CN103940993A - Antigen and kit for detecting anti-human-papillomavirus antibody and application of antigen - Google Patents
Antigen and kit for detecting anti-human-papillomavirus antibody and application of antigen Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
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- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
The invention relates to human papillomavirus (HPV) oncogene protein as an antiagen for detecting an anti-human-papillomavirus antibody, and the antigen has an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3. The invention also provides an immunodetection kit which comprises the antigen for detecting anti-HPV antibody. The invention also provides application of the antigen used for detecting anti-HPV antibody to detect anti-HPV antibody in human blood or saliva. The antigen used for detecting anti-HPV antibody is capable of detecting an HPV antibody in blood or saliva with high sensitivity and high specificity, and thus the antigen helps to provide basis for doctors to accurately diagnose patient cervical intraepithelial neoplasia and cervical carcinoma transformed from HPV virus oncogene, and the immunodetection kit prepared by employing the antigen is capable of rapidly simply accurately detecting the PHV antibody in blood or saliva and is suitable for large-scale popularization and application.
Description
Technical field
The present invention relates to antibody test technical field, be particularly related to anti-human papilloma virus (anti-HPV) (HumanPapillomavirus, HPV) antibody test technical field, specifically refer to a kind of antigen for detection of anti-human papilloma virus (anti-HPV) antibody and related immune detection kit and application, can be used for domestic popular high-risk type Human infectious warts virus (HPV) hypotype HPV16 and HPV18.
Background technology
Cervical carcinoma is the common cancer of female reproductive system, occupies women's malignant tumour second.Large quantity research is found, Human infectious warts virus HPV(Human Papillomavirus) be the arch-criminal who causes cervical carcinoma, can also cause multiple other tumour, comprise genital tract, mammary gland, alimentary canal and respiratory cancer.The HPV propagation in population of China is in recent years growing on and on and is tending towards rejuvenation, therefore early detection, early diagnosis, the early intervention to cervical carcinoma and precancerous lesion seems very necessary.
1949, first Sttauss observed Human infectious warts virus (HPV) particle under Electronic Speculum in wart body leachate.German virologist zurHausen in 1974 etc. propose the imagination that HPV may be relevant with cervical cancer pathogenesis.World cervical carcinoma biological study (IBSCC) mechanism infects with the correlativity of cervical carcinoma and carries out reporting after large sample statistics HPV, in more than 93% cervical cancer tissues, can detect HPV-DNA.Along with deepening continuously of cervical cancer cell biology and molecular biology research, have in report 99.8% cervix cancer biopsy specimen and HPV-DNA can be detected, HPV infects with the generation of cervical intraepithelial neoplasia (CIN) and cervix cancer closely related, particularly high-risk HPV.Find at present more than 120 kind of HPV hypotype, according to pathogenicity size, HPV has been divided into two kinds of low risk and high-risk-types.Low risk mainly causes exophytic condyloma, uterine neck condyloma class pathology and the low cervix intraepithelial neoplasias (CIN I level) of genital tract anal skin and vagina bottom, and Main Subtype has HPV6,11,42,43,44 etc.High-risk-type mainly causes height cervix intraepithelial neoplasias, i.e. CIN II, III level and cervical carcinoma, and Main Subtype has HPV16,18,31,33,35,39,45,51,52,56,58,59 and 68 these 13 kinds of hypotypes.HPV infects hypotype and has diversity and regional disparity.The current research of international cancer research association (IARC) shows, HPV-16, and HPV-18 is modal two strain oncogenic viruses in Patients with Cervical Cancer.2002, United States Medicine association magazine was delivered a common recognition guide of being formulated by 29 authoritative medical institutions, the women that guideline recommendation is borderline lesion for cervical cell picture assay, and HPV detects should serve as first-selected screening technique.
Conventionally the detection HPV method adopting at present has the DNA of external import to detect (HC2, CareHPV), the PCR-fluorescence probe method that home and overseas research and development are produced.They and traditional detection method (Pap smear, acetic acid dyeing VIA, vaginoscopy) are compared, and have higher sensitivity and accuracy.But HC2 and CareHPV detection method adopt sampling in body, with aggressiveness, expense is more expensive, detection time long (HC2 needs at least 6 hours).PCR method adopts sampling in body, and sampling can not evenly, therefore have false negative.Pcr amplification technology itself has easy generation false positive simultaneously.
Ag-Ab detects the feature with high sensitivity and high specific, have at present some taking immune biochemical basis as basic high sensitivity, the Ag-Ab detection method of high specific, for example Chinese patent application CN200710168713.9, CN200810186360.X, CN200910001113.2, CN201010228811.9 and CN01810709.5, and Chinese granted patent ZL03815024.7, ZL200610002546.6.
Therefore, need to provide the more antigen for detection of anti-human papilloma virus (anti-HPV) antibody, the diversification of selecting to be provided for the antigen of HPV antibody test, realizes high sensitivity and the high specific of HPV antibody test.
Summary of the invention
The object of this invention is to provide a kind of antigen for detection of anti-human papilloma virus (anti-HPV) antibody and related immune detection kit and application thereof, should can high sensitivity and detect with high specificity the HPV antibody in blood or saliva for detection of antigen of anti-human papilloma virus (anti-HPV) antibody, thereby can be that doctor's Accurate Diagnosis patient cervical intraepithelial neoplasia becomes and cervix cancer is brought foundation, and then take in time to treat the immunity detection reagent of being made by it and can simply detect exactly the HPV antibody in blood or saliva fast, be suitable for large-scale promotion application.
To achieve these goals, in a first aspect of the present invention, a kind of antigen for detection of anti-human papilloma virus (anti-HPV) antibody is provided, the described antigen for detection of anti-human papilloma virus (anti-HPV) antibody is: n+1 position, n+2 position, n+4 to n+7 position, n+9 to n+12 position and n+18 amino acids are His, Lys, Ala, Ile, Val, Thr, Thr, Tyr, Asp, Ser and Gln successively, wherein n >=0.
The described antigen for detection of anti-human papilloma virus (anti-HPV) antibody is: m+2 position, m+3 position, m+5 position, m+6 position and m+8 to m+12 amino acids are Asp, Ser, Glu, Glu, Asp, Glu, Ile, Asp and Gly successively, wherein m >=0.
In an embodiment therein, the described antigen for detection of anti-human papilloma virus (anti-HPV) antibody has amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or has the amino acid sequence of 80% homology with the amino acid sequence shown in SEQ IDNO:1 or SEQ ID NO:2 or SEQ ID NO:3.
Preferably, the described antigen for detection of anti-human papilloma virus (anti-HPV) antibody adopts chemosynthesis or biological engineering method to obtain.Example: recombinant protein, fusion.
In a second aspect of the present invention, a kind of immunity detection reagent is provided, comprise the above-mentioned antigen for detection of anti-human papilloma virus (anti-HPV) antibody.
In a third aspect of the present invention, a kind of ELISA kit of anti-human papilloma virus (anti-HPV) antibody test is provided, it includes, the above-mentioned antigen coated ELISA Plate for detection of anti-human papilloma virus (anti-HPV) antibody, coated amount is 0.3-0.5 μ g/ hole.
Preferably, described coated amount is 0.4 μ g/ hole.
Preferably, described immunity detection reagent also comprises anti-human IgG antibody two anti-of mark.The two anti-IgG antibody that are used for detecting in human serum, IgM, or IgA. label used can be horseradish peroxidase, alkaline phosphatase, fluorescence molecule FITC(or other fluorescence labeling), chemiluminescence detection.Detection method can be development process, fluorescent method, and chemiluminescence, Electrochemiluminescince carries out qualitative and quantitative analysis.
More preferably, two of described mark anti-be anti-human IgG antibody two anti-of horseradish peroxidase-labeled.Be horseradish peroxidase-labeled goat-anti people (Goat Anti-Human IgG, Fc)
In a third aspect of the present invention, the above-mentioned antigen for detection of anti-human papilloma virus (anti-HPV) antibody or above-mentioned immunity detection reagent application in anti-human papilloma virus (anti-HPV) antibody in human body liquid are provided.
Preferably, above-mentioned Human infectious warts virus is Human infectious warts virus HPV16 or HPV18.
Preferably, described body fluid is blood or saliva.
Function and the protein sequence of HPV cancer protein are known, if but it is higher non-specific by there will be to adopt total length E7 or E2 protein sequence to detect antibody. reason is that HPV has more than 100 kind of hypotype, wherein great majority are low dangerous types, not cancer detection object. but the protein structure of low risk HPV and high-risk-type has a homology to a certain degree. at antibody horizontal, adopt full-length proteins as antigen, can obtain larger cross reaction. the target that the present invention detects is carcinogenic high-risk hypotype, the cancer protein E6 that its transformant produces, the serum IgG reaction that E7 or E2 cause.
The present invention is by synthesizing into polypeptide fragment by the sequence of prediction in theory, or genetic engineering builds recombinant protein fragment as detectable antigens, screen for different blood serum samples. with the high-risk positive patients blood serum sample of HPV, normal human serum sample, and serum of children sample (negative control), carry out experiment sieving for large sample crowd.Experiment sieving multiple albumen segments, polypeptide fragment.Finally select " the immune dominant epitope " that in crowd, show, the epitope that immune performance is strong.Use these antigen fragments can ensure the sensitivity detecting.Pass through the contrast to patient and normal person simultaneously, select specificity stronger strong, the antigen fragment little with low danger HPV cross reaction. carry out on this basis method optimization, develop adaptablely clinically, detect the detection kit product that canceration that HPV causes is reacted.
Beneficial effect of the present invention is: the antigen for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention has the amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3, patient is after by virus infections, can be in serum and saliva of buccal cavity high specific, detect in high sensitivity the specific antibody IgM occurring, IgA or IgG molecule, thereby can be that doctor's Accurate Diagnosis patient cervical intraepithelial neoplasia becomes and cervix cancer is brought foundation, and then take in time to treat, to prevent that cancer from occurring, or cancer diffusion, alleviate in time patient's misery, the immunity detection reagent made from this, can simply detect exactly fast the HPV antibody in blood or saliva, be specially adapted to domestic popular high-risk type Human infectious warts virus (HPV) hypotype HPV16 and HPV18, be suitable for large-scale promotion application.
Brief description of the drawings
Fig. 1 adopts immunity detection reagent of the present invention (HPV16-1) to detect specificity and the susceptibility ROC curve map of patient outcomes.
Fig. 2 adopts the antigen HPV16-1 for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention to detect serum normal distribution, wherein each figure represents a digital point, that is: a routine patient, normal person, or the OD value of immune reagent kit detection of the present invention for children's blood sample, the horizontal line in the middle of every block graphics represents the mean value of each group crowd testing result OD value.
Fig. 3 adopts the antigen HPV18-1 for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention to detect serum normal distribution, wherein each figure represents a digital point, that is: a routine patient, normal person, or the OD value of immune reagent kit detection of the present invention for children's blood sample, the horizontal line in the middle of every block graphics represents the mean value of each group crowd testing result OD value.
Embodiment
In order more clearly to understand technology contents of the present invention, spy enumerates following specific embodiment and describes in detail.But specific embodiment is only used for illustrating, instead of limitation of the present invention.
One, materials and methods
1, reagent and medicine
Horseradish peroxidase-labeled goat-anti people (Goat Anti-Human IgG, Fc), purchased from PrieceProduc t#31413.
Sodium chloride, potassium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate are purchased from Chemical Reagent Co., Ltd., Sinopharm Group.Tween-20 is purchased from AMRESCO.Hyclone is purchased from prosperous Golden Horse biotechnology development corporation, Ltd. of Beijing unit.
2, instrument and consumptive material
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); 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.); WellWASK4MK2 washes plate machine (purchased from Thermo company); ELISA Plate (COSTAR company); Single track, multichannel pipettor (German eppendorf company).
3, main solution system
Coated damping fluid (PBS phosphate solution): potassium dihydrogen phosphate 0.2g, 12 water sodium hydrogen phosphate 2.9g, sodium chloride 8.0g, potassium chloride 0.2g, adding distil water is to 1L, and 4 degree are preserved.
Lavation buffer solution (PBS-Tween20,0.1%): Tween-201mL, joins in 1L dilution buffer liquid (PBS), stirs and evenly mixs, between adjust pH to 7.0～7.2.
Confining liquid: 5%mi lk-PBS-Tween
Serum dilution: 10%FBS-PBS-Tween
TMB shows liquid: Amresc, and CAT NO.64285730, with front TMB background color colour developing A liquid and the mixing of B liquid equal-volume.
O substrate colour developing A liquid: sodium acetate 13.6g, citric acid 1.6g, hydrogen peroxide (30%) 0.3ml, distilled water adds to 500ml.
O substrate colour developing B liquid: disodium ethylene diamine tetraacetate 0.2g citric acid 0.95g glycerine 50ml tetramethyl benzidine 0.2g distilled water adds to 500ml.
Stop buffer (2mol/L H2SO4): distilled water 178.3mL, drips the concentrated sulphuric acid (98%) 21.7mL.
4, the foundation of indirect ELISA method
1) respectively with PBS dilution HPV polypeptide antigen HPV16-1, HPV16-2 and HPV18-1 concentration are 4 μ g/ml, coated costar lath 100 μ l/ holes, and 4 DEG C are spent the night.
2) PBS washs once, pats dry.
3) add the sealing of 5% milk powder-PBS, 250 μ l/ holes, 37 DEG C 3 hours.
4) PBST washs once, pats dry.
5) 10%FBS-PBST dilute serum 1:50 dilution, oscillator mixes.
6) add the serum having diluted, blank, negative control and positive control, 50 μ l/ holes, hatch 1 hour for 37 DEG C.
7) 0.1%tween-PBS washing 5 times, pats dry.
8) add horseradish peroxidase-labeled goat-anti people, with 10%FBS-PBST dilution (1:40000), 100 μ l/ holes, hatch 30 minutes for 37 DEG C.
9) 0.1%tween-PBS washing 5 times, pats dry.
10) TMB colour developing: TMB A liquid and B liquid equal-volume mix, 100 μ l/ holes, 37 DEG C of 20min.
11) 2M H
2sO
4, 50 μ l/ holes stop, with microplate reader 450nm reading.
5, kit composition:
This kit is made up of following components:
1. one of the costar ELISA Plate of the coated antigen for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention
2. horseradish peroxidase-labeled goat-anti people one manages that (10 μ l)
Mono-bottle of PBST of 3.10 times of concentration (10x) (10ml)
One bottle of liquid of 4.TMB nitrite ion A (6ml), one bottle of B liquid (6ml)
5. one bottle of stop buffer (6ml)
6, envelope antigen
The structure of E2 polypeptide antigen is as follows
| HPV16--1 | HKSAIVTLTYDSEWQRDQC | SEQ?I?D?NO：1 |
| HPV16-2 | GDICNTMHYTNWTHIYICEE | SEQ?I?D?NO：2 |
| HPV18-1 | EKTGILTVTYHSETQRTKC | SEQ?I?D?NO：3 |
Three polypeptide of experimental selection (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) are selected as coated, according to the method described above, are coated with respectively, and preparation ELISA kit, and carry out according to the method described above serum detection.
Two, testing result
1, detect a part of Healthy Human Serum by envelope antigen the ELISA Plate of sealing by ELISA method, the raw data results of detection is as follows;
Table one: HPV cancer protein serum antibody: ELISA detects normal person's result table
2,2 antigens of experimental selection (SEQ ID NO:1, SEQ ID NO:3) detect a part of Healthy Human Serum as envelope antigen the ELISA Plate of sealing by ELISA method respectively, and the raw data results of detection is as follows;
Table two: HPV cancer protein serum antibody: ELISA detects normal person's result table
3, detect patients serum by envelope antigen the ELISA Plate of sealing by ELISA method, as shown in Table, wherein part patient is through HC2 pathological examination for patient's pathological diagnosis result, and result is as follows:
Table three: HPV cancer protein serum antibody, ELISA detects patient outcomes table
4,2 antigens of experimental selection (SEQ ID NO:1, SEQ ID NO:3) envelope antigen the ELISA Plate of sealing detect a part of patients serum by ELISA method, and the raw data results of detection is as follows;
Table four: HPV cancer protein serum antibody, ELISA detects patient outcomes table
Three, data analysis
1, according to normal person's detected value, calculate mean value, standard variance, CUTOFF value.Respectively by 31 routine normal human serums at SEQ ID NO:1, reaction OD value on SEQ ID NO:2 and SEQ ID NO:3 is taken the mean, and calculate standard variance SD, this kit selects normal person's mean value to add that the standard variance of twice is as the criterion of HPV yin and yang attribute in the time determining CUTOFF value, and result is as follows:
Cutoff computing formula is: cutoff=mean value OD+2*SD standard variance
Table five: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3ELISA detect tables of data one and add up
2, by cutoff value, feminine gender, the positive of patients serum HPV that judgement detects, what exceed this cutoff value thinks the positive, and as long as a polypeptide test positive think and have HPV to infect, the while contrasts with HC2 diagnostic result.
2.1 detect patients serum in 54 routine serum have HC2 diagnostic result, itself and this kit are detected to HPV result to be contrasted as following table: (in this testing result, adding up positive comprising: (i) to have any one be positive findings to two antigen testing results, and (ii) two antigen testing results are all positive findingses)
Table six: test serum ELISA diagnostic result and HC2 diagnostic result contrast table ("+" expression testing result positive for patient according to table four; "-" represents testing result feminine gender)
The HC2 result of table six result and blood ELISA check result are carried out to demographics analysis, and result is as following table:
Table seven: HC2 result and blood ELISA Detection accuracy number and ratio contrast table
Other gynecological diseases are mainly cervical polyp, the gynecological diseases such as condyloma.
In 2.2 detection patients serums, have 25 routine serum for there is no HC2 examining report, but pathological replacement is CIN, cervical carcinoma, this kit detects HPV result as table eight, and patient's number and blood testing positive rate comparing result are as table nine.
Table eight: test for there is no HC2 diagnostic result according to table four, but have the patient of pathology conclusion and HPV blood ELISA testing result to be summarized as follows:
("+" represents the testing result positive; "-" represents testing result feminine gender)
Table nine: test the CIN without HC2 result according to table four, number and accuracy rate schedule of proportion that the patient blood ELISA of cervical carcinoma detects
2.3 by all CIN I, II, and III, the patient blood ELISA testing result statistical number of person of cervical carcinoma and cervical lesions and positive ratio result are as table eight.
Table ten: test all CIN according to table four, number and accuracy rate schedule of proportion that the patient blood ELISA of cervical carcinoma and cervical lesions detects
Table ten one: test all CIN according to table three, number and accuracy rate schedule of proportion that the patient blood ELISA of cervical carcinoma and cervical lesions detects
Other gynecological diseases are mainly pelvic infecton, ovarian cyst, the gynecological diseases such as fibroid.
3, organize making ROC(ReceiverOperat ing Characteristic according to serum ELISA detection patient's group and normal person simultaneously) curve map, see Fig. 1.
The ROC curve of Fig. 1 is the serum ELISA(HPV16-1 that shows this kit) detect susceptibility and the specific relation of patient HPV, the connecting line at diagonal angle is the respective value of the mutual relationship of specificity and susceptibility.In this inspection, get susceptibility and the specific relation that corresponding both proportionate relationships show to detect by the serum ELISA testing result of antigen HPV16-1 patient HPV, in the time that susceptibility reaches 80%, specific performance reaches 90%.In the time that specificity is 80%, susceptibility also can reach 85%.The serum ELISA detection method that this kit is described has hypersensitivity and high specific.
4 detect serum OD value, patient HC2 pathological examination, patient's state of an illness pathological diagnosis result according to cutoff value comparison kit.
4.1HC2 makes a definite diagnosis totally 54 examples of HPV yin and yang attribute, and this kit blood ELISA antigen detect yin and yang attribute coincide be respectively that HPV16-1 is 41 examples, HPV18-1 is 43 examples.After having 8 examples for CIN and operation for cervical carcinoma in the serum of misfitting with HC2 diagnostic result (this 8 example is kit and detects and have 7 examples positive, and HC2 detects all negative).
4.2 in the cervical carcinoma that there is no HC2 diagnostic result, CIN, and cervical lesions serum amounts to 25 examples, wherein HPV blood ELISA test positive (HPV16-1 is 21 examples, sensitivity is that 84%, HPV18-1 is 23 examples, and sensitivity is 92%) (P value=0.0382<0.05).
4.3 cervical carcinomas that detect in table four experiment, CIN, cervical lesions serum amounts to 79 examples, kit HPV blood ELISA test positive be (diagnostic sensitivity is up to 79.7%, HPV18-1 for HPV16-1,63 examples, 65 examples, diagnostic sensitivity is up to 82.3%).
4.4 have HC2 diagnosis report and for cervical carcinoma, CIN, 48 routine serum (in table seven) of cervical lesions, diagnosing what can accurately detect by HC2 is 40 examples, accuracy rate is 83.3%, and this kit detection HPV can accurately detect 45 examples, and accuracy rate is 93.8%.Visible kit in gynaecology's examination Detection accuracy far above HC2 diagnosis effect.
Fig. 2 adopts the antigen HPV16-1 for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention to detect serum normal distribution, wherein each figure represents a digital point, that is: a routine patient, normal person, or the OD value of immune reagent kit detection of the present invention for children's blood sample, the horizontal line in the middle of every block graphics represents the mean value of each group crowd testing result OD value.
Fig. 3 adopts the antigen HPV18-1 for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention to detect serum normal distribution, wherein each figure represents a digital point, that is: a routine patient, normal person, or the OD value of immune reagent kit detection of the present invention for children's blood sample, the horizontal line in the middle of every block graphics represents the mean value of each group crowd testing result OD value.
Blood enzyme mark immunoassay of the present invention (ELISA), taking immune biochemical basis as basis, provides high sensitivity, the Ag-Ab detection method of high specific.After by virus infections, can in serum and saliva of buccal cavity, there is specific antibody IgM, IgA or IgG molecule in patient.Detect the antibody molecule of the anti-HPV of specificity in patients serum or saliva with enzyme-labeled immunity analytic approach ELISA.The present invention is by HPV virus high-risk HPV 16, the analysis of HPV18 gene order, with bioinformatic analysis to antigenic theoretical prediction, and with experimental technique screening, prove the meaning of clinical practice.By preparing multiple highly purified antigen polypeptides, and carry out repeated screening, checking with domestic population health person serum and infected person anteserum to preparing antigen, obtain and had the object of high specific antigen HPV16-1(SEQ ID No:1), HPV18-1(SEQ ID No:3).And prove to be attached to certain surface of solid phase carriers with these polypeptide antigens, can keep its immune response activity.In the time measuring, the antigen of being examined sample and surface of solid phase carriers is reacted.With washing method the antigen, the antibody complex that on solid phase carrier, form are separated with other materials, be finally that the enzyme amount being combined on immobilization carrier becomes certain ratio with the amount of tested substance in sample.Add after the substrate of enzyme reaction, then detect by developing the color, therefore can do qualitative or quantitative test according to the depth of color reaction.
Blood ELISA detection kit of the present invention and DNA detect (HC2, CareHPV) and compare, and evenly, non-aggressiveness, operates more quick, simple in sampling; Expense is cheap, easily promotes, and is more suitable for Chinese market; False positive and the false negative that can avoid PCR method to bring, the degree of accuracy and the accuracy that have greatly improved testing result; With traditional Pap smear, acetic acid dyeing VIA, vaginoscopy is compared, more accurate, easy, quick.The optimization of ELISA detection method in various parameters and experiment journey, can be used as the instrument of Large-scale Screening for the early screening of cervical carcinoma.
To sum up, antigen for detection of anti-human papilloma virus (anti-HPV) antibody of the present invention can high sensitivity and is detected with high specificity the HPV antibody in blood or saliva, thereby can be that doctor's Accurate Diagnosis patient cervical intraepithelial neoplasia becomes and cervix cancer is brought foundation, and then take in time to treat, to prevent that cancer from occurring, or cancer diffusion, alleviate in time patient's misery, the immunity detection reagent of being made up of it can simply detect the HPV antibody in blood or saliva fast exactly, is suitable for large-scale promotion application.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. for detection of the antigen of anti-human papilloma virus (anti-HPV) antibody, it is characterized in that, described antigen is: n+1 position, n+2 position, n+4 to n+7 position, n+9 to n+12 position and n+18 amino acids are His, Lys, Ala, Ile, Val, Thr, Thr, Tyr, Asp, Ser and Gln successively, wherein n >=0.
2. for detection of the antigen of anti-human papilloma virus (anti-HPV) antibody, it is characterized in that, described antigen is: m+2 position, m+3 position, m+5 position, m+6 position and m+8 to m+12 amino acids are Asp, Ser, Glu, Glu, Asp, Glu, Ile, Asp and Gly successively, wherein m >=0.
3. for detection of the antigen of anti-human papilloma virus (anti-HPV) antibody, it is characterized in that, described antigen is: have amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or have the amino acid sequence of 80% homology with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3.
4. an anti-human papilloma virus (anti-HPV) antibody assay kit, is characterized in that, includes the antigen for detection of anti-human papilloma virus (anti-HPV) antibody described in claim 1-3 any one.
5. an ELISA kit for anti-human papilloma virus (anti-HPV) antibody test, is characterized in that, includes the antigen coated ELISA Plate for detection of anti-human papilloma virus (anti-HPV) antibody described in claim 1-3 any one.
6. ELISA kit according to claim 5, is characterized in that, the amount of coated antigen is 0.3-0.5 μ g/ hole.
7. ELISA kit according to claim 6, is characterized in that, described coated antigen amount is 0.4 μ g/ hole.
8. according to the ELISA kit described in claim 5-7 any one, it is characterized in that, also include anti-human IgG two anti-of horseradish peroxidase-labeled.
9. the application in the anti-human papilloma virus (anti-HPV) antibody of the antigen described in claim 1-3 any one in human body liquid.
10. application according to claim 9, is characterized in that, described body fluid is blood or saliva.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045635.9A CN103940993A (en) | 2013-01-23 | 2013-02-05 | Antigen and kit for detecting anti-human-papillomavirus antibody and application of antigen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108872596A (en) * | 2018-07-05 | 2018-11-23 | 重庆巴而思生物科技有限公司 | A kind of ELISA detection kit of HPV16 L1 antibody |
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| WO1991018294A1 (en) * | 1990-05-11 | 1991-11-28 | Medscand Ab | Synthetic peptides of human papillomaviruses 1, 5, 6, 8, 11, 16, 18, 31, 33 and 56, useful in immunossay for diagnostic purposes |
| US5401627A (en) * | 1988-05-16 | 1995-03-28 | The Scripps Research Institute | Antibodies to human papillomavirus latent proteins, diagnostic systems and methods |
| CN1775802A (en) * | 2004-11-17 | 2006-05-24 | 宋硕 | Human papillomavirus immunogenic polypeptide, and its preparing method and use |
| CN1896105A (en) * | 2005-01-07 | 2007-01-17 | 胡耀雄 | Compounds and methods of early diagnosis of cervical cancer and genital condyloma with hpv, chsp60 tumor suppressor h-ras, k-ras and pten derived peptides modified |
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| US5401627A (en) * | 1988-05-16 | 1995-03-28 | The Scripps Research Institute | Antibodies to human papillomavirus latent proteins, diagnostic systems and methods |
| WO1991018294A1 (en) * | 1990-05-11 | 1991-11-28 | Medscand Ab | Synthetic peptides of human papillomaviruses 1, 5, 6, 8, 11, 16, 18, 31, 33 and 56, useful in immunossay for diagnostic purposes |
| CN1775802A (en) * | 2004-11-17 | 2006-05-24 | 宋硕 | Human papillomavirus immunogenic polypeptide, and its preparing method and use |
| CN1896105A (en) * | 2005-01-07 | 2007-01-17 | 胡耀雄 | Compounds and methods of early diagnosis of cervical cancer and genital condyloma with hpv, chsp60 tumor suppressor h-ras, k-ras and pten derived peptides modified |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108872596A (en) * | 2018-07-05 | 2018-11-23 | 重庆巴而思生物科技有限公司 | A kind of ELISA detection kit of HPV16 L1 antibody |
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