CN106632617A - Antigen for detecting human papillomavirus antibody and related immunoassay kit - Google Patents
Antigen for detecting human papillomavirus antibody and related immunoassay kit Download PDFInfo
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- CN106632617A CN106632617A CN201610854355.6A CN201610854355A CN106632617A CN 106632617 A CN106632617 A CN 106632617A CN 201610854355 A CN201610854355 A CN 201610854355A CN 106632617 A CN106632617 A CN 106632617A
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/084—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a human papillomavirus (HPV) oncogene protein which is used for detecting an HPV antibody as an antigen. The sequence of the antigen is shown in SEQ ID NO: 4. The invention further provides an immunoassay kit containing the antigen and an application of the antigen and the kit. The antigen for detecting the HPV antibody, provided by the invention, can be used for detecting the HPV antibody in blood or saliva in a high-sensitivity, high-specificity and high-stability manner, so that a foundation for accurately diagnosing the cervical intraepithelial neoplasias and cervical cancer, transformed from HPV oncogene, of patients by doctors can be brought, and then, treatment is adopted in time so as to prevent cancer occurrence or cancer diffusion and relieve patient's sufferings in time; and the immunoassay kit prepared from the antigen can be used for rapidly, simply and accurately detecting the HPV antibody in the blood or saliva, thereby being suitable for being popularized and applied on a large scale.
Description
The application be the applying date on 2 22nd, 2013, Application No. 201310056516.3, entitled " be used for
The divisional application of the patent application of the antigen and related immune detection kit of detection anti-human papilloma virus (anti-HPV) antibody ".
Technical field
The present invention relates to antibody test technical field, more 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 high-risk type HPV (HPV) hypotype of domestic prevalence
HPV16 and HPV18.
Background technology
Cervix cancer is the common female malignant of second, and people's papilloma virus (HPV) are the necessary conditions that it occurs.
Although crowd's HPV infection is very universal, such as：35 years old later infection rate of uterine neck high cancer incidence area women is still up to 20.8%.But exceed
80% HPV DNA positive womens will not progress to cancer or precancerous lesion.Therefore HPV DNA detections defect is cannot to differentiate one
Passing infection and the infection that can cause cervical lesionses, cause higher false positive rate.Numerous studies show that HPV oncogene is expressed
Product (cancer protein oncoprotein) has a very high carcinogenic activity, thus these biomarkers have can be used as than HPV DNA
More accurately there is the cervical carcinoma prediction index of specificity.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 carcinoma 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 carcinoma
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 proteins can be with host cell modulin (e.g., P53 and Rb albumen
Deng) interact, TIF is inactivated, cause cellular immortalization and vicious transformation.
Except HPV DNA detections and the detection of HPV carcinogenic proteins, in blood (body fluid) HPV antibody also demonstrate that to HPV infection and
Cervical cancer pathogenesis history has very big correlation.HPV L1 blood antibody available standards ELISA method detects, provides HPV infection and believe
Breath.With the prolongation of HPV infection time, HPV inductions generate 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 sex and 32 male sex couple for once having a condyloma acuminatum medical history patient
The serum IgG antibody of HPV6 types and HPV11 type capsid proteins, and compare with the male sex of 205 agenosomia condyloma acuminatum medical histories,
As a result, the IgG antibody of anti-HPV6 types is 35% in once having the male sex of condyloma acuminatum medical history in serum, in existing disease condyloma acuminatum
For 27%, and it is 10% in control group.This shows that the appearance of anti-HPV6IgG antibody occurs when the evening of condyloma acuminatum stadium is a little
Wait, while the IgG positives also reflect once infected this virus.
Stone etc. reports the infection rate research using the method for 7218 HPV-16 of U.S. general population, as a result
Show women positive rate 17.9%, (Stone, K.M., et al., the Seroprevalence of of male sex's positive rate 7.9%
Human Papillomavirus Type 16Infection in the United States.2002.The Journal
of Infectious Diseases2002；186:1396–402).HPV L1 antibody ELISAs methods are coordinating HPV vaccines
Once it was widely adopted during the research and development and clinical testing of (Merck companies Gardasil preventative vaccines).Luevano etc. uses egg
The blood serum sample of white chip example Patients with Cervical Cancer has carried out high flux detection and 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) the antibody profiling of 54604 kinds of albumen altogether of 13 HPV types, is analyzed, carcinogenic protein in blood is as a result indicated
Antibody has stronger correlation (Luevano, M., et with cervical carcinoma, height pathology, low pathology 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 tests, it is desirable to provide new use
In the antigen of detection anti-human papilloma virus (anti-HPV) antibody.
The content of the invention
In order to solve above-mentioned problem, it is an object of the invention to provide a kind of be used to detect that anti-human papilloma virus (anti-HPV) resists
The antigen and related immune detection kit of body, this be used to detecting anti-human papilloma virus (anti-HPV) antibody antigen can with high sensitivity,
High specific and high stability ground detection blood or the HPV antibody in saliva, such that it is able to for doctor's Accurate Diagnosis patient's uterine neck
Intraepithelial neoplasia (cin) and cervix cancer bring foundation, and then take treatment in time, to prevent cancer from occurring, or cancer diffusion, in time
Mitigate patient's pain, the immunity detection reagent being made from it quickly and easily can exactly detect blood or the HPV in saliva
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 to detect 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
Amino acid sequence, the such as SEQ ID NO for showing:Amino acid sequence, such as SEQ ID NO shown in 2:Amino acid sequence shown in 3, such as
SEQ ID NO:Amino acid sequence, such as SEQ ID NO shown in 4:Amino acid sequence and SEQ ID NO shown in 5:Shown in 1
Amino acid sequence has the amino acid sequence and SEQ ID NO of 80% homology:Amino acid sequence shown in 2 is same with 80%
The amino acid sequence and SEQ ID NO of source property:Amino acid sequence shown in 3 have 80% homology amino acid sequence and
SEQ ID NO:Amino acid sequence shown in 4 have 80% homology amino acid sequence and with SEQ ID NO:Shown in 5
Amino acid sequence has one or more in the amino acid sequence of 80% homology.
It is preferred that described m+2 positions, m+3 positions, m+5 for detecting the antigen of anti-human papilloma virus (anti-HPV) antibody
Position, m+6 positions and m+8-m+12 amino acids are successively Asp, Ser, Glu, Glu, Asp, Glu, Ile, Asp and Gly, its
Middle m >=0.
It is preferred that the described antigen for detecting anti-human papilloma virus (anti-HPV) antibody is using chemical synthesis or bioengineering side
Method is obtained.Example：Recombinant 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 it is above-mentioned for examining
Survey the antigen of anti-human papilloma virus (anti-HPV) antibody.
It is described for detecting anti-human papilloma virus (anti-HPV) antibody it is preferred that the immunity detection reagent also includes carrier
Antigen coat on the carrier.
It is preferred that the immunity detection reagent also includes that the two of the anti-human papilloma virus (anti-HPV) antibody of mark resist.Two anti-use
IgG antibody in detect human serum, IgM, or label used by IgA. can be horseradish peroxidases, alkaline phosphatase,
Fluorescence molecule FITC (or other fluorescence labelings), chemiluminescence detection.Detection method can be development process, and fluorescent method is chemical
Luminous, Electrochemiluminescince carries out qualitative and quantitative analysis.
More preferably, it is the anti-human of horseradish peroxidase-labeled that the two of the anti-human papilloma virus (anti-HPV) antibody of described mark resist
The two of 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
Application of the immunity detection reagent in detection human body fluid in anti-human papilloma virus (anti-HPV) antibody.
It is preferred that the 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:Amino acid sequence, such as SEQ ID NO shown in 1:Amino acid sequence, such as SEQ ID NO shown in 2:Shown in 3
Amino acid sequence, such as SEQ ID NO:Amino acid sequence, such as SEQ ID NO shown in 4:Amino acid sequence and SEQ shown in 5
ID NO:Amino acid sequence shown in 1 has the amino acid sequence and SEQ ID NO of 80% homology:Amino acid sequence shown in 2
Amino acid sequence of the row with 80% homology and SEQ ID NO:Amino acid sequence shown in 3 has the ammonia of 80% homology
Base acid sequence and SEQ ID NO:Amino acid sequence shown in 4 have 80% homology amino acid sequence and with SEQ ID
NO:Amino acid sequence shown in 5 has one or more in the amino acid 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 that doctor's Accurate Diagnosis patient Cervical intraepitheliaI neoplasia and cervix cancer bring foundation,
And then treatment is taken in time, to prevent cancer from occurring, or cancer diffusion, patient's pain is mitigated in time, with immunity inspection made by this
Test agent box, quickly and easily can exactly detect blood or the HPV antibody in saliva, 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 detailed description.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 (plum Teller-support benefit instrument Shanghai Co., Ltd)；85-1 constant temperature blender with magnetic force (Community of Jin Tan County city
Outstanding riel Electrical Appliances Co., Ltd)；MULTISKAN MK3 ELIASAs (Thermo companies)；Ultrapure water preparation device (the U.S.
MILLPORE companies)；GZX-9240MBE digital display air dry ovens (Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.)；
WellWASK4MK2 board-washing machines (are purchased from Thermo companies)；ELISA Plate (COSTAR companies)；Single track, multichannel pipettor (Germany
Eppendorf companies).
3rd, main solution system
Coating buffer solution (PBS phosphate solutions)：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, are added to 1L dilution buffers (PBS)
In, stir and evenly mix, between tune pH value to 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 colors colour developing A liquid and the mixing of B liquid equal-volume.
O substrates colour developing A liquid：Sodium acetate 13.6g, citric acid 1.6g, hydrogen peroxide (30%) 0.3ml, distilled water is added to
500ml。
O substrates 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.
Terminate liquid (2mol/L H2SO4)：Distilled water 178.3mL, is added dropwise the concentrated sulfuric acid (98%) 21.7mL.
4th, the foundation of indirect ELISA method
1) it is with PBS dilutions HPV polypeptide antigen HPV16-1/HPV16-2/HPV16-3/HPV18-1/HPV18-2 concentration
4ug/ml, coating costar lath 100ul/ hole, 4 DEG C overnight.
2) PBS washed once, and pat dry.
3) add 5% milk powder-PBS closing, 250ul/ holes, 37 degree 3 hours.
4) PBST washed once, and pat dry.
5) 10%FBS-PBST dilute serums 1：50 dilutions, oscillator is mixed.
6) serum for having diluted, blank, negative control and positive control are added, 50ul/ holes, 37 degree of incubations 1 are little
When.
7) 0.1%tween-PBS is washed 5 times, is patted dry.
8) horseradish peroxidase-labeled goat-anti people is added, with 10%FBS-PBST (1 is diluted：40000), 100ul/ holes,
37 degree are incubated 30 minutes.
9) 0.1%tween-PBS is washed 5 times, is patted dry.
10) TMB colour developings:TMB A liquid and B liquid equal-volume mix, 100ul/ holes, 37 degree of 20min.
11)2M H2SO4, 50ul/ holes terminate, with ELIASA 450nm readings.
5th, kit forms：
This kit is made up of following components：
1. one piece of the costar ELISA Plates for detecting the antigen of anti-human papilloma virus (anti-HPV) antibody of the present invention are coated with
2. horseradish peroxidase-labeled goat-anti people one manages (10ul)
Mono- bottle of 3.10*PBST (10ml)
One bottle of liquid of 4.TMB nitrite ion A (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
Kit selects 5 polypeptide (SEQ ID NO：1, SEQ ID NO：2, SEQ ID NO：3, SEQ ID NO：4, SEQ
ID NO：5) select as coating, 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
It is 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) ELISA Plate for selecting and closing as coating leads to
ELISA method detection normal human serum is crossed, 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 for being coated with and closing is selected to detect patients serum, the pathological diagnosis of patient by ELISA method as coating
As a result as shown in table three, which part patient is as a result as follows through HC2 pathological examinations：
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) coating is selected simultaneously as coating
The ELISA Plate closed detects patients serum by ELISA method, the pathological diagnosis result of patient as shown in table four, in the middle part of it
Divide patient through HC2 pathological examinations, it is as a result as follows：
Table four：HPV blood ELISA detects patients serum's result table
3rd, data analysis
1st, according to normal person's detected value, mean value, standard variance, CUTOFF values are calculated.Respectively by 44 in table one
Reaction OD value of the normal human serum on HPVE7 polypeptides is taken the mean, and calculates standard variance SD, this kit it is determined that
Normal person's mean value is selected to add the criterion of the standard variance as HPV yin and yang attributes of twice during CUTOFF values, it is as a result as follows：
Cutoff computing formula are：Cutoff=mean value OD+2*SD standard variances
1.1 according to the result of table one, HPVE7 (SEQ ID NO：1, SEQ ID NO：2, SEQ ID NO：3, SEQ ID NO：
4, SEQ ID NO：5) 5 polypeptide detection normal human serum result statistical analyses draw, as a 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 | |
| Mean value | 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 the result of table two, HPVE7 (SEQ ID NO：3 and SEQ ID NO：5) 2 polypeptides detect normal human serum
As a result statistical analysis draws, as a result such as following table：
Table six：2 polypeptide serum ELISA detection data tables of HPV E7
1.3 according to table five and the result of table six, and comparative analysis draws SEQ ID NO：3 and SEQ ID NO：5 this 2 polypeptides
The cutoff values drawn in different experiments are consistent.
2nd, by cutoff values, judge negative, the positive of the patients serum HPV of detection, be more than this cutoff value
Be considered positive, as long as and a polypeptide test positive, that is, think there is HPV infection, at the same with the contrast of HC2 diagnostic results.
2.1 in the patients serum of detection 291 serum have HC2 diagnostic results, have the case 84 that pathological diagnosis is positive.
In pathology positive patients group (clinical diagnosis " goldstandard "), Serum Antibody Detection result and HC2 Molecular Detection results compare (see
Table 7), it is contrasted such as following table with this kit detection HPV results：(statistics includes for positive in this testing result：
I it is positive findings that () two antigen testing results have any one, and (ii) two antigen testing results are all positive findingses)
Table seven：According to the result of table three, for serum ELISA diagnostic results and the HC2 diagnostic result contrast tables of patient
("+" represents that testing result is positive；"-" represents that testing result is negative)
Table eight：According to the result of table four, for serum ELISA diagnostic results and the HC2 diagnostic result contrast tables of patient
("+" represents that testing result is positive；"-" represents that testing result is negative)
The HC2 results of the result of table seven and blood ELISA inspection results are carried out into demographics analysis, as a result such as following table：
Table nine：HC2 results and blood ELISA Detection accuracies number and ratio contrast table
Other gynecological diseases are mainly the gynecological diseases such as pelvic infecton, ovarian cyst, fibroid.
The HC2 results of the result of table eight and blood ELISA inspection results are carried out into demographics analysis, as a result such as following table：
Table ten：HC2 results and blood ELISA Detection accuracies number and ratio contrast table
Other gynecological diseases are mainly the gynecological diseases such as pelvic infecton, ovarian cyst, fibroid.
There are 10 serum to be there is no HC2 examining reports in 2.2 detection patients serums, but pathological replacement is CIN and cervical carcinoma,
This kit detects HPV results, patient's number and positive blood tests rate comparing result such as table.
Table 11：For there is no HC2 diagnostic results, but there is the patient of pathological findings total with HPV blood ELISA testing results
Knot
("+" represents that testing result is positive；"-" represents that testing result is negative)
Table 12：The number table of the patient blood ELISA detections of the CIN without HC2 results, cervical carcinoma 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 carcinoma | 1 | 0 | 1 | 1 |
2.3 tie the patient blood ELISA testing results statistical number of person and positive ratio of all CIN I II III and cervical carcinoma
Fruit such as table 13.
Table 13：The number and accuracy rate ratio of the patient blood ELISA detections of all CIN, cervical carcinoma and cervical lesionses
Table
The aforementioned polypeptides of the present invention are the 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 proteins (respectively SEQ ID NO：6、SEQ
ID NO：Sequence shown in 7) serum is detected, effect on driving birds is not good refers to following experiments data.Or
4th, experiment material and ELISA method are same above first point, simply using polypeptide (SEQ ID NO：3, SEQ ID
NO：5) detect while, also using GST-HPV16-E7 and GST-HPV118E7 total lengths fusion protein as detection antigen, with
During PBS dilution GST-HPV16-E7, GST-HPV18-E7 total length fusion proteins, concentration is 2ug/ml, and testing result is as follows：
1. according to experimental procedure above, detection part patients serum and common human serum, testing result and patient's pathology report
Accuse as shown in the table：
Five. experimental analysis
1. by using the testing result of full-length proteins and before using HPV polypeptide SEQ ID:3 and SEQ ID:5 two kinds of polypeptides
Joint detection results are contrasted, contrast such as following table：
Summarize：Summarize：HPV16-E7, HPV18-E7 total length fusion protein has certain in detection CIN during Patients with Cervical Cancer
Positive patients missing inspection, sensitivity detects poor than polypeptide.There is certain vacation when common gynaecological imflammation patient and general population is detected
Positive rate, sensitiveness detects slightly poor than polypeptide.So, HPV16-E7 and HPV18-E7 full-length proteins are for detection CIN, cervical carcinoma
Etc. being not well suited for.
Blood enzyme mark immunoassay (ELISA) of the present invention is based on immune biochemical basis, there is provided high sensitivity, high
The Ag-Ab detection method of specificity and high stability.Patient can go out after being infected in serum and saliva of buccal cavity
The antibody IgM of existing specificity, IgA or IgG molecules.Detect special in patients serum or saliva with enzyme-labeled immunity analytic approach ELISA
The antibody molecule of the anti-HPV of property.The present invention is by HPV virus high-risk HPVs 16, the analysis of HPV18 gene orders, with biological letter
Breath credit is analysed to antigenic theoretical prediction, and experimentally screening, the meaning of proof clinical practice.It is 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 purpose antigen HPV16-1, HPV16-2 of high sensitivity, 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 sample.With washing method make on solid phase carrier formed antigen,
Antibody complex separates with other materials, is finally incorporated in the amount of tested substance in the enzyme amount on immobilization carrier and sample into one
Fixed ratio.Add enzyme reaction substrate after, then detected by developing the color, thus can according to the depth of color reaction make it is qualitative or
Quantitative analysis.The blood ELISA detection kit of the present invention is compared with DNA detections (HC2, CareHPV), and sampling is uniform, non-to invade
Property is omited, operation is more quick, simply；Expense is cheap, easily promotes, and is more suitable for Chinese market；The vacation that PCR method can be avoided to bring
Positive and false negative, substantially increases accuracy and the degree of accuracy of testing result；VIA is dyeed with traditional Pap smear, acetic acid,
Vaginoscopy is compared, more accurately, easily, quickly.Optimization in ELISA detection method Jing various parameters and experiment journey, can
It is used for the early screening of cervical carcinoma as the instrument of Large-scale Screening.
To sum up, the antigen for detecting anti-human papilloma virus (anti-HPV) antibody of the 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
Foundation is brought with cervix cancer, and then takes treatment in time, to prevent cancer from occurring, or cancer diffusion, patient's pain is mitigated in time
Hardship, the immunity detection reagent being made from it quickly and easily can exactly detect blood or the HPV antibody in saliva, be 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, specification 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 papilloma
The amino acid sequence of the antigen of antiviral antibody is as shown in SEQ ID NO.4.
2. a kind of antigen combination for detecting anti-human papilloma virus (anti-HPV) antibody, it is characterised in that the antigen combination include as
The antigen of the amino acid sequence shown in SEQ ID NO.4；
Preferably, the antigen combination also includes being selected from the group one or more antigen：Such as SEQ ID NO:Amino acid shown in 1
The antigen of sequence, such as SEQ ID NO:The antigen of the amino acid sequence shown in 2, such as SEQ ID NO:Amino acid sequence shown in 3
Antigen, such as SEQ ID NO:The antigen of the amino acid sequence shown in 5.
3. a kind of composition, it is characterised in that described composition contains the antigen described in claim 1 or claim 2 institute
The antigen combination stated, and pharmaceutically acceptable carrier and/or auxiliary material；
Preferably, the composition is reagent, vaccine or pharmaceutical composition.
4. a kind of immunity detection reagent, it is characterised in that including according to claim 1 for detecting anti-human papilloma
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, it is characterised in that the immunity detection reagent also includes carrying
Body, the antigen coat is on the carrier.
6. immunity detection reagent as claimed in claim 4, it is characterised in that the immunity detection reagent also includes mark
The two of the anti-human papilloma virus (anti-HPV) antibody of substance markers resist；
Preferably, the label is selected from the group：Horseradish peroxidase, alkaline 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 purposes of the antigen combination of anti-human papilloma virus (anti-HPV) antibody in reagent preparation, wherein the reagent is used to detect people
Papillomavirus antibody.
8. purposes as claimed in claim 7, it is characterised in that the 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
For detecting the antigen combination or immunity detection reagent according to claim 4 of anti-human papilloma virus (anti-HPV) antibody in detection
Application in human body fluid in anti-human papilloma virus (anti-HPV) antibody；
Preferably, the body fluid is blood or saliva.
10. it is a kind of detection anti-human papilloma virus (anti-HPV) antibody method, it is characterised in that methods described include step：
Antigen combination described in antigen or claim 2 described in claim 1 is contacted with sample to be tested, detects whether to be formed
Antigen antibody complex, wherein, the antibody in the antigen antibody complex is anti-human papilloma virus (anti-HPV) antibody, and the antigen resists
Antigen in nanocrystal composition is the antigen or the antigen in the antigen combination described in claim 2 described in claim 1；
Preferably, methods described is ELISA method.
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| CN110596384A (en) * | 2019-10-12 | 2019-12-20 | 南京黎明生物制品有限公司 | Human papilloma virus 6 type and 11 type immunoassay kit prepared based on Cas protein and gRNA compound |
| CN116162155A (en) * | 2022-11-15 | 2023-05-26 | 北京义翘神州科技股份有限公司 | anti-HPV 6L1 protein antibody and detection method using same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110596384A (en) * | 2019-10-12 | 2019-12-20 | 南京黎明生物制品有限公司 | Human papilloma virus 6 type and 11 type immunoassay kit prepared based on Cas protein and gRNA compound |
| CN110596384B (en) * | 2019-10-12 | 2022-08-12 | 南京黎明生物制品有限公司 | Human papilloma virus 6 type and 11 type immunoassay kit prepared based on Cas protein and gRNA compound |
| CN116162155A (en) * | 2022-11-15 | 2023-05-26 | 北京义翘神州科技股份有限公司 | anti-HPV 6L1 protein antibody and detection method using same |
| CN116162155B (en) * | 2022-11-15 | 2023-09-29 | 北京义翘神州科技股份有限公司 | anti-HPV 6L1 protein antibody and detection method using same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104004067B (en) | 2017-09-01 |
| CN106478784B (en) | 2019-10-25 |
| WO2014127741A1 (en) | 2014-08-28 |
| CN106478784A (en) | 2017-03-08 |
| CN106632617B (en) | 2020-07-03 |
| CN104004067A (en) | 2014-08-27 |
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