immunized with antigen.
4. HPV16E7 monoclonal antibody according to claim 3, is characterized in that, described monoclonal antibody has specificity to HPV16E7, does not have specificity to HPV16L2.
5. a hybridoma cell strain, is characterized in that, described hybridoma cell strain is for generation of HPV16E7 monoclonal antibody according to claim 1, and the preserving number of described hybridoma cell strain is CGMCC5199 or CGMCC5200.
6. the application of HPV16E7 monoclonal antibody according to claim 1 in the diagnostic tool of the human cancer disease that preparation detects and/or auxiliary diagnosis human papilloma virus infection causes.
7. an immunoassay, is characterized in that, described immunoassay adopts HPV16E7 monoclonal antibody according to claim 1.
8. immunoassay according to claim 7, is characterized in that, described immunoassay is ELISA immunoassay, immunochormatography, immunocytochemical stain assay method or immunohistochemical staining assay method.
9. a test kit, is characterized in that, described test kit comprises HPV16E7 monoclonal antibody according to claim 1.
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| CN106366186B (en) * | 2015-07-21 | 2020-03-24 | 艾托金生物医药（苏州）有限公司 | Monoclonal antibody for identifying HPV16 positive tumor cells and application thereof |
| CN105801691B (en) * | 2016-03-29 | 2019-05-03 | 上海市普陀区中心医院 | A kind of HPV16E7 monoclonal antibody and its preparation method and application |
| CN106397583A (en) * | 2016-12-06 | 2017-02-15 | 亳州市新健康科技有限公司 | POCT (point-of-care testing) fluorescent quantitative test kit for HPV16 type E7 protein and application thereof |
| WO2018102982A1 (en) * | 2016-12-06 | 2018-06-14 | 亳州市新健康科技有限公司 | Poct fluorescence quantitative detection kit for hpv16 e7 protein and application thereof |
| CN109053879B (en) * | 2018-08-22 | 2020-12-25 | 深圳市宝安区中心医院 | scFv antibody for treating cervical cancer and application thereof |
| CN110196332A (en) * | 2019-05-22 | 2019-09-03 | 艾托金生物医药（苏州）有限公司 | A kind of method and its application detecting more hypotype HPV E7 albumen |
| CN112430583B (en) * | 2020-10-30 | 2022-02-15 | 武汉呵尔医疗科技发展有限公司 | Monoclonal antibody against HPV E7, cell strain and application thereof |
| CN112362874B (en) * | 2020-10-30 | 2024-02-09 | 武汉呵尔医疗科技发展有限公司 | Cervical cancer screening kit |
| CN112458061B (en) * | 2020-10-30 | 2022-03-22 | 武汉呵尔医疗科技发展有限公司 | Monoclonal antibody against HPV E6, cell strain 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 |
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| US5736318A (en) * | 1995-03-17 | 1998-04-07 | President And Fellows Of Harvard College | Method and kit for evaluating human papillomavirus transformed cells |
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