CN110196332A - A kind of method and its application detecting more hypotype HPV E7 albumen - Google Patents
A kind of method and its application detecting more hypotype HPV E7 albumen Download PDFInfo
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Abstract
The invention discloses a kind of method and its application for detecting more hypotype HPV E7 albumen, are related to Protein Detection field.2 kinds of rabbit resource monoclonal antibodies are mixed and are used as first antibody by the detection method, screening can successfully match with rabbit source monoclonal antibody and identify the source of mouse monoclonal antibody of a variety of hypotype HPV E7 albumen as secondary antibody, one of recognizable HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, HPV68 or a variety of hypotypes.The source of mouse antibody titer that the present invention is prepared is high, and specificity is good, and using HPV E7 protein content in detection method provided by the invention, detection card and immunity detection reagent test sample, sensitivity significantly improves, is simple, convenient.
Description
Technical field
The present invention relates to Protein Detection field more particularly to a kind of method for detecting more hypotype HPV E7 albumen and its answer
With.
Background technique
Human papilloma virus (Human papillomavirus, HPV) is a kind of small DNA virus, has very strong thermophilic squama
The characteristic of columnar epithelium, the main basal layer cell for invading scaly epithelium and the metaplasia cell positioned at uterine neck zone of transformation.By 3 genes
District's groups are at including early stage area (area Early Region, E), late region (area Late Region, L) area and and noncoding region
(Uncoding Region, UCR) or upstream regulatory region (URR).The area E is E6, E7, E1, E2, E3, E4 and E5 totally 7 in order
Gene, wherein E6 and E7 is the Analyses of major carcinogens in mainstream gene of HPV, related with virocyte transformation function and carcinogenicity.It is currently known HPV
Have a type more than 100, according to carcinogenicity, be divided into two class of high-risk HPV and low risk HPV.High-risk HPV infection (such as 16 and 18
Type) lead to most of uterine neck, penis, perineum, vagina, anus and pars oralis pharyngis cancer and precancerous lesion；And non-carcinogenic, low risk
HPV infection (such as 6 and 11 type) will lead to genital wart and recurrent respiratory papillomatous hyperplasia.It is relevant to human tumor
High-risk HPV has forties kinds, mainly includes HPV16 wherein common, and 18,31,33,35,39,45,52,56,58,59,68
Equal types.
Cervical carcinoma is the current cause of disease uniquely specific tumour, it is the result of HPV persistent infection.In development of cancer
HPV DNA is integrated into human cel gene group, and transformant starts continuous expression HPV E6 and E7 albumen into the cell.HPV E6 egg
The white mediation by E6-AP (E6 associated protein), in conjunction with and the cancer suppressor protein p53 that degrades；HPV E7 albumen and suppression
Cancer protein pRb is combined, and so that pRb is discharged nuclear factor E2F, is caused cell hyperproliferation, and cell cycle regulating is out of control, is caused thin
The immortalization of born of the same parents and the generation for promoting cervical carcinoma.In the systematic evolution tree compared based on HPV E7 amino acid sequence similarity
In, there are the populations of two big sequence very high homologies: HPV16 germline includes HPV31 and HPV35；HPV18 germline include HPV45 and
HPV59。
The AV Avantage of Arbor Vita companyTMHPV E6 Test be a kind of quickly test high-risk HPV 16,18,
The Fast Detection Technique of 45 E6 cancer protein.Using product of cell lysis is extracted in Cervical scrapes sample, pass through capillarity
Power is drawn on test strip, to detect the E6 albumen in sample.Using the principle of antibody conjugates, core is the technology
Specifically bind the selection of the monoclonal pairing antibody of E6 albumen.Chinese patent CN105866420B proposes pairing detection
The source of mouse monoclonal antibody pair of HPV18 E7.Chinese patent CN108218983A discloses a kind of list of anti-HPV18 type E7 albumen
Clonal antibody, Test paper and detection kit.In existing HPV detection technique, single HPV hypotype can only be detected mostly,
A variety of monoclonal antibodies are needed to be used in combination when detecting a variety of hypotypes at the same time, and the effect is unsatisfactory for differentiation hypotype, it is practical
Complicated for operation, higher cost.
Since the high-risk hypotype of HPV is more, existing technology have the defects that when detecting a variety of hypotypes at the same time it is certain, because
This, a variety of hypotypes, and the detection technique of high specificity, high sensitivity can be identified simultaneously by needing to develop one kind.
Summary of the invention
In view of the problems of the existing technology, a variety of hypotype HPV E7 albumen can be detected to the present invention simultaneously by providing one kind,
And the detection method of high specificity, high sensitivity.
One aspect of the present invention provides a kind of method using antibody test HPV E7 proteantigen, comprising the following steps:
(1) first antibody of immobilization is contacted with the HPV E7 albumen in sample, forms the first compound；
(2) it contacts the first compound in (1) to form the second compound with secondary antibody；
(3) the second compound in (2) is detected；
The first antibody is the rabbit resource monoclonal antibody for HPV E7 albumen；
The secondary antibody is the source of mouse monoclonal antibody for HPV E7 albumen with detection label；
It is described detection labeled as biotin labeling, colloid gold label, horseradish peroxidase label, radioisotope labeling,
Fluorescein label or nanometer particle to mark；Preferably, the detection is marked labeled as horseradish peroxidase.
Preferably, the HPV be one of HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, HPV68 or
A variety of hypotypes.
Preferably, the first antibody is the rabbit resource monoclonal that HPV E7 albumen difference epitope is directed to comprising 2 kinds
Antibody；The rabbit resource monoclonal antibody includes the rabbit resource monoclonal antibody that clone number is RAB-139 and RAB-034, is selected respectively
From Chinese Patent Application No. be CN2016108593095 and application No. is the monoclonal antibodies disclosed in CN2015104314074；
The weight ratio for cloning number rabbit resource monoclonal antibody for being RAB-139 and RAB-034 is 1:1.
Preferably, the source of mouse monoclonal antibody can successfully match with rabbit resource monoclonal antibody and identify a variety of hypotypes
The source of mouse monoclonal antibody of HPV E7 albumen.
The source of mouse monoclonal antibody be clone number for MAB-1-01, MAB-1-03, MAB-2-01, MAB-2-02,
MAB-4-03, MAB-4-04, MAB-4-05, MAB-4-25, MAB-4-26, MAB-4-27, MAB-4-28, MAB-4-29 and MAB-
One of source of mouse monoclonal antibody of 4-34；Preferably MAB-2-01.
Preferably, the source of mouse monoclonal antibody preparation method the following steps are included:
(A1) animal immune: immunogene is that GST-HPV18E7 recombinant protein is emulsified with complete Freund's adjuvant, subcutaneous or abdominal cavity
Mouse is immunized in injection system, and immunizing dose is 50 μ g/, immune to measure serum titer afterwards three times, and it is highest to choose antibody titer
Mouse is as immune mouse；
(A2) cell fusion and culture: the splenocyte for collecting the immune mouse that (A1) is obtained melts with myeloma cell
It closes, fusion ratio is splenocyte: myeloma cell=5:1, and HAT cultivates based selective culture, coated using His-HPV18E7
ELISA Plate carries out ELISA detection, obtains positive fused cell；
(A3) screen and clone: the positive fused cell for taking (A2) to obtain is subcloned using limiting dilution assay, is obtained
Stable positive cell strain, with His-HPV18 E7, His-HPV16 E7, His-HPV45 E7, His-HPV39/59 E7 mixing
Albumen and His-HPV6/11 E7 mixed protein are antigen, screen positive cell strain, obtain the hybridoma for combining HPV E7 albumen
Cell；
(A4) atoleine sensitized mice, the hybridoma that intraperitoneal injection (A3) obtains the preparation of monoclonal antibody: are used
Suspension, cell dosage be 1x106/ only, mouse ascites are extracted after a week, and ascites is handled through removal fibrin, purified, thoroughly
Analysis detects antibody protein concentration, purity, antibody titer, obtains source of mouse monoclonal antibody；
The amino acid sequence of the GST-HPV18 E7 recombinant protein is as shown in SEQ ID NO:1；
The recombinant protein amino acid sequence of the His-HPV16 E7 is as shown in SEQ ID NO:2；
The recombinant protein amino acid sequence of the His-HPV18 E7 is as shown in SEQ ID NO:3；
The recombinant protein amino acid sequence of the His-HPV39 E7 is as shown in SEQ ID NO:7；
The recombinant protein amino acid sequence of the His-HPV45 E7 is as shown in SEQ ID NO:8；
The recombinant protein amino acid sequence of the His-HPV59 E7 is as shown in SEQ ID NO:12；
The recombinant protein amino acid sequence of the His-HPV6 E7 is as shown in SEQ ID NO:14；
The recombinant protein amino acid sequence of the His-HPV11 E7 is as shown in SEQ ID NO:15.
Specifically, the source of mouse monoclonal antibody preparation method the following steps are included:
(A1) animal immune: taking 4-6 weeks female BAl BIc/C mice to be immunized, immunogene be GST-HPV18E7 albumen with
Complete Freund's adjuvant emulsification, subcutaneously or intraperitoneal injection mode is immune, and immunizing dose only, is immunized for 50 μ g/ and takes tail blood afterwards three times, adopt
Use His-HPV18 E7 recombinant protein (5 μ g/mL) as detection antigen coat ELISA method gradient dilution measurement serum titer, root
Booster immunization is determined whether according to result, chooses the highest mouse of antibody titer as immune mouse；
(A2) cell fusion and culture: splenocyte and the myeloma for collecting the immune mouse that (A1) is obtained according to a conventional method are thin
Born of the same parents SP2/0 is merged, and fusion ratio is splenocyte: myeloma cell's SP2/0=5:1, HAT culture based selective culture is set
In 37 DEG C, 5%CO2It cultivates in constant incubator, is changed full liquid 3 times with HAT culture medium, cell to be fused covers with a microscope view
When wild, ELISA detection is carried out using the coated ELISA Plate of His-HPV18E7, obtains positive fused cell；
(A3) screen and clone: the positive fused cell for taking (A2) to obtain is subcloned using limiting dilution assay, takes one
150 μ l HAT culture solutions are added in every hole in 96 porocyte culture plates of block, gently blow and beat to the positive fused cell that need to be subcloned outstanding
It is floating, it draws 100 μ l cell suspensions and is added in 96 porocyte plates, the doubling dilution since the first hole；Kong Zhongyue is chosen in cell count
There is the hole of 100 cells and be added in the loading slot containing 6mL HAT culture solution, is added to the thin of paving feeder layer by 100 holes μ l/
In born of the same parents' plate, add first three columns；Add HAT culture medium 3mL again, 100 holes μ l/ are added in the cell plates of paving feeder layer, add in three column；Again
HAT culture medium 5mL is added, 100 holes μ l/ are added in the cell plates of paving feeder layer, six column after adding.The positive rate for calculating every block of plate reaches
When to 100%, stable positive cell strain is obtained, with His-HPV18 E7, His-HPV16 E7, His-HPV45 E7, His-
HPV39/59 E7 mixed protein and His-HPV6/11 E7 mixed protein are antigen, screen positive cell strain, obtain and combine HPV
The hybridoma of E7 albumen；
(A4) preparation of monoclonal antibody: the hybridoma that mass propgation (A3) obtains, the BALB/ female of 8-10 week old
Mouse uses atoleine sensitization in advance, and hybridoma suspension is injected intraperitoneally, and cell dosage is 1x106/ only, it extracts after a week small
Mouse ascites fluid, ascites, with the purifying of ProteinA/G affinity column chromatography method, collect protein peak outflow after removal fibrin processing
Liquid is dialysed with phosphate buffer (PBS), with ultraviolet specrophotometer OD260,280 measurement antibody protein concentration, SDS-PAGE
Purity is detected, indirect ELISA detects antibody titer, obtains source of mouse monoclonal antibody.
Preferably, the sample includes but is not limited to that human or animal tissues sample, tumor resection sample, uterine neck fall off carefully
Born of the same parents, liquid basal cell sample；Wherein tumour includes uterine neck, penis, perineum, vagina, anus and pars oralis pharyngis cancer and precancerous lesion etc.
Related neoplasms.
Another aspect of the present invention provides a kind of for detecting the detection plate of HPV E7 albumen, and the detection plate is ELISA Plate
Or lateral flow type detection plate.
Preferably, above-mentioned first antibody is coated in the ELISA Plate；The peridium concentration of first antibody is 1 μ g/mL.
The lateral flow type detection plate includes substrate and test-strips, and test-strips are pasted on substrate, test-strips by filter sample paper,
Chromatographic material, nitrocellulose filter and blotting paper successively overlap composition；
The filter sample paper is antigen sample application zone, and the chromatographic material is above-mentioned secondary antibody combined area, described
Above-mentioned first antibody can be fixed on nitrocellulose filter；The first antibody is fixed on the test-strips, the secondary antibody
It is flowable to be set to the test-strips；" fixation " refer to when being detected described in first detection antibody will not exist with liquid
Flowed in the detection plate, it is described it is " flowable " refer to, the secondary antibody can be with liquid in the detection when being detected
It is flowed on plate.The use concentration of first antibody is 1 μ g/mL, and secondary antibody is 1 μ g/mL using concentration.Another aspect of the present invention is also
Provide a kind of for detecting the kit of HPV E7 albumen, the kit includes above-mentioned detection plate, above-mentioned first
Antibody, above-mentioned secondary antibody.
Preferably, the kit further includes buffer and cell cracking agent,
The buffer includes but is not limited to Tris-HCl buffer, PBS buffer solution etc., the buffer and cell
Lytic reagent is available on the market.
Another aspect of the present invention additionally provides above-mentioned kit and examines in the tumour cell HPV E7 albumen of non-diagnostic purpose
Application in survey.
Compared with prior art, positive and beneficial effect of the invention is:
The present invention provides one kind can detect simultaneously the methods of a variety of hypotype HPV E7 albumen, source of mouse monoclonal antibody and
Its application in detection card and immunity detection reagent.The source of mouse antibody titer that the present invention is prepared is high, can
The specific E7 albumen with more kinds of hypotypes of HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, HPV68 is combined；
Present invention uses the rabbit resource monoclonal antibody and source of mouse monoclonal antibody that can identify more hypotypes respectively, these hypotypes include low danger HPV
With high-risk HPV hypotype, high-risk HPV hypotype is only identified and the low danger HPV hypotype of nonrecognition behind rabbit source and the success of source of mouse antibody conjugates,
On the basis of guaranteeing that detection method is specific, the sensitivity of detection is significantly improved；On the other hand, utilization is provided by the invention
HPV E7 protein content in detection method, detection card and immunity detection reagent test sample, it is simple, convenient.
Detailed description of the invention
Fig. 1 is the ELISA testing result of hybridoma cell strain in embodiment 1.
Fig. 2 is the pairing ELISA testing result of source of mouse monoclonal antibody and rabbit resource monoclonal antibody in embodiment 2.
Fig. 3 is the ELISA testing result that monoclonal antibody identifies high-risk HPV E7 albumen in embodiment 3.
Fig. 4 is the HPV E7 protein subunit analysis detection result that double crush syndrome combines.
Specific embodiment
Following non-limiting embodiment can make those skilled in the art that the present invention be more completely understood, but not with
Any mode limits the present invention.It will be apparent to those skilled in the art that according to present disclosure combination this field skill
Art common sense, amino acid sequence disclosed in the present application can be synthesized according to other methods commonly used in the art, such as be closed by chemistry
At method obtain sequence disclosed in the present application.Following the description be only to this application claims range exemplary theory
Bright, those skilled in the art can make a variety of changes and modification to present invention according to disclosure of that, and its
Should belong to this application claims range among.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, and usually this field is normal
Technological means is advised, or according to normal conditions such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold
Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
ELISA detection method used in the present invention is specific as follows:
(1) envelope antigen, 100 holes μ l/, 4 DEG C overnight；
(2) 1 × PBST (1 × PBS formula are as follows: sodium chloride (NaCl) 5g, potassium chloride (KCl), 0.125g potassium dihydrogen phosphate
(KH2PO4) 0.125g, disodium hydrogen phosphate (Na2HPO4·12H2O) 1.81g, distilled water add to 1000mL；Extremely with 1M HCl tune pH
7.2, it is configured to 1 × PBS.It using in preceding 1 × PBS of Tween-20 to 1000mL that 1mL is added, stirs and evenly mixs, is 1 × PBST
Washing lotion.) washed once, it pats dry；
(3) 5% skimmed milk power/PBS closing is added, 300 holes μ l/, 37 DEG C, 2h, 1 × PBST are washed three times, are patted dry；
(4) every hole is separately added into the antibody or solution (serum, hybridoma for having antibody mediated effect that 100 μ l concentration are 1 μ g/ml
The solution of cell preparation or monoclonal antibody etc.), 37 DEG C of incubation 1h, 1 × PBST washings three times, pat dry；
(5) sheep anti-mouse igg-HRP secondary antibody (being purchased from: Sigma company, article No.: A2554) is added in every hole, using 5%BSA/
PBS 1:10000 is diluted, 100 holes μ l/, and 37 DEG C of incubation 1h, 1 × PBST washings three times, pat dry；
(6) TMB develops the color: TMB A liquid (takes sodium acetate (CH3COONa) 13.6g, citric acid (C6H8O7·H2O) 1.6g, dioxygen
Water (H2O230%) 0.3mL, distilled water add to 500mL) and B liquid (take disodium ethylene diamine tetraacetate (EDTA-Na2) 0.2g, lemon
Acid (C6H8O7·H2O) 0.95g, tetramethyl benzidine (TMB) 0.2g, distilled water add to 500mL) mixing in equal volume, 100 μ l/
Hole, color development at room temperature 5min；
(7) 2M H is added2SO4, 50 holes μ l/ terminate reaction, with microplate reader wavelength be 450nm at detect OD value.
The preparation of embodiment 1:HPV18E7 protein monoclonal antibody
(A1) animal immune: taking nine 4-6 weeks female BAl BIc/C mices to be immunized, and immunogene is GST-HPV18E7 egg
White (amino acid sequence of GST-HPV18E7 recombinant protein is as shown in SEQ ID NO:1) is emulsified with complete Freund's adjuvant, it is subcutaneous or
Intraperitoneal injection mode is immune, and immunizing dose is 50 μ g/, immune to take tail blood afterwards three times, using His-HPV18E7 recombinant protein (5
μ g/mL) as detection antigen coat, ELISA method gradient dilution measures serum titer, determines whether booster immunization according to result,
The highest mouse of antibody titer is chosen as immune mouse.
(A2) cell fusion and culture: splenocyte and the myeloma for collecting the immune mouse that (A1) is obtained according to a conventional method are thin
Born of the same parents SP2/0 is merged, and fusion ratio is splenocyte: myeloma cell's SP2/0=5:1, HAT culture based selective culture is set
In 37 DEG C, 5%CO2It cultivates in constant incubator, is changed full liquid 3 times with HAT culture medium, cell to be fused covers with a microscope view
When wild, ELISA detection is carried out using the coated ELISA Plate of His-HPV18E7, ELISA testing result using OD value greater than 1 as
Positive hole, and rechecked.After continuous test positive twice, determine the positive hole cell for positive fused cell.
(A3) screen and clone: the positive fused cell for taking (A2) to obtain is subcloned using limiting dilution assay, takes one
150 μ l HAT culture solutions are added in every hole in 96 porocyte culture plates of block, gently blow and beat to the positive fused cell that need to be subcloned outstanding
It is floating, it draws 100 μ l cell suspensions and is added in 96 porocyte plates, the doubling dilution since the first hole；Kong Zhongyue is chosen in cell count
There is the hole of 100 cells and be added in the loading slot containing 6mLHAT culture solution, is added to the thin of paving feeder layer by 100 holes μ l/
In born of the same parents' plate, add first three columns；Add HAT culture medium 3mL again, 100 holes μ l/ are added in the cell plates of paving feeder layer, add in three column；Again
HAT culture medium 5mL is added, 100 holes μ l/ are added in the cell plates of paving feeder layer, six column after adding.The positive rate for calculating every block of plate reaches
When to 100%, stable positive cell strain is obtained, with His-HPV16 E7, His-HPV18 E7, His-HPV45 E7, His-
HPV39/59 E7 mixed protein and His-HPV6/11 E7 mixed protein (His-HPV16 E7, His-HPV18 E7, His-
The recombinant protein amino acid of HPV39 E7, His-HPV45 E7, His-HPV59 E7, His-HPV6 E7 and His-HPV11 E7
Sequence is respectively as shown in SEQ ID NO:2,3,7,8,12,14 and 15) it is antigen, positive cell strain is screened in ELISA detection,
ELISA testing result as shown in Figure 1, altogether obtain 13 plants combine immunogen protein hybridoma cell strain 1-01,1-03,2-01,
2-02,4-03,4-04,4-05,4-25,4-26,4-27,4-28,4-29,4-34.Hybridoma cell strain is shifted to cell culture
In plate, expand culture, and freeze.
(A4) preparation of monoclonal antibody: the hybridoma that mass propgation (A3) obtains, the BALB/C of 8-10 week old are female
Property mouse use atoleine sensitization in advance, hybridoma suspension is injected intraperitoneally, cell dosage is 1x106/ only, it sees after a week
It examines, after mouse web portion obviously expands to a certain extent, extracts ascites.After ascites is removed fibrin processing, with
The purifying of ProteinA/G affinity column chromatography method, collects protein peak efflux, is dialysed with phosphate buffer (PBS), with ultraviolet point
Light photometer OD260,280 measurement antibody protein concentration, SDS-PAGE detect purity, and indirect ELISA detects antibody titer, obtains
Source of mouse monoclonal antibody.Identify the IgG hypotype of source of mouse monoclonal antibody: monoclonal antibody purification uses PBS 1:
10000 dilutions are operated according to the subtype identification kit specification of Sigma company, and the hypotype of 13 kinds of source of mouse monoclonal antibodies is shown in
Table 1.
Table 1: source of mouse monoclonal antibody IgG hypotype
| Clone number | Hypotype |
| MAB-1-01 | IgG1 |
| MAB-1-03 | IgG1 |
| MAB-2-01 | IgG2a |
| MAB-2-02 | IgG1 |
| MAB-4-03 | IgG2a |
| MAB-4-04 | IgG2b |
| MAB-4-05 | IgG2b |
| MAB-4-25 | IgG3 |
| MAB-4-26 | IgG1 |
| MAB-4-27 | IgG2b |
| MAB-4-28 | IgG1 |
| MAB-4-29 | IgG1 |
| MAB-4-34 | IgG2a |
Embodiment 2: the pairing detection of source of mouse monoclonal antibody and rabbit resource monoclonal antibody
(1) (Chinese Patent Application No. is that the monoclonal that CN201610859309 is disclosed is anti-to rabbit resource monoclonal antibody RAB-139
Body) and RAB-034 (Chinese Patent Application No. is the monoclonal antibody that CN2015104314074 is disclosed) each 1 μ g/mL mixing packet
Quilt, 100 holes μ l/, 4 DEG C overnight；
(2) 1 × PBST washed once, and pat dry；
(3) 5% skimmed milk power/PBS closing is added, 300 holes μ l/, 37 DEG C, 2h, 1 × PBST are washed three times, are patted dry；
(4) it is separately added into His-HPV18 each 1 μ g/mL of E7 and His-HPV16 E7 recombinant protein, 100 holes μ l/ are as anti-
Original, 37 DEG C of incubation 1h, 1 × PBST washings three times, pat dry；
(5) it is separately added into 13 kinds of monoclonal antibodies, the 1 μ g/ml that embodiment 1 obtains, 100 holes μ l/, 37 DEG C of incubation 1h, 1 ×
PBST is washed three times, is patted dry；
(6) sheep anti mouse-HRP secondary antibody (being purchased from: Sigma company, article No.: A2554) is added in every hole, using 5%BSA/PBS
1:10000 is diluted, 100 holes μ l/, and 37 DEG C of incubation 1h, 1 × PBST washings three times, pat dry；
(7) TMB develops the color: TMB A liquid and B liquid mix in equal volume, 100 holes μ l/, color development at room temperature 5min；
(8) 2M H is added2SO4, 50 holes μ l/ terminate reaction, with microplate reader wavelength be 450nm at detect OD value.
Testing result as shown in Fig. 2, in addition to MAB-4-25, remaining monoclonal antibody can specifically with HPV18E7 albumen
In conjunction with.
Embodiment 3: screening can identify the source of mouse monoclonal antibody of a variety of hypotype HPV E7 albumen
3.1 monoclonal antibodies identify the ELISA testing result of high-risk HPV E7 albumen
According to hybridoma combination HPV recombinant protein subtype data, monoclonal antibody MAB-1-01, MAB-2-01 after purification is chosen,
This 7 kinds of monoclonal antibodies of MAB-2-02, MAB-4-03, MAB-4-27, MAB-4-28 and MAB-4-34 carry out subsequent experimental.
It is detected using ELISA detection method, uses His-HPV16 E7, His-HPV18 E7, His-HPV31 respectively
E7、His-HPV33 E7、His-HPV35 E7、His-HPV39 E7、His-HPV45 E7、His-HPV52 E7、His-HPV56
E7, His-HPV58 E7, His-HPV59 E7, His-HPV68 E7, His-HPV6 E7 and His-HPV11 E7 recombinant protein
(its recombinant protein amino acid sequence is respectively as shown in SEQ ID NO:2-15) is used as antigen, and 1 μ g/mL wrapper sheet, 4 DEG C overnight；
After PBST washing pats dry, the closing of 5% skimmed milk power, 37 DEG C of effect 2h are added；After PBST washing pats dry, it is separately added into above-mentioned 7 kinds
Source of mouse monoclonal antibody (1 μ g/mL) and rabbit resource monoclonal antibody RAB-139 and RAB-034 mixed liquid concentration are each 1 μ g/mL, and
Negative control group (negative control group addition PBS buffer solution) 37 DEG C of reaction 1h are set；After PBST washing pats dry, sheep anti mouse is added
IgG-HRP secondary antibody, TMB colour developing and 2M H2SO4Terminate reaction；It is read at OD450nm.Testing result is as shown in figure 3, each monoclonal
Antibody identifies that HPV hypotype is as shown in table 2.
Table 2: each monoclonal antibody identifies HPV hypotype
| Antibody cloning number | HPV hypotype |
| RAB-139 and RAB-034 | HPV16, HPV18, HPV31, HPV33, HPV52, HPV68 and HPV6 |
| MAB-1-01 | HPV18 and HPV45 |
| MAB-2-01 | HPV18, HPV35, HPV39, HPV45, HPV56, HPV11 |
| MAB-2-02 | HPV18 and HPV45 |
| MAB-4-03 | HPV18 |
| MAB-4-27 | HPV18E7, HPV39, HPV45 and HPV68 |
| MAB-4-28 | HPV18 |
| MAB-4-34 | HPV18 and HPV39 |
The HPV E7 protein subunit analysis that 3.2 double crush syndromes combine
This 5 plants of MAB-1-01, MAB-2-01, MAB-2-02, MAB-4-27 and MAB-4-34 is chosen according to the result in 3.1
Source of mouse monoclonal antibody carries out subsequent experimental.
It is detected using ELISA detection method, each 1 μ g/mL mixing of rabbit resource monoclonal antibody RAB-139 and RAB-034
Coating, 4 DEG C overnight；After PBST washing pats dry, the closing of 5% skimmed milk power, 37 DEG C of effect 2h are added；After PBST washing pats dry, point
The recombinant protein and each 1 μ g/mL of low danger HPV6 and HPV11E7 of each self-identifying of above-mentioned 5 plants of source of mouse monoclonal antibodies are not added,
100 holes μ l/ are as antigen, 37 DEG C of reaction 1h；After PBST washing pats dry, it is separately added into above-mentioned 5 plants of source of mouse monoclonal antibodies (1 μ g/
ML), 37 DEG C of reaction 1h；After PBST washing pats dry, sheep anti mouse-HRP secondary antibody, TMB colour developing and 2M H are added2SO4 is terminated；
It is read at OD450nm, testing result is as shown in Figure 4.When using rabbit source mixing monoclonal antibody coating, each monoclonal antibody identifies HPV
Hypotype is as shown in table 3.
Table 3: each monoclonal antibody identifies HPV hypotype
| Antibody cloning number | HPV hypotype |
| MAB-1-01 | HPV18 and HPV45 |
| MAB-2-01 | HPV18, HPV35 and HPV45 |
| MAB-2-02 | HPV18 and HPV45 |
| MAB-4-27 | HPV18 |
| MAB-4-34 | HPV18 |
Testing result shows that source of mouse monoclonal antibody MAB-2-01 provided by the invention can match with rabbit resource monoclonal antibody
To success, and the E7 albumen of a variety of high-risk HPV hypotypes can be effectively identified simultaneously, and the low danger HPV hypotype of nonrecognition, guaranteeing to examine
On the basis of survey method specificity, the sensitivity of detection is significantly improved.
Embodiment 4: a kind of for detecting the detection plate of HPV E7 albumen
4.1 ELISA Plate
First antibody (each 1 μ g/ of rabbit resource monoclonal antibody RAB-139 and RAB-034 is coated in commercially available conventional ELISA Plate
ML), 100 hole μ l/, 4 DEG C overnight, and 1 × PBST washing pats dry, and obtains the ELISA Plate for being coated with first antibody.
4.2 lateral flow type detection plates
Lateral flow type detection plate includes substrate and test-strips, and test-strips are pasted on substrate, and test-strips are by filter sample paper, chromatography material
Material, nitrocellulose filter and blotting paper successively overlap composition；
The filter sample paper is antigen sample application zone, and the chromatographic material is above-mentioned secondary antibody combined area, described
Above-mentioned first antibody can be fixed on nitrocellulose filter.
Embodiment 5: a kind of for detecting the kit of HPV E7 albumen
5.1 detection kits containing ELISA Plate
The kit include 4.1 in embodiment 4 described in ELISA Plate, independent packaging secondary antibody (source of mouse monoclonal is anti-
Body MAB-2-01), cell cracking agent (50mM Tris-HCl, 150mM NaCl, 1%NP-40,0.5% NaTDC,
0.1%SDS) with its other buffer solution (PBS buffer solution or Tris-HCl buffer etc.).
In addition, the kit further includes specification, the application method of the kit is recorded in the description.
The detection kit of 5.2 detection plates containing lateral flow type
The kit include 4.2 in embodiment 4 described in lateral flow type detection plate, independent packaging first antibody (rabbit source is single
Clonal antibody RAB-139 and RAB-034, concentration is than the mixed liquor for 1:1), (source of mouse monoclonal is anti-for the secondary antibody of independent packaging
Body MAB-2-01), cell cracking agent (50mM Tris-HCl, 150mM NaCl, 1%NP-40,0.5% NaTDC,
0.1%SDS) with its other buffer solution (PBS buffer solution or Tris-HCl buffer etc.).
In addition, the kit further includes specification, the application method of the kit is recorded in the description.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Aituojin Biological Medicine (Suzhou) Co., Ltd.
<120>a kind of method and its application for detecting more hypotype HPV E7 albumen
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Claims (10)
1. a kind of method using antibody test HPV E7 proteantigen, which comprises the following steps:
(1) first antibody of immobilization is contacted with the HPV E7 albumen in sample, forms the first compound；
(2) it contacts the first compound in (1) to form the second compound with secondary antibody；
(3) the second compound in (2) is detected；
The first antibody is the rabbit resource monoclonal antibody for HPV E7 albumen；
The secondary antibody is the source of mouse monoclonal antibody for HPV E7 albumen with detection label；
The detection is labeled as biotin labeling, colloid gold label, horseradish peroxidase label, radioisotope labeling, fluorescence
Element label or nanometer particle to mark.
2. the method according to claim 1, wherein the HPV be HPV18, HPV35, HPV39, HPV45,
One of HPV56, HPV58, HPV68 or a variety of hypotypes.
3. the method according to claim 1, wherein the first antibody is to be directed to HPV E7 egg comprising 2 kinds
The rabbit resource monoclonal antibody of Bai Butong epitope；
The rabbit resource monoclonal antibody includes the rabbit resource monoclonal antibody that clone number is RAB-139 and RAB-034.
4. the method according to claim 1, wherein the source of mouse monoclonal antibody can be with rabbit resource monoclonal
Antibody successfully matches and identifies the source of mouse monoclonal antibody of a variety of hypotype HPV E7 albumen.
5. the method according to claim 1, wherein the preparation method of the source of mouse monoclonal antibody include with
Lower step:
(A1) animal immune: immunogene is that GST-HPV18E7 recombinant protein is emulsified with complete Freund's adjuvant, subcutaneous or intraperitoneal injection
Mouse is immunized in mode, and immunizing dose is 50 μ g/, immune to measure serum titer afterwards three times, chooses the highest mouse of antibody titer
As immune mouse；
(A2) cell fusion is with culture: the splenocyte for collecting the immune mouse that (A1) is obtained is merged with myeloma cell, is melted
Composition and division in a proportion rate is splenocyte: myeloma cell=5:1, and HAT cultivates based selective culture, using the coated enzyme mark of His-HPV18E7
Plate carries out ELISA detection, obtains positive fused cell；
(A3) screen and clone: the positive fused cell for taking (A2) to obtain is subcloned using limiting dilution assay, is stablized
Positive cell strain, with His-HPV18E7, His-HPV16E7, His-HPV45E7, His-HPV39/59E7 mixed protein and
His-HPV6/11E7 mixed protein is antigen, screens positive cell strain, obtains the hybridoma for combining HPV E7 albumen；
(A4) preparation of monoclonal antibody: using atoleine sensitized mice, and the hybridoma that intraperitoneal injection (A3) obtains hangs
Liquid, cell dosage are 1x106/ only, mouse ascites are extracted after a week, and ascites is through the processing of removal fibrin, purifying, dialysis, inspection
Antibody protein concentration, purity, antibody titer are surveyed, source of mouse monoclonal antibody is obtained；
The amino acid sequence of the GST-HPV18E7 recombinant protein is as shown in SEQ ID NO:1；
The recombinant protein amino acid sequence of the His-HPV16E7 is as shown in SEQ ID NO:2；
The recombinant protein amino acid sequence of the His-HPV18E7 is as shown in SEQ ID NO:3；
The recombinant protein amino acid sequence of the His-HPV39E7 is as shown in SEQ ID NO:7；
The recombinant protein amino acid sequence of the His-HPV45E7 is as shown in SEQ ID NO:8；
The recombinant protein amino acid sequence of the His-HPV59E7 is as shown in SEQ ID NO:12；
The recombinant protein amino acid sequence of the His-HPV6E7 is as shown in SEQ ID NO:14；
The recombinant protein amino acid sequence of the His-HPV11E7 is as shown in SEQ ID NO:15.
6. the method according to claim 1, wherein the sample is human or animal tissues sample, tumour is cut
Except sample, cervical exfoliated cell, liquid basal cell sample.
7. a kind of for detecting the detection plate of HPV E7 albumen, which is characterized in that the detection plate is that ELISA Plate or lateral flow type are examined
Drafting board.
8. detection plate according to claim 7, which is characterized in that be coated with described in claim 1 in the ELISA Plate
First antibody；
The lateral flow type detection plate includes substrate and test-strips, and test-strips are pasted on substrate, and test-strips are by filter sample paper, chromatography
Material, nitrocellulose filter and blotting paper successively overlap composition；
The filter sample paper is antigen sample application zone, and the chromatographic material is secondary antibody combined area described in claim 1, institute
First antibody described in claim 1 can be fixed on the nitrocellulose filter stated.
9. a kind of for detecting the kit of HPV E7 albumen, which is characterized in that the kit includes described in claim 7
Detection plate, first antibody described in claim 1, secondary antibody described in claim 1.
10. kit according to claim 8, which is characterized in that the kit further includes that buffer and cell are split
Solve reagent.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114935649A (en) * | 2022-06-15 | 2022-08-23 | 北京热燕生物科技有限公司 | HPV (human papilloma virus) detection kit |
| CN115028689A (en) * | 2022-06-15 | 2022-09-09 | 北京热燕生物科技有限公司 | Bispecific detection kit for specifically detecting HPV6 and HPV11 |
| CN117447561A (en) * | 2023-10-26 | 2024-01-26 | 四川大学华西医院 | Preparation and application of human papillomavirus 16 type E7 protein |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103130890A (en) * | 2011-11-28 | 2013-06-05 | 常小迦 | Human papillomavirus (HPV) 16E7 monoclonal antibody and relevant hybridoma cell strain and application thereof |
| CN103254307A (en) * | 2012-02-16 | 2013-08-21 | 艾托金生物医药（苏州）有限公司 | HPV18E7 monoclonal antibody, hybridoma cell strain and application |
| WO2016045563A1 (en) * | 2014-09-26 | 2016-03-31 | 艾托金生物医药（苏州）有限公司 | Cervial cancer-related hpv e7 protein monoclonal antibody and use thereof |
| US20160154007A1 (en) * | 2010-12-27 | 2016-06-02 | Japan Health Sciences Foundation | Monoclonal antibody recognizing human papillomavirus (hpv) l2 protein and method for measuring hpv-neutralizing antibody titer using the same |
| CN105866420A (en) * | 2015-01-22 | 2016-08-17 | 艾托金生物医药（苏州）有限公司 | Method and apparatus for detecting immunogen |
| CN106366186A (en) * | 2015-07-21 | 2017-02-01 | 艾托金生物医药（苏州）有限公司 | Monoclonal antibody for recognizing HPV16 positive tumor cells, and applications thereof |
| CN107192830A (en) * | 2017-05-31 | 2017-09-22 | 珠海美华医疗科技有限公司 | Monoclonal antibody, Test paper and the detection kit of anti-HPV16 types E7 albumen |
| CN107556379A (en) * | 2016-07-01 | 2018-01-09 | 艾托金生物医药（苏州）有限公司 | Identify monoclonal antibody and its application of high-risk HPV E7 albumen |
| CN107868127A (en) * | 2016-09-28 | 2018-04-03 | 艾托金生物医药（苏州）有限公司 | A kind of monoclonal antibody for being used to detect Oncoprotein Expression in histopathologic slide |
| CN108586607A (en) * | 2018-04-13 | 2018-09-28 | 郑州大学 | The preparation method and applications of anti-HPV16 L1 protein monoclonal antibodies |
| CN109021097A (en) * | 2017-06-08 | 2018-12-18 | 艾托金生物医药（苏州）有限公司 | A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45 |
| CN109575130A (en) * | 2018-12-03 | 2019-04-05 | 艾托金生物医药（苏州）有限公司 | A kind of monoclonal antibody and its preparation and application detecting HPV18 E7 albumen |
-
2019
- 2019-05-22 CN CN201910430877.7A patent/CN110196332A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160154007A1 (en) * | 2010-12-27 | 2016-06-02 | Japan Health Sciences Foundation | Monoclonal antibody recognizing human papillomavirus (hpv) l2 protein and method for measuring hpv-neutralizing antibody titer using the same |
| CN103130890A (en) * | 2011-11-28 | 2013-06-05 | 常小迦 | Human papillomavirus (HPV) 16E7 monoclonal antibody and relevant hybridoma cell strain and application thereof |
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