CN108761091A - Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof - Google Patents
Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof Download PDFInfo
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- CN108761091A CN108761091A CN201810715149.6A CN201810715149A CN108761091A CN 108761091 A CN108761091 A CN 108761091A CN 201810715149 A CN201810715149 A CN 201810715149A CN 108761091 A CN108761091 A CN 108761091A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention discloses test strips, test cards and preparation method thereof that a kind of double-antibody method quickly detects HPV antibody, test strips (card) include sample pad, bonding pad, analyzing film and the water absorption pad set gradually along chromatography direction, it is provided with nanoparticle marker on the bonding pad, detection band and nature controlling line are provided on analyzing film；The nanoparticle marker is selected from the antibody for the anti-human igg that colloidal gold or latex mark, and detection band includes HPV Major capsid protein L1s；Alternatively, the nanoparticle marker is selected from the HPV Major capsid protein L1s that colloidal gold or latex mark, detection band includes the antibody of anti-human igg.Test strips (card) are not only easy to use, quick in the present invention, it is of low cost, and accuracy, safety and the sensitivity detected is higher, to the scene of can carrying out of the HPV16/18 L1 IgG antibodies in human body, quickly detection, it is suitable for hospital, disease control, basis health unit detection and family to test oneself, is suitable for promoting.
Description
Technical field
The invention belongs to field of immunoassay detection, and in particular to one kind is quickly detected based on colloidal gold immunochromatographimethod technology
The method and test strip (card) of HPV antibody.
Background technology
Cervical carcinoma is a kind of common gynecologic malignant tumor, occupies second in the incidence of female tumor, the whole world is annual
New cases be about 470,000 people, and cause 300,000 people dead, be the women die major reason for being only second to breast cancer.Cervical carcinoma
Pathogenesis etiology it is related with many factors, wherein with the sense of human nipple warty viral (human papillomavirus, HPV)
Dye is most important factor, it was reported that 99.8% cervical carcinoma merges HPV infection, and HPV negative patients hardly happen cervical carcinoma.
Effective remedy measures there is no to HPV infection at present, prevent HPV infection and carry out periodic detection to infection population to be current palace
The primary treatment regimen of neck cancer.
HPV belongs to papillomavirus section, is a kind of double-strand closed hoop DNA virus for biting mucous membrane and skin.Up to the present,
Report that the HPV of discovery has more than 120 kinds, wherein it is about more than 40 kinds related with the infection of human reproduction road, it is divided into according to its carcinogenicity
High-risk-type (carcinogenic type) HPV, being potentially carcinogenic property and low risk HPV, with HPV16, HPV18, HPV31, HPV33 in high-risk HPV
The onset relation of type and cervical carcinoma is the closest, especially most commonly seen with HPV16, HPV18.It is reported that in cervical carcinoma this two
The recall rate of the HPV of type is up to 70% or more.HPV16 types are more common in cervical squamous cell carcinoma, and HPV18 types are more common in adenocarcinoma of the uterine cervix,
And low risk HPV mostly has found in the benign lesions such as condyloma.
High-risk HPV infection is typically not have Symptomatic, the women of persistent infection HPV, suffers from the risk higher of cervical carcinoma.It is fixed
Phase detection high-risk HPV is most important for preventing cervical carcinoma, but because lacking highly sensitive and specificity HPV diagnostic reagents,
It focuses mostly on to the detection of HPV and is detected in the DNA for carrying out histopathology or HPV viruse in hospital at present.
10 open reading frames of HPV genome encodings are respectively E1~E8 and L1~L2, and wherein HPV L1 are main
Capsid protein.HPV L1 are just to be exposed to the antigen of body immune system before HPV viruse invasion cell, compared to other viral eggs
It is white to be easier to be identified and removed by immune system, it is that current research and development HPV vaccines are predominantly targeting antigen.Studies have reported that
Detection of the HPV6L1IgG antibody in patients with condyloma acuminatum peripheral blood, but it merely relates to enzyme linked immunosorbent assay.
The report for not having detection human body high-risk HPV L1IgG antibody also at present, also has not seen and is with immunochromatography technique
The test strips (card) of the double-antibody method detection high-risk HPV L1IgG antibody on basis.Test strips (card) are identifying and are capturing sample
The specific coating protein pair on detection band and nature controlling line is also needed in this after detection target (high-risk HPV L1IgG antibody)
Testing result gives display and verification, quickly, is accurately detected to realize.
Invention content
The purpose of the present invention is to provide a kind of double-antibody method quickly detect the test strips of HPV antibody, test card and its
Preparation method.
In order to achieve the above objectives, present invention employs following technical schemes：
A kind of double-antibody method quickly detects the test paper of HPV antibody, which includes the sample set gradually along chromatography direction
Product pad, bonding pad, analyzing film and water absorption pad are provided with nanoparticle marker on the bonding pad, inspection are provided on analyzing film
Measuring tape and nature controlling line；The nanoparticle marker is selected from the antibody for the anti-human igg that colloidal gold or latex mark, and detection band includes
HPV Major capsid protein L1s, nature controlling line include the antibody combined with anti-human IgG antibodies, and HPV Major capsid protein L1s are Jiang Rengong
HPV16 the or HPV18 Major capsid protein L1 genes of neck cancer cell strain Hela are through obtained from prokaryotic expression；Or
Person, the nanoparticle marker are selected from the HPV Major capsid protein L1s that colloidal gold or latex mark, and detection band includes anti-human
The antibody of IgG, nature controlling line include the HPV major capsid eggs of the antibody of anti-HPV Major capsid protein L1s, colloidal gold or latex label
White L1 is HPV16 the or HPV18 Major capsid protein L1 genes by human cervical carcinoma cell lines Hela through prokaryotic expression
It carries out afterwards obtained from colloidal gold or latex label.
Preferably, the antibody of the anti-HPV Major capsid protein L1s is mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or globefish
It is one or more in the polyclonal antibody of mouse source HPV Major capsid protein L1s.
Preferably, the antibody of the anti-human igg is mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source anti-human igg
It is one or more in polyclonal antibody, colloidal gold or latex label can be carried out.
Preferably, the antibody combined with anti-human IgG antibodies refer to can be formed with the antibody of anti-human igg it is immune compound
The antibody of object, if for example, anti-human IgG antibodies' selection is mouse anti-human IgG antibodies, then what is combined with the anti-human IgG antibodies is anti-
Body is rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source dynamics.
A kind of double-antibody method quickly detects the test strips (card) of HPV antibody, including above-mentioned test paper and for fixed branch
Support the substrate of the test paper.
Preferably, the substrate is selected from PVC board or gets stuck, and the sample pad is selected from glass fibre membrane or polymer PET.
Above-mentioned double-antibody method quickly detects the preparation method of the test paper of HPV antibody, includes the following steps：
Step 1：The preparation of bonding pad
1.1) preparation of colloidal gold or latex labelled protein (nanoparticle marker)
It mixes and reacts 10~30 minutes with binding protein after the pH of colloidal gold solution or latex is adjusted to 5.5~7.0,
Then BSA is added, and obtains labelled protein through mixing, standing and centrifugation successively, the binding protein is selected from the anti-of anti-human igg
Body or HPV Major capsid protein L1s, HPV Major capsid protein L1s are by HPV16 the or HPV18 masters of human cervical carcinoma cell lines Hela
Want capsid protein L 1 gene through obtained from prokaryotic expression；
1.2) it is coated on glass fibre membrane or polymer PET after the labelled protein dispersion dilution prepared step 1.1), then
It is dry, it is spare；
Step 2：The preparation of analyzing film
2.1) prepared by detection band (T lines)
According to the labelled protein on the bonding pad, the coating protein of selection detection band：If the labelled protein on bonding pad
Antibody containing anti-human igg, the then coating protein for detecting band are HPV Major capsid protein L1s；If the labelled protein on bonding pad
Containing HPV Major capsid protein L1s, then the coating protein for detecting band is the antibody of anti-human igg；The coating protein of selection is fixed
On nitrocellulose filter；
2.2) prepared by nature controlling line (C lines)
According to the labelled protein on the bonding pad, the coating protein of nature controlling line is selected：If the labelled protein on bonding pad
Antibody containing anti-human igg, then the coating protein of nature controlling line is the corresponding antibody combined with anti-human IgG antibodies；If bonding pad
On labelled protein contain HPV Major capsid protein L1s, then the coating protein of nature controlling line is the anti-of anti-HPV Major capsid protein L1s
Body；The coating protein of selection is fixed on the nitrocellulose filter.
Preferably, in the step 1, the dosage of colloidal gold solution is 10mL, the antibody or HPV major capsids of anti-human igg
The dosage of albumen L1 is 50~100 μ g；Coating control is in 0.1~0.5mL/cm2, dry condition is：18 at 37~40 DEG C~
24 hours.
Preferably, in the step 2, by for detection band and Quality Control line options coating protein respectively according to 1~4mg/
Nitrocellulose filter is coated in after mL dilutions, then cured processing obtains detection band and nature controlling line.
Above-mentioned double-antibody method quickly detects the preparation method of the test strips (card) of HPV antibody, includes the following steps：
According to above-mentioned test paper along chromatography direction set gradually sample pad, bonding pad, analyzing film and water absorption pad, sample pad,
Bonding pad and analyzing film obtained by water absorption pad and above-mentioned steps 1~2 are sequentially arranged on bottom plate (PVC board), then basis
The specification of test strips is divided, and test strips are obtained；Alternatively, first obtained by sample pad, water absorption pad and step 1~2 bonding pad and
Analyzing film, certain length and width is cut into according to the specification of test card, is then sequentially arranged in getting stuck, is obtained test card.
Beneficial effects of the present invention are embodied in：
Immunochromatography and dual-antigen sandwich method are applied to the Test paper of HPV antibody by the present invention, with the antibody of anti-human igg
It is dual anti-original with HPV Major capsid protein L1s (HPV L1), is being combined using one of antigen (passing through nanoparticle label)
The HPV L1IgG antibody in sample is captured at pad, and the HPV antibody of capture is fixed with place in detection using another antigen
(colour developing), to complete the detection to high-risk HPV L1IgG antibody.Meanwhile invention not only avoids use same combination
The antigen-antibody binding reaction in site completes capture and colour developing, and passes through the HPV L1 genes to human cervical carcinoma cell lines Hela
Expressed using prokaryotic expression system, the HVP L1 of expression have higher immunogenicity, and solve high-risk-type (HPV16,
18) the problem of HVP Major capsid protein L1s are easy to happen non-specific binding, improves detection sensitivity and accuracy.In addition,
HPV L1 used in the present invention are the main species specificity albumen of HPV, and the group without containing complete poisoning intrusion and duplication
Point, do not have pathogenic.Therefore, test strips provided by the invention, test card are not only easy to use, quick, of low cost, and
The accuracy of detection, safety and sensitivity are higher, and HPV (HPV16,18) the L1IgG antibody generated to human body can show
Field, quickly detection are suitable for hospital, disease control, basis health unit detection and family and test oneself, be suitable for popularization.
Description of the drawings
Fig. 1 is the structural schematic diagram of the quickly test strips (card) of detection HPV antibody；In figure：1 is PVC board, and 2 be detection band,
3 be nature controlling line, and 4 be water absorption pad, and 5 be NC films, and 6 be gold-labelled pad, and 7 be sample pad.
Fig. 2 is amplification electrophoresis pattern.
Fig. 3 is recombinant protein electrophoresis pattern.
Specific implementation mode
The present invention is described in further details with reference to the accompanying drawings and examples.
(1) HPV L1 albumen and its Antibody preparation
(1) prepared by HPV L1 albumen：Build HPV16L1 full genomes and the full bases of HPV18L1 respectively using gene clone technology
Because being expressed in expression vector, and using Escherichia coli
1.1, amplimer
According to HPV16L1 gene reference sequences (Gene ID:And HPV18L1 gene reference sequences (Gene 1489082)
ID:1489090) HPV L1 gene specific primers, are designed, HPV16L1 and HPV18L1 gene (sequence designs are expanded with PCR method
Deadline is in May, 2016, the endonuclease recognized site containing limitation)：
HPV16L1 forward primers-SphI：5`-CATGCATGC CAGGTGACTTTTATTTACATC-3`
HPV16L1 reverse primers-SacI：5`-CGAGCTC CAGCTTACGTTTTTTGCGTTT-3`
HPV18L1 forward primers-SphI：5`-CATGCATGC GCTTTGTGGCGGCCTAGT-3`
HPV18L1 reverse primers-SacI：5`-CGAGCTC CTTTCTGGCACGTACACGC-3`
1.2, template and amplification
With human cervical carcinoma cell lines Hela (in August, 2016 is obtained from China typical culture collection center) cell of extraction
Total DNA is template, HPV16L1 and HPV18L1 gene magnifications are carried out using above-mentioned special primer.
PCR amplification system is：4 μ L of 10 × Taq Buffer, 5 μ L, 0.25 μ L, dNTP Mixture of Taq enzyme, DNA profiling
2.5ng, 1 μ L of forward primer (25 μm), 1 μ L of reverse primer (25 μm), sterile purified water are mended to 50 μ L.
Reaction condition is：95 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 1min 40s are recycled 30 times；72 DEG C of 10min are recycled 1 time.
Pcr amplification product is about 1500bp, as shown in Fig. 2, consistent with ncbi database HPV16L1 genes, HPV18L1 gene sizes.
1.3, construction of recombinant plasmid
PCR amplification gene and pQE80L plasmids (Beijing Hua Yue ocean biology) are subjected to the bis- enzymes of SphI and SacI respectively
It cuts, digestion condition is as follows：
522 μ L of μ L, SacI of μ L, SphI of pcr amplification product 300ng or pQE80L plasmid 100ng, 10 × K buffer,
Sterile purified water is mended to 50 μ L, is reacted 8 hours in 37 DEG C.
It is attached after digestion products are recycled, linked system and condition are：10 × Ligase buffer 1 μ L, T4DNA
0.5 μ L of Ligase (350U/ μ L), 7.5 μ L of digestion amplified production, 1 μ L of digestion pQE80L plasmids react 8 hours in 16 DEG C.Weight
Group plasmid size is 6.2kb.
1.4, it HPV L1 protein expressions and isolates and purifies
1.4.1, recombinant plasmid is expressed in prokaryotic expression system BL21
By recombinant plasmid transformed coli strain BL21 (Beijing Hua Yue ocean biology), conversion process is：In centrifuge tube
After connection product is mixed with 50 μ L BL21 competence bacterial strains, 30min is placed on ice, is placed rapidly after 42 DEG C of water-bath heat shock 45s
The sterile LB medium that 700 μ L are free of antibiotic (ampicillin) is added into centrifuge tube on ice, after 2min, 37 after mixing
DEG C culture 60min, 5000rpm centrifuge 1min and receive bacterium, leave and take 100 μ L or so supernatant and gently blows and beats resuspension and fungus block and are applied to containing phase
It answers on the LB culture mediums of antibiotic, screening positive clone, identifies.
The coli strain BL21 of work(is converted into LB liquid medium, in 37 DEG C of cultures to OD600=0.6~
0.8, the IPTG that final concentration of 1mM is added carries out induced expression 8h.
1.4.2, with affinitive layer purification HPV16L1 albumen, HPV18L1 albumen
By the BL21 thalline of induced expression HPV16L1 or HPV18L1 through ultrasonic treatment and centrifugation.By nickel on supernatant
Column (QIAGEN Ni-NTA Spin Kit), with elution, collects expression egg after the rinsing liquid rinsing in kit
In vain, the size for determining purifying protein is analyzed through SDS-PAGE, it is finally pure with escherichia expression system expression and affinity chromatography
Change technology acquires recombination HPV16L1 albumen and recombination HPV18L1 albumen with His labels, about 50KD, such as figure respectively
Shown in 3, it is consistent with ncbi database Protein Information.
(2) HPV L1 Antibody preparations
Assistant is immunized with Freund respectively in the recombination HPV16L1 albumen of a concentration of 0.5~2mg/mL and recombination HPV18L1 albumen
Agent is according to volume ratio 1:1 mixes simultaneously immune BALB/c mouse, the recombination HPV16L1 albumen of every mouse immune or recombination
HPV18L1 protein contents are 100 μ g, and the 14th and 28 day after initial immunity is similarly to measure booster immunization twice, first
The 35th day acquisition mice serum after immune, more grams of HPV16L1 and HPV18L1 albumen are prepared using ProteinG affinity purifications
Grand antibody.Affinity purification process is：By serum with combination buffer (20mM phosphoric acid is received, pH7.0) upper prop in ProteinG columns
In (Invitrogen Protein G), after combination buffer rinsing, is eluted, washed with eluent (100mM glycine, pH2.7)
De- liquid uses neutralization buffer (1M Tris-HCl, pH9.0) by the polyclonal antibody being collected into (HPV16L1 or HPV18L1 immediately
Polyclonal antibody) it is neutralized to neutrality.
(2) HPV L1 antibody titers detect
1) first with recombination HPV16L1 albumen and recombination HPV18L1 albumen respectively with the amount of 1 μ g coating enzyme linked immunological, 96 hole
Plate, coating plate are incubated overnight at 4 DEG C.
2) each hole is washed 6 times with the PBST washing buffers (adding Tween20 in PBS) containing 0.05% Tween-20,
100 μ L of confining liquid (the PBST solution for containing 5% skimmed milk power) are added per hole to close 2 hours.
3) 100 μ L are added per hole and dilute 200 times of polyclonal antibody sample with PBST solution, react 1 hour in 37 DEG C, so
Afterwards each hole is washed with PBST solution 4 times, 5 minutes every time.
4) sheep anti-mouse igg antibody of 100 μ L horseradish peroxidases is added per hole, 37 DEG C are reacted 1 hour, are then used
PBST solution washs each hole 6 times, to remove unbonded enzyme labelled antibody.
5) 100 μ L TMB developing solutions are added per hole, room temperature carries out chromogenic reaction half an hour.
6) reaction terminating liquid (2M sulfuric acid solutions) color development stopping reaction of 50 μ L is added per hole, is surveyed after five minutes with microplate reader
Determine the absorption value at 450nm wavelength, and records result.
The serum antibody for determining 6 processes immune mouse and a non-immunized Negative control mice altogether, wherein 3
Only by absorption value of the serum antibody at 450nm wavelength of recombination HPV16L1 protein immunization mouse be respectively 0.726,1.258 and
0.915,3 absorption value of the serum antibody at 450nm wavelength by recombination HPV18L1 protein immunization mouse is respectively 0.612,
0.834 and 0.793, and absorption value of the Negative control mice serum antibody at 450nm wavelength is 0.049.Thus judge, by weight
Group HPV L1 protein immunizations mouse can activate the immune system of mouse, and generate the spy of corresponding HPV16L1 and HPV18L1 albumen
Heterogenetic antibody has higher immunogenicity and binding specificity.
(3) antibody of anti-human igg
For example, commercially available goat anti-human igg antibody (Zhong Shan Golden Bridge).
(4) the quickly preparation of detection HPV antibody test strips (card)
1) prepared by colloidal gold：By gold chloride (HAuCl4) it is made into the chlorauric acid solution of mass fraction 1%, take 1mL gold chlorides
Solution is added in 99mL deionized waters, is heated to that the minor official acid sodium solution of Chinese holly of 2mL mass fractions 1% is added after boiling immediately, rapidly
It stirs evenly, continues ebuillition of heated 10 minutes, be cooled to room temperature, supplement deionized water obtains colloidal gold solution to 100mL.
2) preparation of colloid gold label object：The colloidal gold solution that 10mL is prepared is taken, with the solution of potassium carbonate of 0.1M by pH
Value is adjusted to 5.5~7.0, and anti-human IgG antibodies or HPV L1 albumen (recombination HPV16L1 albumen or recombination HPV18L1 eggs is added
50~100 μ g in vain), mixing react at room temperature 10~30 minutes, and BSA is then added to final concentration (mass fraction) 0.5~1%, mixes
It is even, stand 5~10 minutes, 4 DEG C, 2000rpm centrifuge 20 minutes, discard precipitation.Supernatant centrifuges 60 points in 4 DEG C, 12000rpm
Clock discards supernatant, and precipitation is dissolved in the PBS (including mass fraction 0.2%BSA) of 1mL, obtains colloid gold label object.
3) preparation of gold-labelled pad (bonding pad for being covered with colloid gold label object)：Colloid gold label object is taken (to include matter with PBS
Measure score 0.2%BSA) dilution 1~2 times, using spraying instrument uniformly spray on glass fibre membrane, quantity for spray be 0.1~
0.5mL/cm2, 30% humidity obtains gold-labelled pad 6 hereinafter, 37~40 DEG C of dryings 18~24 hours.Gold-labelled pad 6 is used into Fresco Bag
Sealing, built-in drier, room temperature preserve.Opening and use should be below 30% humidity.
4) coating of band and nature controlling line is detected：
A1, HPV L1 albumen (recombination HPV16L1 albumen or recombination HPV18L1 albumen) is diluted to a concentration of 1 with PBS~
4mg/mL crosses (or being sprayed on NC films 5) on nitrocellulose filter (NC films 5), as detection band.From 6 millimeters of band of detection
Place, draws a line (or being sprayed on NC films), as nature controlling line again with the antibody of 1~4mg/mL combined with anti-human IgG antibodies.Make
Detection band 2 and nature controlling line 3 are solidificated on NC films.
A2, anti-human IgG antibodies are diluted to a concentration of 1~4mg/mL with PBS, are drawn on nitrocellulose filter (NC films 5)
Line (or being sprayed on NC films 5), as detection band.At from 6 millimeters of band of detection, with the HPV L1 protein polyclones of 1~4mg/mL
Antibody draws a line (or being sprayed on NC films 5) and is used as nature controlling line again.Detection band 2 and nature controlling line 3 is set to be solidificated on NC films.
B, cure：After the drying of NC films, it is soaked in the PBS of the pH7.2 containing 1%BSA and is closed (4 DEG C, 1 hour).
It then takes out NC films to be washed 3 times with PBS, hang, in drying at room temperature 3 hours or more, then be put in ventilation in 30 DEG C of aeration cabinets
It is 1 hour dry.
5) assembling of test strips (card)：
Test strips (card) mainly include by sample pad 7, the gold-labelled pad obtained through above step 6, (solidification has detection to NC films 5
Band 2 and nature controlling line 3) and the substrate of test paper and fixed support test paper that constitutes of water absorption pad 4.
Wherein, substrate using PVC board 1 (test strips) or gets stuck (test card)；Absorbent filter can be used in water absorption pad 4, absorbs
Detect surplus liquid in sample；Glass fibre membrane may be used in sample pad 7, contacts detected sample.
By taking test strips as an example, concrete structure is referring to Fig. 1：
(1) A1 classes test strips：In PVC board 1, the various pieces of test paper are sequentially arranged along chromatography direction, wherein under
Trip end is water absorption pad 4, and upstream end is sample pad 7, and stage casing has detection band 2 (containing recombination HPV16L1 albumen or recombination to cure
HPV18L1 albumen) and the NC films 5 of nature controlling line 3 (containing the antibody that is combined with anti-human IgG antibodies) as detection reaction and analyze
The gold-labelled pad 6 for the anti-human IgG antibodies for being adsorbed with colloid gold label is placed in area between NC films 5 and sample pad 7.
(2) A2 classes test strips：In PVC board 1, the various pieces of test paper are sequentially arranged along chromatography direction, wherein under
Trip end is water absorption pad 4, and upstream end is sample pad 7, and there are detection band 2 (containing anti-human IgG antibodies) and nature controlling line 3 in stage casing to cure
The NC films 5 of (containing HPV16L1 or HPV18L1 polyclonal antibodies) are as detection reaction and analysis area, in NC films 5 and sample pad 7
Between place be adsorbed with colloid gold label recombination HPV16L1 albumen or recombinate HPV18L1 albumen gold-labelled pad 6.
(3) test paper is attached in PVC board 1, cuts into the belt strip of 4mm wide；Wherein, NC films 5 are first attached to PVC board 1
On, then water absorption pad 4 and gold-labelled pad 6 are attached in PVC board 1, and partly overlaps respectively with 5 both ends of NC films, finally by sample
Product pad 7 is attached in PVC board 1, and is partly overlapped with gold-labelled pad 6.Test strips hermetically drying preserves, spare.
(5) HPV L1IgG antibody tests
(1) measuring samples are detected using above-mentioned test strips
1.1) sample process：Serum, blood plasma or whole blood sample are taken, dilutes 0~100 times with sample loading buffer, sample-adding buffering
Liquid is water, 0.9% sodium chloride solution of mass fraction, 0.05~0.3M phosphate buffers or 0.05~0.2M boric acid salt buffers
Liquid；
1.2) serum, blood plasma or 50~100 μ L of whole blood sample Jing Guo above-mentioned processing are added to the sample pad 7 of test strips
On, 5~20 minutes are stood, observation detection band 2 and 3 color of nature controlling line；
1.3) result interpretation：Detection band 2 and nature controlling line 3 develop the color for the positive；Detection band 2 does not develop the color and nature controlling line 3 develops the color,
As a result it is feminine gender；It detects band 2 and nature controlling line 3 does not develop the color or other phenomenons, prompt test strips failure.
(2) testing principle
After detected sample is loaded onto in sample pad 7, HPV L1IgG antibody in sample with it is anti-human on bonding pad
The antibody or HPV L1 antigens of IgG form immune complex, due to capillary effect, the compound to 4 direction swimming of water absorption pad,
The antibody of this compound and the HPV L1 antigens or anti-human igg that are coated on detection band 2 occurs that association reaction is immunized, and forms gold mark
Anti-human IgG antibodies-HPV L1IgG antibody-HPV L1 antigens or gold mark HPV L1 antigen-HPV L1IgG antibody-anti-human igg are anti-
Body triplet compound and be trapped within detection band 2 on, be progressively enriched with to form deeper aubergine band；Due to capillarity after
Continuous swimming forward, colloid gold label anti-human IgG antibodies or colloid gold label HPV L1 antigens and be coated on nature controlling line 3 with it is anti-
The immune response that specificity occurs for the antibody or HPV L1 polyclonal antibodies that human IgG antibody combines is trapped, and is progressively enriched in matter
Deeper aubergine band is formed on control line 3, extra unbonded substance continues chromatography to water absorption pad 4, therefore is detecting
The band for all occurring developing the color with 2 and nature controlling line 3 is judged as positive findings (there are HPV L1IgG antibody in sample)；If to be checked
Do not contain HPV L1IgG antibody in sample, the antibody or HPV L1 antigens of the anti-human igg of colloid gold label not with detection band 2
On HPV L1 antigens or anti-human igg antibody occur be immunized association reaction, so detection band 2 on be not in colour developing item
Band, and the antibody of the anti-human igg of colloid gold label or HPV L1 antigens continue swimming forward, be coated on nature controlling line 3 with
The immune response that specificity occurs for the antibody or HPV L1 polyclonal antibodies that anti-human IgG antibodies combine is trapped, and is progressively enriched with
On nature controlling line 3, therefore there is colour developing band only on nature controlling line 3 and be judged as that (it is anti-that HPV L1IgG are not present in negative findings in sample
Body).
(3) sample collection is illustrated
3.1, serum sample：Take 1~5mL of whole blood in serum collection pipe, stand 1~2 hour, 3000~5000rpm from
The heart 10~30 minutes, takes supernatant to obtain the final product.
3.2, plasma sample：Take 1~5mL of whole blood in sodium citrate or heparin sodium anticoagulant tube, 1000~3000rpm centrifugations
10~30 minutes, take supernatant to obtain the final product.
3.3, whole blood sample：Fetching point or about 50~100 μ L of ear-lobe fresh blood to get.
(4) experiment is found, for the sample Jing Guo HPV DNA tests positives, can be obtained by above-mentioned test strips
The testing result of corresponding HPV L1IgG antibodies positives, reliability and sensitivity are higher.
In short, the present invention provides it is a kind of can field quick detection HPV antibody test strips (card), it is easy to use, quick,
Efficiently, accurately, the case where easy observation of testing result is distinguished, can be used for judging infection HPV viruse is the screening of cervical carcinoma and is controlled
It treats and quick auxiliary diagnosis is provided, can be used for the self diagnosis of High-risk Population of Cervical Carcinoma caused by HPV, prevent, while also can be used
In HPV infection person or the timely of vaccine recipient, line treatment effect assessment, state of an illness detection and Index for diagnosis, it is suitble to family certainly
Inspection and clinical quick diagnosis and base's epidemiological survey large-scale application.
Sequence table
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<213>Artificial synthesized ()
<400> 4
cgagctcctt tctggcacgt acacgc 26
Claims (10)
1. a kind of double-antibody method quickly detects the test paper of HPV antibody, it is characterised in that：The test paper includes that edge chromatographs direction successively
Sample pad (7), bonding pad, analyzing film and the water absorption pad (4) of setting are provided with nanoparticle marker on the bonding pad, point
Detection band (2) and nature controlling line (3) are provided on analysis film；The nanoparticle marker marks anti-human selected from colloidal gold or latex
The antibody of IgG, detection band (2) includes HPV Major capsid protein L1s, and nature controlling line (3) includes resisting of being combined with anti-human IgG antibodies
Body, HPV Major capsid protein L1s are to pass through HPV16 the or HPV18 Major capsid protein L1 genes of human cervical carcinoma cell lines Hela
Obtained from prokaryotic expression；Alternatively, the nanoparticle marker is selected from colloidal gold or the HPV of latex label is main
Capsid protein L 1, detection band (2) include the antibody of anti-human igg, and nature controlling line (3) includes the antibody of anti-HPV Major capsid protein L1s,
The HPV Major capsid protein L1s of colloidal gold or latex label are by the main clothing of the HPV16 of human cervical carcinoma cell lines Hela or HPV18
Glutelin L1 genes are carried out after prokaryotic expression obtained from colloidal gold or latex label.
2. a kind of double-antibody method quickly detects the test paper of HPV antibody according to claim 1, it is characterised in that：It is described anti-
The antibody of HPV Major capsid protein L1s is mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source HPV Major capsid protein L1s
Polyclonal antibody in it is one or more.
3. a kind of double-antibody method quickly detects the test paper of HPV antibody according to claim 1, it is characterised in that：It is described anti-
The antibody of human IgG be mouse source, rabbit source, Yang Yuan, Ma Yuan, camel source or cavy source anti-human igg polyclonal antibody in one kind or
A variety of, the antibody of the anti-human igg of the colloidal gold or latex label is the polyclonal antibody progress colloidal gold or glue of the anti-human igg
Obtained from breast label.
4. a kind of double-antibody method quickly detects the test paper of HPV antibody according to claim 1, it is characterised in that：It is described with
The antibody that anti-human IgG antibodies combine refers to the antibody that immune complex can be formed with the antibody of anti-human igg.
5. a kind of double-antibody method quickly detects the test strips or test card of HPV antibody, it is characterised in that：Including test paper and use
In the fixed substrate for supporting the test paper, the test paper includes the sample pad (7) set gradually along chromatography direction, bonding pad, analysis
Film and water absorption pad (4) are provided with nanoparticle marker on the bonding pad, and detection band (2) and Quality Control are provided on analyzing film
Line (3)；The nanoparticle marker is selected from the antibody for the anti-human igg that colloidal gold or latex mark, and detection band (2) includes HPV
Major capsid protein L1, nature controlling line (3) include the antibody combined with anti-human IgG antibodies, and HPV Major capsid protein L1s are Jiang Rengong
HPV16 the or HPV18 Major capsid protein L1 genes of neck cancer cell strain Hela are through obtained from prokaryotic expression；Or
Person, the nanoparticle marker are selected from the HPV Major capsid protein L1s that colloidal gold or latex mark, and detection band (2) includes anti-
The antibody of human IgG, nature controlling line (3) include the antibody of anti-HPV Major capsid protein L1s, and the HPV that colloidal gold or latex mark is main
Capsid protein L 1 is HPV16 the or HPV18 Major capsid protein L1s gene by human cervical carcinoma cell lines Hela through prokaryotic expression system
It is carried out after system expression obtained from colloidal gold or latex label.
6. a kind of double-antibody method quickly detects the test strips or test card of HPV antibody according to claim 5, feature exists
In：The substrate is selected from PVC board (1) or gets stuck, and the sample pad (7) is selected from glass fibre membrane or polymer PET.
7. a kind of double-antibody method as described in claim 1 quickly detects the preparation method of the test paper of HPV antibody, feature exists
In：Include the following steps：
Step 1：The preparation of bonding pad
1.1) preparation of nanoparticle marker
It is mixed with binding protein after the pH of colloidal gold solution or latex is adjusted to 5.5~7.0 and reacts 10~30 minutes, then
BSA is added, and obtains nanoparticle marker through mixing, standing and centrifugation successively, the binding protein is selected from anti-human igg
Antibody or HPV Major capsid protein L1s, HPV Major capsid protein L1s are by the HPV16 or HPV18 of human cervical carcinoma cell lines Hela
Major capsid protein L1 gene is through obtained from prokaryotic expression；
1.2) it is coated on glass fibre membrane or polymer PET after the nanoparticle marker dispersion dilution prepared step 1.1), so
After dry；
Step 2：The preparation of analyzing film
2.1) prepared by detection band
According to the nanoparticle marker on the bonding pad, the coating protein of selection detection band (2)：If the nanometer on bonding pad
Particulate labels contain the antibody of anti-human igg, then the coating protein for detecting band (2) is HPV Major capsid protein L1s；If bonding pad
On nanoparticle marker contain HPV Major capsid protein L1s, then detect band (2) coating protein be anti-human igg antibody；
The coating protein of selection is fixed on nitrocellulose filter；
2.2) prepared by nature controlling line
According to the nanoparticle marker on the bonding pad, the coating protein of nature controlling line (3) is selected：If the nanometer on bonding pad
Particulate labels contain the antibody of anti-human igg, then the coating protein of nature controlling line (3) is corresponding to be combined with anti-human IgG antibodies
Antibody；If the nanoparticle marker on bonding pad contains HPV Major capsid protein L1s, the coating protein of nature controlling line (3) is
The antibody of anti-HPV Major capsid protein L1s；The coating protein of selection is fixed on the nitrocellulose filter.
8. preparation method according to claim 7, it is characterised in that：In the step 1, the dosage of colloidal gold solution is
10mL, the antibody of anti-human igg or the dosage of HPV Major capsid protein L1s are 50~100 μ g；Coating control is in 0.1~0.5mL/
cm2, dry condition is：37~40 DEG C, 18~24 hours.
9. preparation method according to claim 7, it is characterised in that：In the step 2, detection band (2) and matter will be directed to
It controls after the coating protein that line (3) selects is diluted according to 1~4mg/mL respectively and is coated in nitrocellulose filter, then cured place
Reason obtains detection band (2) and nature controlling line (3).
10. a kind of double-antibody method quickly detects the test strips of HPV antibody or the preparation method of test card, it is characterised in that：Packet
Include following steps：
Step 1：The preparation of bonding pad
1.1) preparation of nanoparticle marker
It is mixed with binding protein after the pH of colloidal gold solution or latex is adjusted to 5.5~7.0 and reacts 10~30 minutes, then
BSA is added, and obtains nanoparticle marker through mixing, standing and centrifugation successively, the binding protein is selected from anti-human igg
Antibody or HPV Major capsid protein L1s, HPV Major capsid protein L1s are by the HPV16 or HPV18 of human cervical carcinoma cell lines Hela
Major capsid protein L1 gene is through obtained from prokaryotic expression；
1.2) it is coated on glass fibre membrane or polymer PET after the nanoparticle marker dispersion dilution prepared step 1.1), so
After dry；
Step 2：The preparation of analyzing film
2.1) prepared by detection band
According to the nanoparticle marker on the bonding pad, the coating protein of selection detection band (2)：If the nanometer on bonding pad
Particulate labels contain the antibody of anti-human igg, then the coating protein for detecting band (2) is HPV Major capsid protein L1s；If bonding pad
On nanoparticle marker contain HPV Major capsid protein L1s, then detect band (2) coating protein be anti-human igg antibody；
The coating protein of selection is fixed on nitrocellulose filter；
2.2) prepared by nature controlling line
According to the nanoparticle marker on the bonding pad, the coating protein of nature controlling line (3) is selected：If the nanometer on bonding pad
Particulate labels contain the antibody of anti-human igg, then the coating protein of nature controlling line (3) is corresponding to be combined with anti-human IgG antibodies
Antibody；If the nanoparticle marker on bonding pad contains HPV Major capsid protein L1s, the coating protein of nature controlling line (3) is
The antibody of anti-HPV Major capsid protein L1s；The coating protein of selection is fixed on the nitrocellulose filter；
Step 3：By the bonding pad and analyzing film and water absorption pad (4) obtained by sample pad (7), above-mentioned steps 1~2, install successively
On bottom plate, is then divided according to the specification of test strips, obtain test strips；Alternatively, first by sample pad (7), 1~2 institute of above-mentioned steps
Bonding pad and analyzing film and water absorption pad (4) obtained, certain length and width is cut into according to the specification of test card, then according to
It is secondary be mounted on get stuck in, obtain test card.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810715149.6A CN108761091A (en) | 2018-06-29 | 2018-06-29 | Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof |
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| CN201810715149.6A CN108761091A (en) | 2018-06-29 | 2018-06-29 | Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof |
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