CN106610434A - ELISA detection kit for HPV16 type E7 protein - Google Patents

ELISA detection kit for HPV16 type E7 protein Download PDF

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CN106610434A
CN106610434A CN201610107162.4A CN201610107162A CN106610434A CN 106610434 A CN106610434 A CN 106610434A CN 201610107162 A CN201610107162 A CN 201610107162A CN 106610434 A CN106610434 A CN 106610434A
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antibody
hpv16
albumen
holes
micropore
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苗进超
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Frey Meade Biological Medicine Technology (tianjin) Co Ltd
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

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Abstract

The invention relates to an ELISA detection kit for an HPV16 type E7 protein. The kit includes: an enzyme labeled plate coated with an HPV16 type E7 antibody, wherein the coating amount of the antibody in each micro hole of the enzyme labeled plate is 0.05 to 0.5 [mu]g; and an HPV16 type E7 antibody labeled with HRP, wherein the concentration is 0.05 to 0.4 [mu]g/ml. The kit is used for directly detecting the expression and the expression quantity of high risk type HPV related carcinogenic proteins, so as to clearly determine the infection degree of high risk type HPV and be convenient for subsequent treatment.

Description

A kind of ELISA detection kit of HPV16 types E7 albumen
Technical field
The present invention relates to a kind of detection kit, more particularly to a kind of ELISA detection kit of HPV16 types E7 albumen.
Background technology
Cervical carcinoma is the second common cancer diagnosis in women, and in the case of 99.7% with high-risk human papilloma Virus infection is related, and there are every year about 400000 cervical carcinomas in the whole world, and nearly 200000 people is dead.HPV have more than 100 kinds it is different Separation strains, are broadly separated into height according to them with cervical carcinoma or with benign cervical lesionses or the relevance of atypical hyperplasia Danger and low danger hypotype.Low dangerous type HPV includes HPV6,11,42,43,44 etc., often causes the benign lesion bag such as external genital organs condyloma Include in epithelium of cervix uteri low pathology (CIN I), high-risk type HPV include HPV16,18,31,33,35,39,45,51,52,56, 58th, 59, the hypotype such as 68, CP8304, related to the generation of cervical carcinoma and epithelium of cervix uteri inner height pathology (CIN II/III), especially It is HPV16 and 18 types, wherein HPV16 accounts for more than 50%.
HPV (Human Papillomavirus, HPV) is a kind of thermophilic epithelial virus, has 3 genes District's groups are into including early stage area (Early Region, E area), late region (Late Region, L area) area and and noncoding region (Uncoding Region, UCR) or upstream regulatory region (URR).E areas are in order E6, E7, E1, E2, E3, E4 and E5 totally 7 Gene, participates in duplication, transcription, coding virus protein, the gene of the high copy number of maintenance intracellular virus of viral DNA, wherein E6 and E7 are the Analyses of major carcinogens in mainstream genes of HPV, relevant with virocyte transformation function and carcinogenicity.E6 and E7 albumen makes tumor suppressor Proteins p53 and pRB are passivated, and cell cycle control and inhibited apoptosis are released respectively.Therefore, for determining tumour in HPV The best approach of state is to measure the E6/E7 albumen in tumour cell.
At present HPV (HPV) predominantly detects method including in situ hybridization, direct trappings of DNA and all kinds of PCR method.Real-time fluorescence quantitative PCR (qPCR, Real- that the mid-90 in last century grows up on the basis of traditional PCR Time Quantitative PCR) technology realizes leaps of the PCR from qualitative to quantitative, with its high specificity, sensitivity it is high, The advantages of reproducible, quantitative accurate, fast speed, totally-enclosed reaction, becomes the important tool in molecular diagnosis research.
But said method simply detects whether to have infected HPV (HPV) on gene level, as correlation Whether is the expression of carcinogenic protein, and Real-Time Fluorescent Quantitative PCR Technique can not provide accurately judgement, and the method needs are supporting Instrument real-time fluorescence quantitative PCR instrument, expensive, testing cost is high.
The content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided for detecting high-risk human mammilla papillomavirus (HPV) ELISA detection kit of 16 hypotype E7 carcinogenic proteins.Directly it is loaded after cervical exfoliated cell cracking, direct detection is high Whether and expression the expression of danger type HPV correlation carcinogenic protein, has clearly hence for the gradient of infection of high-risk HPV Judge, facilitate follow-up treatment.Exactly current detection method (including Real-Time Fluorescent Quantitative PCR Technique) can not for this point Match in excellence or beauty.
Kit of the present invention adopts double antibody sandwich method, using the high-risk-type in antibody test human body cervical exfoliated cell HPV16 type E7 albumen.After first cervical exfoliated cell is cracked, in being directly added into the ELISA Plate for being coated with specific antibody, Jing temperature After educating and washing, enzyme labelled antibody is added, then substrate is added Jing after incubating and wash, with ELIASA OD values are detected.OD values and E7 eggs Bai Hanliang is proportionate, and is achieved in the quantitative determination to the HPV16 type E7 albumen in human body cervical exfoliated cell.
The technical solution used in the present invention is：
The invention provides a kind of ELISA Plate of HPV16 types E7 Protein Detection, it is anti-that the ELISA Plate is coated with HPV16 types E7 Body.
Preferably, the package amount of each micropore interior antibody of the ELISA Plate is 0.05～0.5 μ g.
Preferably, the preparation method of the ELISA Plate, comprises the steps：HPV16 type E7 antibody is taken, will by 100 μ L/ holes Coating buffer (coating buffer：0.5～5mg of antibody adds coating buffer solution 1000ml；Being coated with buffer solution is：Na2CO31.59g/L, NaHCO32.93g/L) add in micropore, 4 DEG C overnight (16～18h)；Using cleaning solution (Na2HPO4 1.44g/L、KH2PO4 0.24g/L, NaCl 8g/L, KCl 0.2g/L, Tween-200.5ml/L, proclin300 0.3ml/L) washed, 300 μ L/ holes, wash five times；Confining liquid (Na is added toward micropore by 300 μ l/ holes2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L, KCl 0.2g/L, BSA 10g/L, sucrose 25g/L, proclin300 0.3ml/L), placing 1～2h for 37 DEG C is carried out Closing；After drying shed (18-26 DEG C) is drained, vacuum sealing (2-8 DEG C) obtains being coated with the microwell plate of HPV16 type E7 antibody, does Dry preservation.
Present invention also offers a kind of ELISA detection kit of HPV16 types E7 albumen, the kit includes：
It is coated with the ELISA Plate of HPV16 type E7 antibody：The package amount of each micropore interior antibody of ELISA Plate is 0.05～0.5 μg；
The HPV16 type E7 antibody of HRP marks：Concentration is 0.05～0.4 μ g/ml.
Further, described kit also includes human body cervical exfoliated cell lysate.
Preferably, described human body cervical exfoliated cell lysate includes：Live on 0.05%～1% (percent by volume) surface Property agent；10mM～1M buffer solutions；1mM～10mM protease inhibitors.(can reach more preferable cracking effect with reference to thawing Really)
It is highly preferred that described surfactant is Tween 20, in Triton-100 or dodecyl sodium sulfate Plant or two or more.Described buffer solution be phosphate-buffered saline, Tris-HCL or carbonate solution, institute The protease inhibitors stated is one or more in cystatin, PMSF or Antipain.
It is highly preferred that described human body cervical exfoliated cell lysate is prepared by the method for comprising the steps：With 10mM～1M buffer solutions configure 0.05%～1% (percent by volume) surfactant；With add before final concentration of 1mM～ 10mM protease inhibitors.It is highly preferred that method system of the described human body cervical exfoliated cell lysate by comprising the steps It is standby to obtain：1%Tween 20 is configured with 50mM PBS solutions；With adding final concentration of 5mM protease inhibitors PMSF before.
Further, the preparation method of the described ELISA Plate for being coated with HPV16 type E7 antibody, comprises the steps：Take HPV16 type E7 antibody, by 100 μ L/ holes by coating buffer (coating buffer：0.5～5mg of antibody adds coating buffer solution 1000ml；Coating Buffer solution is：Na2CO31.59g/L, NaHCO32 .93g/L) add in micropore, 4 DEG C are overnight (16～18h)；Using cleaning solution (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、Tween-200.5ml/L、 Proclin300 0.3ml/L) washed, 300 μ L/ holes, wash five times；Confining liquid is added by 300 μ l/ holes toward micropore (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、BSA 10g/L、sucrose 25g/L、 Proclin300 0.3ml/L), place 1～2h for 37 DEG C and closed；After drying shed (18-26 DEG C) is drained, vacuum sealing (2- 8 DEG C), obtain being coated with the microwell plate of HPV16 type E7 antibody, kept dry.
Specifically, the main constituents of the kit are as follows：
1. the above-mentioned ELISA Plate for being coated with HPV16 type E7 antibody：The package amount of each micropore interior antibody is 0.05～0.5 μ G, bar shaped microwell plate (12 × 8 hole), in being placed in framework；
2. HPV16 types E7 albumen calibration object 0,1,2,3,4,5：Calibration object is freeze-dried powder, is separately added into 1ml ddH2O is molten Solution, the linear concentration of calibration object solution is respectively 0,10,20,40,80,160ng/ml；
3. HPV16 types E7 albumen quality-control product 1,2：Quality-control product is freeze-dried powder, adds 1ml ddH2O dissolves, and concentration is respectively 10 and 80ng/ml；
4. the HPV16 type E7 antibody that HRP is marked：Directly use, concentration be 0.05～0.4 μ g/ml, 1 bottle, every bottle of 12ml；
5. above-mentioned human body cervical exfoliated cell lysate；
6. substrate solution：One-component TMB nitrite ions；(being purchased from Beijing Suo Laibao Science and Technology Ltd)
7. concentrated cleaning solution (20 ×)：Concentrated cleaning solution containing detergent and preservative, uses ddH2O dilutes 20 times；
8. terminate liquid：Measure 80ml ddH2O is poured in container, measures the 10.87ml concentrated sulfuric acids, is slowly poured into along wall In water, and ceaselessly stir, be settled to 100ml, mix, room temperature preservation.
9. sealed membrane：The adhesive membrane of microwell plate is sealed during incubation.
Present invention also offers the preparation method of mentioned reagent box, comprises the steps：
1. the ELISA Plate of coating HPV16 type E7 antibody is prepared
HPV16 type E7 antibody is taken, by 100 μ L/ holes by coating buffer (coating buffer：0.5～5mg of antibody adds coating buffer solution 1000ml；Being coated with buffer solution is：Na2CO31.59g/L, NaHCO32.93g/L) add in micropore, 4 DEG C overnight (16～18h)； Using cleaning solution (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、Tween-200.5ml/ L, proclin300 0.3ml/L) washed, 300 μ L/ holes, wash five times；Confining liquid is added by 300 μ l/ holes toward micropore (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、BSA10g/L、sucrose 25g/L、 Proclin300 0.3ml/L), place 1～2h for 37 DEG C and closed；After drying shed (18-26 DEG C) is drained, vacuum sealing (2- 8 DEG C), obtain being coated with the microwell plate of HPV16 type E7 antibody, kept dry.
2. standard items, quality-control product
HPV16 type E7 albumen calibration object 0,1,2,3,4,5：Calibration object is freeze-dried powder, is separately added into 1ml ddH2O dissolves, The linear concentration of calibration object solution is respectively 0,10,20,40,80,160ng/ml；
HPV16 type E7 albumen quality-control product 1,2：Quality-control product is freeze-dried powder, adds 1ml ddH2O dissolves, and concentration is respectively 10 And 80ng/ml；
3. the HPV16 type E7 antibody that HRP is marked
It is marked using Over-voltage protection, HRP stabilizers is added afterwards, mixed, concentration is 0.05～0.4 μ g/ml, 2- 8 DEG C of preservations.
4. human body cervical exfoliated cell lysate
0.05%～1% (percent by volume) surfactant is configured with 10mM～1M buffer solutions；It is dense eventually with adding before Spend for 1mM～10mM protease inhibitors.
Described surfactant be Tween 20, in Triton-100 or dodecyl sodium sulfate one or two with On.Described buffer solution be phosphate-buffered saline, Tris-HCL or carbonate solution, described protease Inhibitor is one or more in cystatin, PMSF or Antipain.
5. concentrated cleaning solution (20 ×)
28.8g Na2HPO4·12H2O、4.8g KH2PO4、160g NaCl、4g KCl、10ml Tween-20、6ml Proclin300 adds ddH2O, is settled to 1000ml, mixes, room temperature preservation.
6. terminate liquid
Measure 80ml ddH2O to pour in container, measure the 10.87ml concentrated sulfuric acids, be slowly poured into water along wall, not Stir with stopping, be settled to 100ml, mix, room temperature preservation.
7. substrate solution, sealed membrane, are directly commercially available.
The detection method of the kit, comprises the steps：
Enough human body cervical exfoliated cells are obtained, in adding lysate, be vortexed 2～10min of concussion, is put into -20 DEG C of ice Case freeze thawing 1～3 time, shakes again 2～10min, and 10～30min of centrifugation (13000rpm) is collected supernatant, directly detected；
1. required ELISA Plate is taken out from 4 DEG C of refrigerators, room temperature places 15min；
2. 100ul calibration objects/quality-control product/sample, mixing is added to be placed in 37 DEG C of 30～60min of incubation per hole；
3. liquid in hole is dried, adds 300 μ l cleaning solutions, stationary incubation 1min liquid in hole to be dried, in blotting paper per hole On pat dry, so repeatedly 3～5 times；
4. 100 μ l enzyme labelled antibody solution are added per hole, 37 DEG C of 30～60min of incubation are placed in；
5. liquid in hole is dried, adds 300 μ l cleaning solutions, stationary incubation 1min liquid in hole to be dried, in blotting paper per hole On pat dry, so repeatedly 3～5 times；
6. 100ul substrate solutions are added per hole, 37 DEG C of incubation 15min are placed in；
7. 50ul terminate liquids are added per hole, is mixed, be placed in ELIASA and read OD values；
With calibration object concentration as abscissa, OD values are ordinate, and using 4-parameter logistic fit calibration curve side is obtained Journey, according to calibration curve equation HPV16 type E7 protein contents in sample to be checked are calculated.
The result interpretation of kit
The present invention is had the advantage that:
Although having infected HPV (HPV) on some clinical patient gene levels, related carcinogenic protein table Even do not express up to amount is very low, such case can eliminate HPV (HPV) by the immune system of patient itself, without the need for Raise fear and unnecessary treatment, mitigate patient mental's pressure and economic pressures.Based on this, direct detection of the present invention is high Whether and expression the expression of danger type HPV correlation carcinogenic protein, has clearly hence for the gradient of infection of high-risk HPV Judge, facilitate follow-up treatment.The invention forms complementation with current gene level detection method so that testing result is further smart Really, therapeutic scheme is further clear and definite, patient's early recovery.Simultaneously the method only needs to conventional ELIASA, and testing cost is low, can make For the prefered method of cervical carcinoma screening.
Description of the drawings
Fig. 1 be embodiment 4 with calibration object concentration as abscissa, OD values be ordinate, obtained using 4-parameter logistic fit To calibration curve.
Specific embodiment
With reference to specific embodiment, the invention will be further described, but does not limit protection scope of the present invention.
The preparation method of embodiment 1, the ELISA Plate of coating HPV16 type E7 antibody, comprises the steps：
HPV16 type E7 antibody is taken, by 100 μ L/ holes by coating buffer (coating buffer：Antibody 5mg adds coating buffer solution 1000ml；Being coated with buffer solution is：Na2CO31.59g/L, NaHCO32.93g/L) add in micropore, 4 DEG C overnight (18h)；Using Cleaning solution (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、Tween-200.5ml/L、 Proclin300 0.3ml/L) washed, 300 μ L/ holes, wash five times；Confining liquid is added by 300 μ l/ holes toward micropore (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、BSA 10g/L、sucrose 25g/L、 Proclin300 0.3ml/L), place 2h for 37 DEG C and closed；After drying shed (26 DEG C) is drained, vacuum sealing (4 DEG C) is obtained To the microwell plate of coating HPV16 type E7 antibody, kept dry.
Embodiment 2, configuration human body cervical exfoliated cell lysate and cracking cervical samples
1%Tween 20 is configured with 50mM PBS solutions；With adding final concentration of 5mM protease inhibitors PMSF before. By 500ul lysates add cervical exfoliated cell collecting pipe in, be vortexed concussion 2min, be placed in -20 DEG C of refrigerator 10min, it is rearmounted In 37 DEG C of baking oven 2min, shake again, be centrifuged (20min, 13000rpm, 4 DEG C), collect supernatant and detected.
Embodiment 3
A kind of preparation method of the ELISA detection kit of HPV16 types E7 albumen,
1. it is coated with the ELISA Plate of HPV16 type E7 antibody：The package amount of each micropore interior antibody be 0.5 μ g, bar shaped micropore Plate (12 × 8 hole), in being placed in framework；Preparation method is with embodiment 1.
2. HPV16 types E7 albumen calibration object 0,1,2,3,4,5：Calibration object is freeze-dried powder, is separately added into 1ml ddH2O is molten Solution, the linear concentration of calibration object solution is respectively 0,10,20,40,80,160ng/ml；
3. HPV16 types E7 albumen quality-control product 1,2：Quality-control product is freeze-dried powder, adds 1ml ddH2O dissolves, and concentration is respectively 10 and 80ng/ml；
4. the HPV16 type E7 antibody that HRP is marked：It is marked using Over-voltage protection, HRP stabilizers is added afterwards, is mixed It is even, 2-8 DEG C preservation, concentration be 0.4 μ g/ml, 1 bottle, every bottle of 12ml；
5. human body cervical exfoliated cell lysate；Preparation method is with embodiment 2.
6. substrate solution：One-component TMB nitrite ions；(being purchased from Beijing Suo Laibao Science and Technology Ltd)
7. concentrated cleaning solution (20 ×)：28.8g Na2HPO4·12H2O、4.8g KH2PO4、160g NaCl、4g KCl、 10ml Tween-20,6ml proclin300 add ddH2O, is settled to 1000ml, mixes, room temperature preservation.
8. terminate liquid：Measure 80ml ddH2O to pour in container, measure the 10.87ml concentrated sulfuric acids, slowly pour into along wall In water, and ceaselessly stir, be settled to 100ml, mix, room temperature preservation.
9. sealed membrane：The adhesive membrane of microwell plate is sealed during incubation.
Embodiment 4, a certain cervical samples of detection
The coating of ELISA Plate and the cracking of cervical samples are with embodiment 1～2.1. required ELISA Plate is taken out from 4 DEG C of refrigerators, Room temperature places 15min；2. 100ul calibration objects/quality-control product/sample, mixing is added to be placed in 37 DEG C of incubation 60min per hole；3. dry Liquid in hole, adds 300 μ l cleaning solutions, stationary incubation 1min to dry liquid in hole, pat dry on blotting paper, so weight per hole It is multiple 5 times；4. 100 μ l enzyme labelled antibody solution are added per hole, 37 DEG C of incubation 60min are placed in；5. liquid in hole is dried, is added per hole 300 μ l cleaning solutions, stationary incubation 1min dries liquid in hole, pats dry on blotting paper, is so repeated 5 times；6. add per hole 100ul substrate solutions, are placed in 37 DEG C of incubation 15min；7. 50ul terminate liquids are added per hole, is mixed, be placed in ELIASA and read OD values；
With calibration object concentration as abscissa, OD values are ordinate, and calibration curve is obtained such as using 4-parameter logistic fit Shown in Fig. 1.It is 43.47ng/ml to calculate HPV16 types E7 protein content in sample to be checked according to calibration curve, according to result interpretation, The sample is the positive, and patient needs active treatment.
A part of Healthy People cervical exfoliated cell is detected with kit of the present invention (such as the detection kit of embodiment 3), is examined Survey data result as follows；
Table one：Detection normal person's cervical exfoliated cell data result
Table two：Detection patient's cervical exfoliated cell data result
Data analysis
1st, according to normal person's detected value, mean value, standard variance, Cutoff values are calculated.Respectively by 83 normal person palaces Neck cast-off cells testing result is taken the mean, and calculates standard variance SD, and this kit is it is determined that select during Cutoff values normal For each person value adds the criterion of the standard variance as HPV16E7 protein expression yin and yang attributes of twice, as a result as follows：
Cutoff computing formula are：Cutoff=mean value+2*SD standard variances
Table three：Detection data table one is counted
Mean value 4.06
SD standard variances 5.48
Cutoff 15.02ng/ml
2nd, by Cutoff values, the yin and yang attribute of sample HPV 16E7 protein expressions to be checked is judged, more than this Cutoff value Be considered positive, that is, think to express HPV 16E7 albumen, at the same with pathological data contrast.
3rd, table three：Judge that ("+" represents testing result sun to the patient's cervical exfoliated cell diagnostic result of table two according to Cutoff values Property；“-”
Represent that testing result is negative)
Table four：Accuracy rate ratio table is formulated according to the patient's cervical exfoliated cell diagnostic result yin and yang attribute of table three
The patient's cervical exfoliated cell detected in table two is tested amounts to 19, kit HPV 16E7 Protein Detections knot For positive for 14, diagnostic sensitivity is up to 78.9% to fruit；CIN II above case samples are wherein directed to, diagnostic sensitivity is high Up to 80%.

Claims (9)

1. a kind of ELISA Plate of HPV16 types E7 Protein Detection, it is characterised in that：The ELISA Plate is coated with HPV16 type E7 antibody.
2. a kind of ELISA Plate of HPV16 types E7 Protein Detection according to claim 1, it is characterised in that：The ELISA Plate it is every The package amount of individual micropore interior antibody is 0.05～0.5 μ g.
3. the preparation method of ELISA Plate described in claim 1 or 2, it is characterised in that：Comprise the steps：Take HPV16 types E7 to resist Body, by 100 μ L/ holes by coating buffer (coating buffer：0.5～5mg of antibody adds coating buffer solution 1000ml；Being coated with buffer solution is： Na2CO31.59g/L, NaHCO32.93g/L) add in micropore, 4 DEG C overnight (16～18h)；Using cleaning solution (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、Tween-20 0.5ml/L、proclin300 0.3ml/ L) washed, 300 μ L/ holes, washed five times；Confining liquid (Na is added toward micropore by 300 μ l/ holes2HPO4 1.44g/L、 KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、BSA 10g/L、sucrose 25g/L、proclin300 0.3ml/ L), 37 DEG C of 1～2h of placement are closed；After drying shed (18-26 DEG C) is drained, vacuum sealing (2-8 DEG C) is coated with The microwell plate of HPV16 type E7 antibody, kept dry.
4. a kind of ELISA detection kit of HPV16 types E7 albumen, it is characterised in that：The kit includes：
It is coated with the ELISA Plate of HPV16 type E7 antibody：The package amount of each micropore interior antibody of ELISA Plate is 0.05～0.5 μ g；
The HPV16 type E7 antibody of HRP marks：Concentration is 0.05～0.4 μ g/ml.
5. the ELISA detection kit of a kind of HPV16 types E7 albumen according to claim 4, it is characterised in that：Described Kit also includes human body cervical exfoliated cell lysate.
6. the ELISA detection kit of a kind of HPV16 types E7 albumen according to claim 5, it is characterised in that：Described Human body cervical exfoliated cell lysate includes：0.05%～1% surfactant；10mM～1M buffer solutions；1mM～10mM albumen Enzyme inhibitor.
7. the ELISA detection kit of a kind of HPV16 types E7 albumen according to claim 6, it is characterised in that：Described Surfactant is Tween 20, one or more in Triton-100 or dodecyl sodium sulfate；Described buffering Liquid is phosphate-buffered saline, Tris-HCL or carbonate solution, and described protease inhibitors is One or more in cystatin, PMSF or Antipain.
8. a kind of ELISA detection kit of the HPV16 types E7 albumen according to any one of claim 4-7, its feature exists In：The preparation method of the described ELISA Plate for being coated with HPV16 type E7 antibody, comprises the steps：HPV16 type E7 antibody is taken, By 100 μ L/ holes by coating buffer (coating buffer：0.5～5mg of antibody adds coating buffer solution 1000ml；Being coated with buffer solution is：Na2CO3 1.59g/L, NaHCO32.93g/L) add in micropore, 4 DEG C overnight (16～18h)；Using cleaning solution (Na2HPO4 1.44g/L、 KH2PO40.24g/L, NaCl 8g/L, KCl 0.2g/L, Tween-200.5ml/L, proclin3000.3ml/L) washed Wash, 300 μ L/ holes, wash five times；Confining liquid (Na is added toward micropore by 300 μ l/ holes2HPO4 1.44g/L、KH2PO4 0.24g/L, NaCl 8g/L, KCl 0.2g/L, BSA 10g/L, sucrose 25g/L, proclin300 0.3ml/L), 37 DEG C Place 1～2h to be closed；After drying shed (18-26 DEG C) is drained, vacuum sealing (2-8 DEG C) obtains coating HPV16 types E7 and resists The microwell plate of body, kept dry.
9. a kind of preparation method of the ELISA detection kit of HPV16 types E7 albumen, it is characterised in that：Comprise the steps：
1. the ELISA Plate of coating HPV16 type E7 antibody is prepared
HPV16 type E7 antibody is taken, by 100 μ L/ holes by coating buffer (coating buffer：0.5～5mg of antibody adds coating buffer solution 1000ml；Being coated with buffer solution is：Na2CO31.59g/L, NaHCO32.93g/L) add in micropore, 4 DEG C overnight (16～18h)； Using cleaning solution (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、Tween-20 0.5ml/ L, proclin300 0.3ml/L) washed, 300 μ L/ holes, wash five times；Confining liquid is added by 300 μ l/ holes toward micropore (Na2HPO4 1.44g/L、KH2PO4 0.24g/L、NaCl 8g/L、KCl 0.2g/L、BSA 10g/L、sucrose 25g/L、 Proclin300 0.3ml/L), place 1～2h for 37 DEG C and closed；After drying shed (18-26 DEG C) is drained, vacuum sealing (2- 8 DEG C), obtain being coated with the microwell plate of HPV16 type E7 antibody, kept dry.
2. standard items, quality-control product
HPV16 type E7 albumen calibration object 0,1,2,3,4,5：Calibration object is freeze-dried powder, is separately added into 1ml ddH2O dissolves, calibration object The linear concentration of solution is respectively 0,10,20,40,80,160ng/ml；
HPV16 type E7 albumen quality-control product 1,2：Quality-control product is freeze-dried powder, adds 1ml ddH2O dissolves, and concentration is respectively 10 Hes 80ng/ml；
3. the HPV16 type E7 antibody that HRP is marked
Be marked using Over-voltage protection, afterwards add HRP stabilizers, mix, concentration be 0.05～0.4 μ g/ml, 2-8 DEG C Preserve.
4. human body cervical exfoliated cell lysate
0.05%～1% (percent by volume) surfactant is configured with 10mM～1M buffer solutions；It is final concentration of with adding before 1mM～10mM protease inhibitors.
Described surfactant is Tween 20, one or more in Triton-100 or dodecyl sodium sulfate. Described buffer solution be phosphate-buffered saline, Tris-HCL or carbonate solution, described albumen enzyme level Agent is one or more in cystatin, PMSF or Antipain.
5. concentrated cleaning solution (20 ×) 28.8g Na2HPO4·12H2O、4.8g KH2PO4、160g NaCl、4g KCl、10ml Tween-20,6ml proclin300 adds ddH2O, is settled to 1000ml, mixes, room temperature preservation.
6. terminate liquid
Measure 80ml ddH2O is poured in container, measures the 10.87ml concentrated sulfuric acids, is slowly poured into water along wall, and is ceaselessly stirred It is dynamic, 100ml is settled to, mix, room temperature preservation.
7. substrate solution, sealed membrane, are directly commercially available.
CN201610107162.4A 2016-02-26 2016-02-26 ELISA detection kit for HPV16 type E7 protein Pending CN106610434A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107192830A (en) * 2017-05-31 2017-09-22 珠海美华医疗科技有限公司 Monoclonal antibody, Test paper and the detection kit of anti-HPV16 types E7 albumen
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

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1553191A (en) * 2003-06-05 2004-12-08 北京新科基业生物科技有限责任公司 Reagent box, for diagnosing Down's syndrome in early gestational period by pregnancy correlative protein-A
CN1836166A (en) * 2003-08-25 2006-09-20 Mtm实验室公司 Method for detecting neoplastic disorders in a solubilized body sample
CN101058578A (en) * 2006-04-21 2007-10-24 中国医学科学院药物研究所 Dextrorotation deoxidization tylophorinine, preparation method thereof, pharmaceutical composition and use thereof
CN101152179A (en) * 2006-09-28 2008-04-02 中国医学科学院药物研究所 Application of isoniazide as histone deacetylase inhibitors
CN101538574A (en) * 2009-04-27 2009-09-23 黄祖瑚 Codon-optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof
CN101607985A (en) * 2008-12-24 2009-12-23 中国科学院生物物理研究所 The monoclonal antibody of anti-people CEA comprises its composition, and uses thereof
CN102435734A (en) * 2011-09-08 2012-05-02 大连医科大学 Kit used for evaluating ovarian cancer primary chemotherapeutic sensitivity, and application thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1553191A (en) * 2003-06-05 2004-12-08 北京新科基业生物科技有限责任公司 Reagent box, for diagnosing Down's syndrome in early gestational period by pregnancy correlative protein-A
CN1836166A (en) * 2003-08-25 2006-09-20 Mtm实验室公司 Method for detecting neoplastic disorders in a solubilized body sample
CN101058578A (en) * 2006-04-21 2007-10-24 中国医学科学院药物研究所 Dextrorotation deoxidization tylophorinine, preparation method thereof, pharmaceutical composition and use thereof
CN101152179A (en) * 2006-09-28 2008-04-02 中国医学科学院药物研究所 Application of isoniazide as histone deacetylase inhibitors
CN101607985A (en) * 2008-12-24 2009-12-23 中国科学院生物物理研究所 The monoclonal antibody of anti-people CEA comprises its composition, and uses thereof
CN101538574A (en) * 2009-04-27 2009-09-23 黄祖瑚 Codon-optimized cholera enterotoxin CTB gene and cholera nucleic acid vaccine thereof
CN102435734A (en) * 2011-09-08 2012-05-02 大连医科大学 Kit used for evaluating ovarian cancer primary chemotherapeutic sensitivity, and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LINDA A. SELVEY, ET AL.: "An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18", 《 JOURNAL OF VIROLOGICAL METHODS》 *
傅俊江: "《精编医学分子生物学实验指导》", 31 January 2012 *
王晓佳: "《蛋白质技术在病毒学研究中的应用》", 31 August 2015 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107192830A (en) * 2017-05-31 2017-09-22 珠海美华医疗科技有限公司 Monoclonal antibody, Test paper and the detection kit of anti-HPV16 types E7 albumen
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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