CN106478784A - For detecting antigen and the related immune detection kit of anti-human papilloma virus (anti-HPV) antibody - Google Patents
For detecting antigen and the related immune detection kit of anti-human papilloma virus (anti-HPV) antibody Download PDFInfo
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Abstract
The present invention relates to a kind of human papillomavirus' HPV oncogene protein is as antigen, for detecting the antibody of anti-human papilloma virus (anti-HPV) HPV, its sequence such as SEQ ID NO:Shown in 5.Additionally provide the immunity detection reagent including above-mentioned antigen.And the application of above-mentioned antigen and test kit.The antigen for detecting anti-HPV antibody of the present invention can detect HPV antibody in blood or saliva with high sensitivity, high specific and high stability, such that it is able to have the Cervical intraepitheliaI neoplasia of HPV viral oncogenes conversion and cervical cancer to bring foundation for doctor Accurate Diagnosis patient, and then take treatment in time, to prevent cancer from occurring, or cancer diffusion, mitigate patient in time painful, the immunity detection reagent being made from quickly and easily can detect the HPV antibody in blood or saliva exactly, is suitable to large-scale promotion application.
Description
The application is is on 2 22nd, 2013 the applying date, Application No. 201310056516.3, invention entitled " be used for
The divisional application of the patent application of the antigen of detection anti-human papilloma virus (anti-HPV) antibody and related immune detection kit ".
Technical field
The present invention relates to antibody test technical field, particularly to anti-human papilloma virus (anti-HPV) (Human
Papillomavirus, HPV) antibody test technical field, specifically refer to a kind of for detecting anti-human papilloma virus (anti-HPV) antibody
Antigen and related immune detection kit, are used especially for domestic popular high-risk type human papillomavirus (HPV) hypotype
HPV16 and HPV18.
Background technology
Cervical cancer is the common female malignant of second, and people's papilloma virus (HPV) are the essential conditions that it occurs.
Although crowd's HPV infection is very universal, such as：35 years old later infection rate of cervix uteri high cancer incidence area women is still up to 20.8%.But exceed
80% HPV DNA positive women will not progress to cancer or precancerous lesion.Therefore HPV DNA detection defect is to differentiate one
Passing infection and the infection that can cause cervical lesionses, lead to higher false positive rate.Numerous studies show, HPV oncogene is expressed
Product (cancer protein oncoprotein) has very high carcinogenic activity, and therefore have can be used as ratio HPV DNA for these biomarkers
More accurately there is specific cervical cancer prediction index.The expression water of high-risk HPV E2, E6, E7 cancer protein (oncoprotein)
Gentle precancerous lesions of uterine cervix different phase is directly related.Brand-new cervical cancer Molecular screening can be built using this characteristic to refer to
Mark.
Research to HPV mechanism of carcinogenesis shows, the early expression albumen E6 of high-risk HPV, E7 albumen sending out in cervical cancer
Play an important role in life.In the cervical epithelial cellses of the high of cervical cell pathological changes and cancer patient HPV E6 and E7 albumen typically in
The E6 of overexpression, continuous expression and overexpression, E7 carcinogenic protein can be with host cell modulin (e.g., P53 and Rb albumen
Deng) interact, so that tumor-inhibiting factor is inactivated, lead to cellular immortalization and vicious transformation.
Except HPV DNA detection and the detection of HPV carcinogenic protein, in blood (body fluid) HPV antibody also demonstrate that to HPV infection and
Cervical cancer pathogenesis history has very big dependency.HPV L1 blood antibody available standards ELISA method detects, provides HPV infection to believe
Breath.With the prolongation of HPV infection time, HPV induction creates anti-HPV antibody, therefore can detect that anti-HPV antibody in serum.
Wikstrom etc. have detected the existing disease patients with condyloma acuminatum of 47 male and 32 male couple once having condyloma acuminatum medical history patient
HPV6 type and the serum IgG antibody of HPV11 type capsid protein, and compare with the male of 205 agenosomia condyloma acuminatum medical histories,
As a result, in serum, the IgG antibody of anti-HPV6 type is 35% in the male once having condyloma acuminatum medical history, in existing disease condyloma acuminatum
For 27%, and it is 10% in matched group.This shows that the appearance of anti-HPV6 IgG antibody occurs when the evening of condyloma acuminatum stadium is a little
Wait, the IgG positive also reflects and once infected this virus simultaneously.
Stone etc. is reported and is studied for the infection rate of 7218 HPV-16 of U.S. general population using the method, result
Show women positive rate 17.9%, male's positive rate 7.9% (Stone, K.M., et al., Seroprevalence of
Human Papillomavirus Type 16Infection in the United States.2002.The Journal
of Infectious Diseases 2002；186:1396–402).HPV L1 antibody ELISA method is in cooperation HPV vaccine
Once it was widely adopted during the research and development of (Merck company Gardasil preventative vaccine) and clinical trial.Luevano etc. uses egg
The blood serum sample of white chip example Patients with Cervical Cancer has carried out high throughput testing analysis (Luevano, M., et al., High-
Throughput Profiling of the Humoral Immune Responses Against Thirteen Human
Papillomavirus Types by Proteome Microarrays.2010.Virology.2010；405(1):31–
40), analyze the antibody profiling of 54604 kinds of albumen altogether of 13 HPV types, result indicates carcinogenic protein in blood
Antibody has stronger dependency (Luevano, M., et with cervical cancer, height pathological changes, low pathological changes and asymptomatic ordinary people
al.,High-Throughput Profiling of the Humoral Immune Responses Against
Thirteen Human Papillomavirus Types by Proteome
Microarrays.2010.Virology.2010；405(1):31–40).
Therefore, in order to realize high sensitivity, high specific and the high stability of HPV antibody test, it is desirable to provide new use
Antigen in detection anti-human papilloma virus (anti-HPV) antibody.
Content of the invention
In order to solve above-mentioned problem, it is an object of the invention to provide one kind is used for detecting that anti-human papilloma virus (anti-HPV) resists
The antigen of body and related immune detection kit, this be used for detecting anti-human papilloma virus (anti-HPV) antibody antigen can with high sensitivity,
HPV antibody in high specific and high stability ground detection blood or saliva, such that it is able to for doctor's Accurate Diagnosis patient's cervix uteri
Intraepithelial neoplasia and cervical cancer bring foundation, and then take treatment in time, to prevent cancer from occurring, or cancer diffusion, in time
Mitigate patient painful, the immunity detection reagent being made from quickly and easily can detect the HPV in blood or saliva exactly
Antibody, is suitable to large-scale promotion application.
To achieve these goals, in a first aspect of the present invention, there is provided one kind is used for detecting anti-human papilloma virus (anti-HPV)
The antigen of antibody, is characterized in, the described antigen for detecting anti-human papilloma virus (anti-HPV) antibody is selected from such as SEQ ID NO:1 institute
The aminoacid sequence that shows, such as SEQ ID NO:Aminoacid sequence shown in 2, such as SEQ ID NO:Aminoacid sequence shown in 3, such as
SEQ ID NO:Aminoacid sequence shown in 4, such as SEQ ID NO:Aminoacid sequence shown in 5 and SEQ ID NO:Shown in 1
Aminoacid sequence has aminoacid sequence and the SEQ ID NO of 80% homology:It is same that aminoacid sequence shown in 2 has 80%
The aminoacid sequence of source property and SEQ ID NO:Aminoacid sequence shown in 3 have 80% homology aminoacid sequence and
SEQ ID NO:Aminoacid sequence shown in 4 have 80% homology aminoacid sequence and with SEQ ID NO:Shown in 5
Aminoacid sequence has one or more of aminoacid sequence of 80% homology.
It is preferred that the described m+2 position of the antigen for detecting anti-human papilloma virus (anti-HPV) antibody, m+3 position, m+5
Position, m+6 position and m+8-m+12 amino acids are Asp, Ser, Glu, Glu, Asp, Glu, Ile, Asp and Gly successively, its
Middle m >=0.
It is preferred that the described antigen for detecting anti-human papilloma virus (anti-HPV) antibody adopts chemosynthesis or biological engineering side
Method obtains.Example：Recombiant protein, fusion protein.
In a second aspect of the present invention, there is provided a kind of immunity detection reagent, it is characterized in, including above-mentioned for examining
Survey the antigen of anti-human papilloma virus (anti-HPV) antibody.
It is preferred that described immunity detection reagent also includes carrier, described for detecting anti-human papilloma virus (anti-HPV) antibody
Antigen coat on the carrier.
It is preferred that described immunity detection reagent also include the anti-human papilloma virus (anti-HPV) antibody of labelling two resist.Two anti-use
To detect that the IgG antibody in human serum, IgM, or label used by IgA. can be horseradish peroxidases, alkali phosphatase,
Fluorescence molecule FITC (or other fluorescent labeling), chemiluminescence detection.Detection method can be development process, fluorescent method, chemistry
Luminous, Electrochemiluminescince carries out qualitative and quantitative analysis.
More preferably, the two of the anti-human papilloma virus (anti-HPV) antibody of described labelling resist is the anti-human of horseradish peroxidase-labeled
The two of human papillomavirus antibody resist.
In a third aspect of the present invention, there is provided the above-mentioned antigen for detecting anti-human papilloma virus (anti-HPV) antibody or above-mentioned
Immunity detection reagent application in anti-human papilloma virus (anti-HPV) antibody in detection human body fluid.
It is preferred that described body fluid is blood or saliva.
The beneficial effects of the present invention is：The antigen for detecting anti-human papilloma virus (anti-HPV) antibody of the present invention is selected from such as
SEQ ID NO:Aminoacid sequence shown in 1, such as SEQ ID NO:Aminoacid sequence shown in 2, such as SEQ ID NO:Shown in 3
Aminoacid sequence, such as SEQ ID NO:Aminoacid sequence shown in 4, such as SEQ ID NO:Aminoacid sequence shown in 5 and SEQ
ID NO:Aminoacid sequence shown in 1 has aminoacid sequence and the SEQ ID NO of 80% homology:Aminoacid sequence shown in 2
Row have aminoacid sequence and the SEQ ID NO of 80% homology:Aminoacid sequence shown in 3 has the ammonia of 80% homology
Base acid sequence and SEQ ID NO:Aminoacid sequence shown in 4 have 80% homology aminoacid sequence and with SEQ ID
NO:Aminoacid sequence shown in 5 has one or more of aminoacid sequence of 80% homology, and patient is being infected
Afterwards, can in serum and saliva of buccal cavity high specific, high sensitivity, detect the specific antibody of appearance with high specificity
IgM, IgA or IgG molecule, such that it is able to be doctor's Accurate Diagnosis patient's Cervical intraepitheliaI neoplasia and cervical cancer brings foundation,
And then take treatment in time, to prevent cancer from occurring, or cancer diffusion, mitigate the immunity inspection that patient is painful, makes with this in time
Test agent box, quickly and easily can detect the HPV antibody in blood or saliva exactly, be suitable to large-scale promotion application.
Specific embodiment
In order to be more clearly understood that the technology contents of the present invention, spy enumerates specific examples below and describes in detail.So
And, specific embodiment is merely for illustration, rather than limitation of the present invention.
First, materials and methods
1st, reagent and medicine
Horseradish peroxidase-labeled goat-anti people (Goat Anti-Human IgG, FC), purchased from Priece Product#
31413.
Sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate are purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Tween-20 is purchased from AMRESCO.Hyclone is purchased from henry Golden Horse biotechnology development corporation, Ltd. of Beijing unit.
2nd, instrument and consumptive material
Electronic balance (prunus mume (sieb.) sieb.et zucc. Teller-support benefit instrument Shanghai company limited)；85-1 constant temperature blender with magnetic force (Community of Jin Tan County city
Outstanding riel Electrical Appliances Co., Ltd)；MULTISKAN MK3 microplate reader (Thermo company)；Ultrapure water preparation device (the U.S.
MILLPORE company)；GZX-9240MBE digital display air dry oven (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.)；
WellWASK4MK2 washes trigger (purchased from Thermo company)；ELISA Plate (COSTAR company)；Single track, multichannel pipettor (Germany
Eppendorf company).
3rd, main solution system
It is coated buffer (PBS phosphate solution)：Potassium dihydrogen phosphate 0.2g, 12 water disodium hydrogen phosphate 2.9g, sodium chloride
8.0g, potassium chloride 0.2g, plus distilled water is to 1L, 4 degree of preservations.
Lavation buffer solution (PBS-Tween 20,0.1%)：Tween-20 1mL, is added to 1L dilution buffer (PBS)
In, stirring and evenly mixing, adjust pH value between 7.0～7.2.
Confining liquid：5%milk-PBS-Tween
Serum dilution：10%FBS-PBS-Tween
TMB shows liquid：Amresc, 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：Disodiumedetate 0.2g citric acid 0.95g glycerol 50ml tetramethyl benzidine 0.2g
Distilled water adds to 500ml.
Terminate liquid (2mol/L H2SO4)：Distilled water 178.3mL, Deca concentrated sulphuric acid (98%) 21.7mL.
4th, the foundation of indirect ELISA method
1) with PBS dilution HPV polypeptide antigen HPV16-1/HPV16-2/HPV16-3/HPV18-1/HPV18-2 concentration it is
4ug/ml, is coated costar lath 100ul/ hole, 4 DEG C overnight.
2) PBS washed once, and pats dry.
3) add 5% milk powder-PBS closing, 250ul/ hole, 37 degree 3 hours.
4) PBST washed once, and pats dry.
5) 10%FBS-PBST dilute serum 1：50 dilutions, agitator mixes.
6) serum, blank, negative control and the positive control having diluted, 50ul/ hole are added, 37 degree of incubations 1 are little
When.
7) 0.1%tween-PBS washs 5 times, pats dry.
8) add horseradish peroxidase-labeled goat-anti people, dilute (1 with 10%FBS-PBST：40000), 100ul/ hole,
37 degree are incubated 30 minutes.
9) 0.1%tween-PBS washs 5 times, pats dry.
10) TMB colour developing:TMB A liquid and the mixing of B liquid equal-volume, 100ul/ hole, 37 degree of 20min.
11)2M H2SO4, 50ul/ hole terminates, with microplate reader 450nm reading.
5th, kit forms：
This test kit is made up of following components：
1. it is coated one piece of the costar ELISA Plate of the antigen for detecting anti-human papilloma virus (anti-HPV) antibody of the present invention
2. horseradish peroxidase-labeled goat-anti people one manages (10ul)
Mono- bottle of 3.10*PBST (10ml)
One bottle of nitrite ion A liquid of 4.TMB (6ml), one bottle of B liquid (6ml)
5. one bottle of terminate liquid (6ml)
6th, envelope antigen
Polypeptide antigen is as follows
| HPV16-1 | KCDSTLRLCVQSTHVDIRTLE | SEQ ID NO：1 |
| HPV16-2 | PTLHEYMLDLQPETTDLYCYEQLNDSSEEE | SEQ ID NO：2 |
| HPV16-3 | LNDSSEEEDEIDGPAGQAEPDRAH | SEQ ID NO：3 |
| HPV18-1 | IDGVNHQHLPARRAEPQR | SEQ ID NO：4 |
| HPV18-2 | SDSEEENDEIDGVNHQHLPARRAEPQRH | SEQ ID NO：5 |
2nd, testing result
Test kit selects 5 polypeptide (SEQ ID NO：1, SEQ ID NO：2, SEQ ID NO：3, SEQ ID NO：4, SEQ
ID NO：5) as being coated selection, serum is detected by ELISA method.
1st, with envelope antigen and the ELISA Plate closed detects Healthy Human Serum, the original number of detection by ELISA method
As follows according to result；
Table one：HPV blood ELISA detects normal human serum result table
2nd, 2 polypeptide (SEQ ID NO are selected：3, SEQ ID NO：5) select as being coated and the ELISA Plate closed is led to
Cross ELISA method detection normal human serum, the raw data results of detection are as follows；
Table two：HPV blood ELISA detects normal human serum result table
3rd, 5 polypeptide (SEQ ID NO are selected：1, SEQ ID NO：2, SEQ ID NO：3, SEQ ID NO：4, SEQ ID
NO：5) ELISA Plate being coated and closing is selected to detect patients serum, the pathological diagnosis of patient by ELISA method as being coated
, as shown in table three, through HC2 pathological examination, result is as follows for wherein some patients for result：
Table three：HPV blood ELISA detects patients serum's result table
4th, 3 polypeptide (SEQ ID NO are selected：1, SEQ ID NO：3, SEQ ID NO：5) conduct is coated selection and is coated simultaneously
The ELISA Plate closed by ELISA method detect patients serum, the pathological diagnosis result of patient as shown in table four, in the middle part of it
Divide patient through HC2 pathological examination, result is as follows：
Table four：HPV blood ELISA detects patients serum's result table
3rd, data analysiss
1st, according to normal person's detected value, meansigma methodss, standard variance, CUTOFF value are calculated.Respectively by 44 in table one
Reaction OD value on HPVE7 polypeptide for the normal human serum is taken the mean, and calculates standard variance SD, and this test kit is determining
Normal person's meansigma methodss are selected to add the criterion as HPV yin and yang attribute for the standard variance of twice during CUTOFF value, result is as follows：
Cutoff computing formula is：Cutoff=meansigma methodss OD+2*SD standard variance
1.1 according to table one result, HPVE7 (SEQ ID NO：1, SEQ ID NO：2, SEQ ID NO：3, SEQ IDNO：4,
SEQ ID NO：5) 5 polypeptide detection normal human serum result statistical analysiss draw, result such as following table：
Table five：5 polypeptide serum ELISA detection data tables of HPV E7
| SEQ ID NO：1 | SEQ ID NO：2 | SEQ ID NO：3 | SEQ ID NO：4 | SEQ ID NO：5 | |
| Meansigma methodss | 0.292 | 0.279 | 0.382 | 0.301 | 0.383 |
| Standard variance | 0.098 | 0.100 | 0.111 | 0.076 | 0.111 |
| cutoff | 0.487 | 0.480 | 0.604 | 0.452 | 0.605 |
1.2 according to table two result, HPVE7 (SEQ ID NO：3 and SEQ ID NO：5) 2 polypeptides detect normal human serum
Result statistical analysiss draw, result such as following table：
Table six：2 polypeptide serum ELISA detection data tables of HPV E7
1.3 draw SEQ ID NO according to the result of table five and table six, relative analyses：3 and SEQ ID NO：5 this 2 polypeptides
The cutoff value drawing in different experiments is consistent.
2nd, by cutoff value, judge detection patients serum HPV negative, positive, exceed this cutoff value be
Be considered positive, as long as and a polypeptide test positive, that is, think there is HPV infection, contrast with HC2 diagnostic result simultaneously.
2.1 in the patients serum of detection 291 serum have HC2 diagnostic result, have the case 84 that pathological diagnosis are positive.
In pathology positive patients group (clinical diagnosises " goldstandard "), Serum Antibody Detection result and HC2 Molecular Detection result compare (see
Table 7), it is carried out contrast such as following table with this test kit detection HPV result：(count in this testing result as positive inclusion：
I () two Detection of antigen results have any one is positive findingses, and (ii) two Detection of antigen results are all positive findingses)
Table seven：According to the result of table three, for serum ELISA diagnostic result and the HC2 diagnostic result contrast table of patient
("+" represent that testing result is positive；"-" represents that testing result is negative)
Table eight：According to the result of table four, for serum ELISA diagnostic result and the HC2 diagnostic result contrast table of patient
("+" represent that testing result is positive；"-" represents that testing result is negative)
The HC2 result of table seven result and blood ELISA inspection result are carried out demographics analysis, result such as following table：
Table nine：HC2 result and blood ELISA Detection accuracy number and ratio contrast table
Other gynaecopathias are mainly pelvic inflammatory disease, ovarian cyst, the gynaecopathia such as hysteromyoma.
The HC2 result of table eight result and blood ELISA inspection result are carried out demographics analysis, result such as following table：
Table ten：HC2 result and blood ELISA Detection accuracy number and ratio contrast table
Other gynaecopathias are mainly pelvic inflammatory disease, ovarian cyst, the gynaecopathia such as hysteromyoma.
There are 10 serum to be there is no HC2 examining report in 2.2 detection patients serums, but pathological replacement be CIN and cervical cancer,
This test kit detects HPV result, patient's number and positive blood tests rate comparing result such as table.
Table 11：For there is no HC2 diagnostic result, but there is the patient of pathological findings total with HPV blood ELISA testing result
Knot ("+" represent that testing result is positive；"-" represents that testing result is negative)
Table 12：The CIN of no HC2 result, the number table of the patient blood ELISA detection of cervical cancer and cervical lesionses
| Pathology | Number (10) | SEQ ID NO：1 is positive | SEQ ID NO：3 is positive | SEQ ID NO：5 is positive |
| CINⅠ | 2 | 2 | 1 | 1 |
| CIN II, III | 7 | 4 | 5 | 5 |
| Cervical cancer | 1 | 0 | 1 | 1 |
The patient blood ELISA testing result statistical number of person of all CIN I II III and cervical cancer and positive ratio are tied by 2.3
Fruit such as table 13.
Table 13：The number of patient blood ELISA detection of all CIN, cervical cancer and cervical lesionses and accuracy rate ratio
Table
The aforementioned polypeptides of the present invention are selections through inventor and are examined in patient samples and normal human sample
Test card, and directly adopt GST-HPV16-E7 and GST-HPV118E7 total length fusion protein (respectively SEQ ID NO：6、SEQ
IDNO：Sequence shown in 7) detection serum, effect on driving birds is not good, refer to following experiments data.Or
4th, experiment material and ELISA method, with above first point, simply adopt polypeptide (SEQ ID NO：3, SEQ ID
NO：5) detect while, also adopt GST-HPV16-E7 and GST-HPV118E7 total length fusion protein as detection antigen, with
During PBS dilution GST-HPV16-E7, GST-HPV18-E7 total length fusion protein, concentration is 2ug/ml, and testing result is as follows：
1. according to experimental procedure, detection part patients serum and common human serum, testing result and patient's pathology report above
Accuse as shown in the table：
Five. experimental analysiss
1. by the testing result using full-length proteins and before using HPV polypeptide SEQ ID:3 and SEQ ID:5 two kinds of polypeptides
Joint detection results contrast, contrast such as following table：
Summarize：Summarize：HPV16-E7, HPV18-E7 total length fusion protein, in detection CIN, has certain during Patients with Cervical Cancer
Positive patients missing inspection, it is poor that susceptiveness detects than polypeptide.There is certain vacation when detecting common gynecological inflammation patient and general population
Positive rate, it is slightly poor that sensitivity detects than polypeptide.So, HPV16-E7 and HPV18-E7 full-length proteins are for detection CIN, cervical cancer
Etc. being not well suited for.
Blood enzyme mark immunoassay (ELISA) of the present invention, based on immune biochemical basis, provides high sensitivity, high
The Ag-Ab detection method of specificity and high stability.Patient, after being infected, can go out in serum and saliva of buccal cavity
Existing specific antibody IgM, IgA or IgG molecule.Detected special in patients serum or saliva with enzyme-labeled immunity analytic process ELISA
The antibody molecule of the anti-HPV of property.The present invention is by HPV virus high-risk HPV 16, the analysis of HPV18 gene order, with biological letter
Breath credit analysis is to antigenic theoretical prediction, and experimentally screening, the meaning of proof clinical practice.Multiple high by preparing
The antigen polypeptide of purity, and repeated screening, checking are carried out to preparing antigen with domestic people healthy person serum and infected person anteserum,
Obtained high sensitivity, purpose antigen HPV16-1, HPV16-2 of high specific and high stability, HPV16-3, HPV18-1,
HPV18-2.And prove to be attached to certain surface of solid phase carriers with these polypeptide antigens, its immune response activity can be kept.Surveying
Regularly, make to be reacted with the antigen of surface of solid phase carriers by inspection specimen.With washing method make on solid phase carrier formed antigen,
Antibody complex is separated with other materials, and the enzyme amount being finally incorporated on immobilization carrier becomes one with the amount of tested substance in specimen
Fixed ratio.After adding the substrate of enzyme reaction, then by colour developing detecting, thus can according to the depth of color reaction make qualitative or
Quantitative analyses.The blood ELISA detection kit of the present invention and DNA detection (HC2, CareHPV) are compared, and sampling uniformly, non-is invaded
Slightly property, operation is more quick, simply；Expense is cheap, easily promotes, is more suitable for Chinese market；The vacation that PCR method is brought can be avoided
The positive and false negative, substantially increase degree of accuracy and the accuracy of testing result；Dye VIA with traditional Pap smear, acetic acid,
Colposcopy is compared, more accurately, easily, quickly.ELISA detection method, can through the optimization in various parameters and experiment journey
Instrument as Large-scale Screening is used for the early screening of cervical cancer.
To sum up, the antigen for detecting anti-human papilloma virus (anti-HPV) antibody of the present invention can with high sensitivity, high specific and
HPV antibody in high stability ground detection blood or saliva, such that it is able to for doctor's Accurate Diagnosis patient's Cervical intraepitheliaI neoplasia
Bring foundation with cervical cancer, and then take treatment in time, to prevent cancer from occurring, or cancer diffusion, mitigate patient's pain in time
Hardship, the immunity detection reagent being made from quickly and easily can detect the HPV antibody in blood or saliva exactly, is suitable to big
Scale popularization and application.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that still can make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, description is considered as illustrative rather than limit
Property processed.
Claims (10)
1. a kind of antigen for detecting anti-human papilloma virus (anti-HPV) antibody it is characterised in that described for detecting anti-human papillary tumor
The aminoacid sequence of the antigen of antiviral antibody is as shown in SEQ ID NO.5.
2. a kind of antigen combination for detecting anti-human papilloma virus (anti-HPV) antibody it is characterised in that described antigen combination include as
The antigen of the aminoacid sequence shown in SEQ ID NO.5；
Preferably, described antigen combination also includes being selected from the group one or more antigen：As SEQ ID NO:Aminoacid shown in 1
The antigen of sequence, such as SEQ ID NO:The antigen of the aminoacid sequence shown in 2, such as SEQ ID NO:Aminoacid sequence shown in 3
Antigen, such as SEQ ID NO:The antigen of the aminoacid sequence shown in 4；It is highly preferred that described antigen combination also includes SEQ ID
NO:The antigen of the aminoacid sequence shown in 3.
3. a kind of compositionss are it is characterised in that described compositionss contain antigen or claim 2 institute described in claim 1
The antigen combination stated, and pharmaceutically acceptable carrier and/or adjuvant；
Preferably, described compositionss are reagent, vaccine or pharmaceutical composition.
4. a kind of immunity detection reagent it is characterised in that include according to claim 1 for detecting anti-human papillary tumor
The antigen combination for detecting anti-human papilloma virus (anti-HPV) antibody described in the antigen of antiviral antibody or claim 2.
5. immunity detection reagent as claimed in claim 4 is it is characterised in that described immunity detection reagent also includes carrying
Body, described antigen coat is on the carrier.
6. immunity detection reagent as claimed in claim 4 is it is characterised in that described immunity detection reagent also includes labelling
The two of the anti-human papilloma virus (anti-HPV) antibody of substance markers resist；
Preferably, described label is selected from the group：Horseradish peroxidase, alkali phosphatase, fluorescent labelling, chemiluminescence mark
Note.
7. antigen for detecting anti-human papilloma virus (anti-HPV) antibody according to claim 1, according to claim 2
For detecting the antigen combination of the anti-human papilloma virus (anti-HPV) antibody purposes in reagent preparation, wherein said reagent is used for detecting people
Human papillomavirus antibody.
8. purposes as claimed in claim 7 is it is characterised in that described reagent is additionally operable to diagnose Cervical intraepitheliaI neoplasia or uterus
Neck cancer.
9. antigen for detecting anti-human papilloma virus (anti-HPV) antibody according to claim 1, according to claim 2
Antigen combination or immunity detection reagent according to claim 4 for detecting anti-human papilloma virus (anti-HPV) antibody are detecting
Application in anti-human papilloma virus (anti-HPV) antibody in human body fluid；
Preferably, described body fluid is blood or saliva.
10. a kind of method of detection anti-human papilloma virus (anti-HPV) antibody is it is characterised in that methods described includes step：
Antigen described in claim 1 or the antigen combination described in claim 2 are contacted with sample to be tested, detects whether to be formed
Antigen antibody complex, wherein, the antibody in described antigen antibody complex is anti-human papilloma virus (anti-HPV) antibody, and described antigen resists
Antigen in nanocrystal composition is the antigen described in claim 1 or the antigen in the antigen combination described in claim 2；
Preferably, methods described is ELISA method.
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| CONRAD BLEUL,ET AL: "Human Papillomavirus Type 18 E6 and E7 Antibodies in Human Sera: Increased Anti-E7 Prevalence in Cervical Cancer Patients", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104004067B (en) | 2017-09-01 |
| CN106478784B (en) | 2019-10-25 |
| WO2014127741A1 (en) | 2014-08-28 |
| CN106632617B (en) | 2020-07-03 |
| CN104004067A (en) | 2014-08-27 |
| CN106632617A (en) | 2017-05-10 |
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