CN108872593A - It is a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine - Google Patents
It is a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine Download PDFInfo
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- CN108872593A CN108872593A CN201810726862.0A CN201810726862A CN108872593A CN 108872593 A CN108872593 A CN 108872593A CN 201810726862 A CN201810726862 A CN 201810726862A CN 108872593 A CN108872593 A CN 108872593A
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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
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention discloses a kind of monoclonal antibodies and its application for specifically binding HPV16 E6 albumen.The invention also discloses the kits and preparation method thereof of application monoclonal antibody assessment HPV16 subgroup vaccine immune effect of the invention, the kit is using blocking ELISA principle preparation, be conducive to improve accuracy rate, reduce false positive, provides guidance for assessment clinic HPV16 subgroup vaccine immune effect.
Description
Technical field
The present invention relates to field of biological detection, and in particular to HPV16 immune antiboidy in a kind of detection HPV vaccine recipient's body
The ELISA kit of content instructs for vaccine immunity situation.
Background technique
Human papilloma virus (Human papillomavirus, HPV) is that one kind belongs to papovaviridae
(Papovaviridae) papilloma vacuolating virus A belongs to, and is spherical DNA virus, can cause the scaly epithelium of human skin mucous membrane
Proliferation.Show as the symptoms such as verruca vulgaris, genital wart (condyloma acuminatum).As the disease incidence of condyloma acuminatum in venereal disease rapidly rises
With increasing for cervical carcinoma, cancer of anus etc., HPV infection is had attracted more and more attention from people.But not all HPV genotype is equal
Cancer is induced, according to its hypotype difference, low risk and two kinds of high-risk-type can be divided into.Low risk HPV mainly causes verruca vulgaris, toe
Wart, verruca plana etc., and the HPV infection of high-risk-type mainly causes genital wart and cervical carcinoma.It is currently understood that the most in-depth HPV is carcinogenic
Example be women cervical carcinoma, Harald zur Hausen in 2008 is to lead to the pathogen of cervical carcinoma because demonstrating HPV viruse
And obtain Nobel Prize in Physiology or Medicine.
Cervix cancer is the common female malignant of second, and annual about 500,000 women in the whole world are diagnosed as uterine neck
Cancer, wherein therefore dead more than the women of half.The generation of almost all of cervical carcinoma is all that high-risk HPV persistent infection is led
It causes, 16 type of high-risk HPV and 18 types are related with 70% cervical cancer pathogenesis.It was gratifying that 2006 and 2007 first
Two kinds of preventative tetravalences and divalent HPV are succeeded in developing by Merck (Merck) and Britain's GlaxoSmithKline PLC (GSK) drugmaker
Vaccine, the preventative HPV of nine valences that MSD Corp. of the U.S. develops is also on April 28th, 2018 in Discussion on Chinese Listed.
These vaccines include the antigenic determinant from HPV early protein (e.g., E6, E7), this is because E6 and E7
The equal continuous expression in HPV infection thumping majority cervical carcinoma and precancerous lesion, and do not express in the normal tissue.Meanwhile E6,
The continuous expression of E7 to induce and maintain cancer cell malignant phenotype be it is required, cancer cell antigen loss in be less likely to escape
Immune response is crossed, results of animal shows that prevention can be generated by the vaccine of target antigen of the early protein of papillomavirus
Effect can generate therapeutic effect again.Therefore, E6, E7 albumen just become antigen-specific therapies HPV chronic infection and related diseases
Become the promising target such as vaccine for cervical cancer, but the immune response ratio E7 albumen that the HPV early protein E6 of research discovery recently induces
It is more common, and there is stronger immunogenicity.
HPV vaccine examines listing in succession at present, lacks in practical application for assessing the simple and easy to do of vaccine immunity effect
Sensitive and accurate Serology test is with recombinant protein ELISA envelope antigen, with highly sensitive anti-HPV16 monoclonal
Antibody is that competition antibody establishes blocking ELISA detection method, to identify antibody present in HPV vaccine recipient's serum.Extremely
The present has no the practical application of Serology test or Kit in clinical examination.Therefore affinity height, high specificity are badly in need of in this field
Anti- HPV antibody and its kit by the amount of antibody present in individual serum after detection vaccine immunity determine that vaccine is imitated
Valence.
Summary of the invention
In view of the deficiencies of the prior art, the present invention intends to provide a kind of Dan Ke for HPV16 E6 albumen
The ELISA kit for being used to detect HPV16 neutralizing antibody content of its preparation of grand antibody and application.
The first aspect of the present invention provides a kind of monoclonal antibody for specifically binding HPV16 E6 albumen, it is by mouse
Hybridoma cell line CCTCC NO：C2018153 secretion.
The second aspect of the present invention provides a kind of mouse hybridoma cell system for secreting monoclonal antibody of the invention
CCTCC NO：C2018153.
The third aspect of the present invention provides monoclonal antibody of the invention in preparation for assessing HPV16 vaccine immunity effect
Application in the blocking ELISA kit of fruit.
The fourth aspect of the present invention provide it is a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine,
Wherein, the kit includes monoclonal antibody of the invention, and the monoclonal antibody is for detecting HPV16 vaccine recipient's blood
The content of anti-HPV16 immune antiboidy in liquid.
In a preferred embodiment of the invention, in the kit, in conjunction with the monoclonal antibody and
The antigen of anti-HPV16 immune antiboidy is by SEQ ID NO in HPV16 vaccine recipient's blood：Nucleic acid sequence encoding shown in 2
HPV16 E6 recombinant protein, peridium concentration lug/ml, the monoclonal antibody are the monoclonal antibodies marked with HRP, and
And it is diluted when in use with 1: 4000 dilution ratio.
In a preferred embodiment of the invention, when being detected with the kit, the blocking rate for being detected sample is big
In or be equal to 40%, the sample can be judged to the positive, that is, with the presence of HPV16 antibody, immune effect is good；If by
The blocking rate of sample sheet is less than or equal to 30%, and the sample can be judged to feminine gender, i.e. nonreactive HPV16 antibody exists, and builds
Discuss renewed vaccination vaccine；If the blocking rate of tested sample between 30~40%, is determined as doubtful, need to detect again.
The fifth aspect of the present invention provides a kind of method for preparing blocking ELISA kit of the invention, the method
Include the following steps：
(1) HPV16 E6 recombinant protein is prepared, by SEQ ID NO：Nucleic acid sequence encoding shown in 2；
(2) using the recombinant protein of step (1) acquisition as immunogene, monoclonal antibody is prepared by hybridoma technology；
(3) the recombinant protein coated elisa plate for obtaining step (1)；
(4) monoclonal antibody obtained with HRP markers step (2)；
To which preparation is to block ELISA as the blocking ELISA of HPV16 immune antiboidy in principle detection vaccine immunity person serum
Kit.
In a preferred embodiment of the invention, in step (1), when expanding E6 protein gene, to codon
It optimizes, in favor of obtaining immunogene.
In a preferred embodiment of the invention, in step (3), the peridium concentration of recombinant protein is 1ug/ml.
In a preferred embodiment of the invention, the monoclonal antibody marked with HRP is dilute with 1: 4000 when in use
The ratio of releasing is diluted.
The beneficial effects of the invention are as follows：
HPV16 E6 monoclonal antibody has neutralization to HPV16 hypotype, detects HPV16 by indirect ELISA
The content situation of neutralizing antibody in subgroup vaccine inoculator's body provides guidance foundation for vaccine inoculation, clinical application, treatment etc..
Detailed description of the invention
By referring to the following drawings combination detailed description of non-limiting embodiments, other feature of the invention,
Objects and advantages will become more apparent：
Fig. 1 is gel electrophoresis figure of the display by the HPV16 E6 gene order of PCR amplification.
Fig. 2 is to connect HPV16 E6 gene with expression vector PET30a, is verified by PCR, finally obtained correct
The gel electrophoresis figure of the sequencing of transformant.
Fig. 3 is the SDS-PAGE figure that HPV16 E6 protein expression situation is detected by SDS-PAGE.
Fig. 4 is display HPV16 E6 protein purification result SDS-PAGE figure.
Fig. 5 is the figure that HPV16 E6 protein expression situation in tissue is detected by WB (Western blotting) mode.
Fig. 6 is the ability for detecting antibody of the invention by indirect ELISA and inhibiting HPV16 positive serum combination antigen
Curve graph.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following implementation will be helpful to this field
Technical staff further understands the present invention, but the invention is not limited in any way.
Embodiment
Test agents useful for same and instrument：HAT culture medium (be purchased from Sigma company), HT culture medium (being purchased from Sigma company),
RPMI1640 culture medium (being purchased from GIBCO company), sheep anti-mouse igg-HRP antibody (being purchased from Sigma company), fetal calf serum (are purchased from
GIBCO company), DMSO (be purchased from Sigma company), 50%PEG (being purchased from Sigma company, molecular weight 1450), DMEM culture medium
(being purchased from GIBCO company), TMB (Sigma company).
The preparation of the monoclonal antibody of the anti-HPV16 E6 antigen of embodiment 1
1.HPV16 E6 gene cloning
Entrust Sangon Biotech (Shanghai) Co., Ltd.) limited liability company's synthesis HPV16 E6 protein sequence, to ensure albumen success
Expression, synthesis while, carry out codon optimization.
The synthesis of 1.1HPV16 E6 gene order
Student on commission's work synthesizes HPV16 E6 protein sequence, and to ensure albumen successful expression, synthesis while carries out codon
Optimization.
Former DNA sequence dna：
The DNA sequence dna of optimum synthesis：
PCR amplification the primer sequence is as follows：
Upstream primer：5'-TTTGAATTCATGCATCAGAAACGTACCGC-3'(SEQ ID NO：3)
Downstream primer：5'-TTTCTCGAGCAGCTGGGTCTCTCTCCTGGTACGG-3'(SEQ ID NO：4) PCR instrument
(ABI, 2720), PCR program are：94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 30s expand 30 circulations, last 72 DEG C of extensions
10min, amplified production is with detected through gel electrophoresis, as a result as shown in Figure 1, size is consistent with target gene.PCR product followed by
Double enzyme site EcoR I/Xho I (precious biology, the article No. of introducing：1040S, 1094s) double digestion is carried out, digestion products are through core
Sour purification kit (Suo Laibao science and technology, article No.：D2500-01 genetic recombination) is carried out after purification.
1.2 genetic recombination：By by digestion purifying 6 μ L of HPV16 E6 gene, 2.5 μ L of pET30a expression vector, 10 ×
Ligase buffer1 μ L and T4 ligase (precious biology, article No.：2011a) 0.5 μ L mixing is placed on 16 DEG C of connection 12h, then
Connection product is converted in competent escherichia coli cell DH5 α, and is coated on containing 25 μ g/ml kanamycins (solarbio)
LB plate after recombination bacterium colony is formed, is inoculated with liquid LB with single bacterium colony；Bacterium solution PCR identification is carried out to it, as a result as shown in Figure 2.
Positive plasmid conversion expression bacterial strain BL21, the culture of picking positive clone molecule are simultaneously sequenced.
2.HPV16 the expression and purification of E6 albumen
By the IPTG inducing expression of the 37 degree of 1mM of recombinant bacterial strain built, inducing expression sample is collected in different time points
Product, while doing empty carrier strain control.Protein expression situation is detected by SDS-PAGE, as a result as shown in Figure 3.Determine albumen
After matter expression, ultrasonic disruption thalline distinguishes supernatant and precipitating, detects protein in what manner by SDS-PAGE
Expression.Testing result shows that protein is expressed with insoluble inclusion bodies, and renaturing inclusion bodies are handled and purified, obtain mesh
Albumen, as a result as shown in Figure 4.
3. immune animal
It is emulsified by antigen and isometric Freund's complete adjuvant (sigma) of above-mentioned HPV16 E6 albumen, to female after emulsification
Property BALB/c mouse dorsal sc injection；It uses Freund's incomplete adjuvant emulsification antigen after 2 weeks instead, is injected after emulsification；It is spaced at least one moon
Afterwards, it is immunized again using Freund's incomplete adjuvant emulsification antigen；Booster immunization is carried out by intraperitoneal injection antigen in fusion first three days.
4. cell fusion
4.1 myeloma (SP2/0) cell activation：
It thaws and recovers and SP2/0 cell is commercialized, be then resuspended in nutrient solution (RPMI-1640 adds fetal calf serum),
It is placed in 37 DEG C, incubator culture under the conditions of 5%CO2, is passed on after 3-5d；
It collects cell and is suspended in 1640 basal liquids, 0.5~1 × 10 are taken after counting6It is a to be injected in BALB/c mouse back
Portion is subcutaneous, persistently cultivates 9~10d.Diameter about 0.8cm is increased to dorsal tumors volume, draws neck to put to death mouse, 75% alcohol
Sterile working takes tumor after impregnating 5min.
Tumor mass to be cut to be placed in the homogenizer of sterilizing, 1640 basal liquids of addition add 1640 liquid of 10mL after being fully ground,
2min is stood, the cell suspension for drawing upper layer is placed in another centrifuge tube, then adds 1640 liquid of 10mL, repeats grinding twice；It will
The cell suspension of above-mentioned acquirement removes supernatant in 1000r/min centrifugation 10min, is then resuspended in basic 1640 liquid of 30mL.
In another centrifuge tube be added 15mL lymphocyte separation medium, by above-mentioned cell suspension be carefully placed in separating liquid it
On；Subsequent 1200r/min is centrifuged 15min, and suction pipe draws the white cellular layer for being located at interface densification, cleans cell using 1640 liquid
It is resuspended in after 2 times in 1640 liquid of 10mL, it is spare after counting.
The preparation of 4.2 immune spleen cells：
BALB/c mouse one of booster immunization is taken, eye socket sacrificed by exsanguination (collects serum, as positive serum), in 75% wine
5-10min disinfection is impregnated in essence, is then fixed in dissection plate and is dissected, take out spleen and is broken, and sterilizing is placed in
In homogenizer；Grinding and cell suspension preparation method are spare after counting with described in SP2/0.
The preparation of 4.3 feeder cells：
A non-immune BALB/c mouse, eye socket bloodletting are taken, collection serum is negative serum.2 are injected into mouse peritoneal
1640 basal liquid of~3mL, suction is placed in spare in another centrifuge tube after piping and druming, contains peritoneal macrophage in the liquid.Ibid grasp
Splenocyte suspension is prepared, and is put into peritoneal macrophage pipe.1000r/min centrifugation 10min removes supernatant, and cell is trained with HAT
It supports after base suspends, is placed in 37 DEG C, it is stand-by in 5%CO2 incubator.
4.4 fusions and selectivity culture：
By 1-2 × 107A SP2/0 and 108A immunocyte mixes in 50mL centrifuge tube, 1000r/min, is centrifuged 8min.
Abandon after net supernatant and the centrifuge tube equipped with cell mixture be placed in 37 DEG C of water-baths, be then added pre-temperature to 37 DEG C 50%
PEG0.8mL (Sigma), stands 30s after stirring.1640 basal liquid 10mL of 37 DEG C of pre-temperatures are added after standing.1000r/ after mixing
Min is centrifuged 5min, abandons supernatant in 37 DEG C of placement 5-8min.It is then mixed with feeder cells suspension, point kind is in 96 well culture plates
In, the hole 250uL/ is cultivated in 37 DEG C, 5%CO2 incubator.It changes HT culture medium and continues to cultivate within the 4th day after fusion.It is to be fused thin
Born of the same parents' colony is long to culture hole 1/4, when culture medium slightly turns yellow, carries out antibody test.
5. the screening of hybridoma positive colony and the cloning of cell
5.1 screen positive hybridoma cell with indirect ELISA, and its step are as follows：
It is coated with known antigens：With coating buffer by the coating antigen diluent of purifying to 1-10ug/mL；It is every into micropore
Hole adds 100uL, gently shakes up, 4 DEG C of refrigerator overnights or 37 DEG C of 1h；Get rid of liquid in hole；Washing 3 times, every time 2~3 minutes.
Not by the position of antigen coat in sealase mark hole：Into micropore, every hole adds 200uL confining liquid (5% defatted milk
Powder or 0.1% BSA (solarbio), gently shake up, 37 DEG C of 1h；Get rid of liquid in hole；Washing buffer is hole-specifically filled it up with,
2~3min is stood, liquid in hole is got rid of, pats dry, first washed 3 times with washing buffer with this method.Sample-adding：Hybridoma to be measured is every
Hole takes 50uL supernatant to be sequentially added into enzyme mark hole, gently shakes up 37 DEG C of 1h, and washing pats dry.
Enzyme mark antiantibody：First with dilution by enzyme mark secondary antibody (solarbio HRP mark sheep anti mouse secondary antibody) normally
Bright to be diluted to working concentration, every hole is added 100uL, gently shakes up, set 37 DEG C of 1h；It is washed out, pats dry.Add TMB developing solution：
Every hole adds the developing solution 100uL of fresh configuration, gently shakes up, and 37 DEG C, 10min.Terminate reaction：Every hole add terminate liquid (2M sulfuric acid,
Purchased from solarbio) 50uL.
Determine result：It can directly detect by an unaided eye in white background as a result, being determined according to shade.
The cloning (limiting dilution assay) of 5.2 hybridomas
Mouse feeder cells layer is prepared before clone；The hybridoma that will be cloned gently is blown down out of culture hole, uses blood
Cell counting board living cell counting number；Cell is diluted to 5,10,30 cells/mls with complete medium；
The cell suspension of above three concentration is separately added into 96 well culture plates of the feeder cells prepared, 100uL/
Hole makes corresponding every hole contain 0.5,1 and 3 cell respectively.It cultivates to fluid infusion one in the 4th day and drips, the examines in each hole for 5-6 days
The growing state of cell, and record；
The detection of specific antibody：7~9d after clone, when cell clone covers with the 1/3-1/2 visual field, Ji Kejian
It surveys；The cell in positive hole can be moved to 24 well culture plates, when the cell well-grown in 24 orifice plates, can intraperitoneal inoculation mouse gather and process
Ascites obtains a strain of hybridoma strain and (obtains deposit number in China typical culture collection center on June 24th, 2018
CCTCC NO：C2018153).
6. a large amount of preparations of monoclonal antibody
Logarithmic growth phase cell is washed and has been hanged with serum free medium, counts 2~5 × 105, 1ml.The cell abdomen of suspension
The mouse of paraffin oil sensitization is used in chamber injection in advance.Start to collect ascites after 7d.The ascites of taking-up in 4 DEG C of centrifugation 4000rpm,
10min.The intermediate ascites of careful suction is collected in centrifuge tube, 4 DEG C or -20 DEG C preservations.With HiTrap rProtein AFF
(GE company) affinity chromatography by specification antibody purification from ascites.SDS-PAGE glue identifies that purity, BCA method measurement concentration are
4mg/ml.The antibody of purifying is stored in -20 DEG C.It is 1: 80000 with indirect ELISA measurement potency.
The atopic of the monoclonal antibody of the anti-HPV16 E6 antigen of embodiment 2 is tested
The present embodiment tests monoclonal antibody prepared by the present invention to the atopic of HPV16 E6 albumen.
The tissue (assaypositive tissue comes from Medical School of Zhengzhou University, and friend's friendship is given) of two cervical carcinoma HPV positives is selected,
Suitable RIPA is added and is cracked into the tissue sample that final protein concentration is 1-5mg/mL, detects this with the method for immunoblotting
The identification specificity of the monoclonal antibody of invention, immunoblot experiment process are as follows：Every kind of histone loading 10ng is carried out
13.5% polyacrylamide gel electrophoresis.Gel protein band is transferred in Bio-Rad electrotransfer system according to a conventional method
On pvdf membrane (Millipore company).Film is placed in the TBS-T confining liquid containing 5% skimmed milk power and is stayed overnight for 4 DEG C.It is added about
1mg/mL is incubated overnight by 1: the 1000 diluted above-mentioned monoclonal antibody being prepared at 4 DEG C.Film is washed with TBS-T solution
Afterwards, 1: 5000 diluted sheep anti mouse secondary antibody (Beijing Suo Laibao Science and Technology Ltd, SE131) is added, is incubated at room temperature 1h.Again
TBS-T washes film, the super quick developing solution (Beijing Suo Laibao Science and Technology Ltd, PE0010) of ECL is added, with ChemiDoc MP polychrome
The acquisition of fluoroscopic imaging systems (Bio-Rad) progress chemiluminescence image data.As a result as shown in figure 5, showing list of the invention
Clonal antibody being capable of specific recognition HPV16 E6 albumen.
The monoclonal antibody of the anti-HPV16 E6 antigen of embodiment 3 inhibits the aptitude tests of HPV16 positive serum combination antigen
The present embodiment inhibits HPV16 positive serum to combine with indirect ELISA method detection monoclonal antibody of the invention
The ability of antigen.
Experimental procedure is as follows：
1. 50 μ l sample diluting liquid PBST are added in each detection hole and control wells respectively.
2. it is corresponding right that the positive control of 50 μ l (hpv positive serum standard items) and negative control (PBST) are added respectively
It to be replaced according to the suction nozzle in hole, paying attention to different controls, with anti-cross-contamination.
3. the test sample of 50 μ l is added in remaining detection hole respectively, notice that the suction nozzle of different test samples will divide
It opens, to prevent pollution.
4. flicking ELISA Plate or being vibrated with oscillator, the solution in reaction plate is mixed.
5. ELISA Plate is closed, 37 degree of incubation 1h.
6. getting rid of the liquid in reacting hole, and washed 3 times with the cleaning solution PBST diluted, paying attention to will when washing every time
Cleaning solution is filled it up with into reacting hole, discard the cleaning solution in reacting hole and pats dry reaction plate.It can also be machine-washed with board-washing and wash 3 times, often
A reacting hole should add the cleaning solution of 300 μ l or so.Pay attention to being careful when board-washing, in order to avoid the cross contamination between sample.
7. the anti-HPV16 ELIAS secondary antibody of 100 μ l Fresh (the HPV monoclonal antibody of preparation, label then obtain) is added respectively
In reacting hole, it is incubated for 30 minutes with 37 degree of strip of paper used for sealing capping plate.
8. after board-washing, the substrate solution (TMB) of 100 μ l is added in reacting hole respectively, and in be protected from light, room temperature condition decentralization
It sets 10 minutes.Add behind the first hole i.e. Timing.
9. the terminate liquid (2M sulfuric acid) that 50 μ l are added in each reacting hole terminates reaction.Paying attention to will be by adding ELIAS secondary antibody
Sequence plus terminate liquid.
10. measuring the absorbance value of sample and control at 450nm.
As a result as shown in table 1 and Fig. 6.
Table 1 detects the ability that the antibody inhibits HPV16 positive serum combination antigen by indirect ELISA.
It can be seen that by table 1 and Fig. 6 as the amount of monoclonal antibody increases it and inhibits positive serum combination antigen
Ability gradually increases.Show that the anti-HPV16 subclass antibodies in the monoclonal antibody and positive serum have good blocking ability.
Composition, preparation and the application of the blocking ELISA kit of embodiment 4HPV16 antibody test
After the debugging of square matrix titration experiments, the best peridium concentration for measuring hpv16 E6 antigen is 1ug/ml, will be resisted
The monoclonal antibody of HPVE6 does HRP (purchased from sigma) label, and optimal labelled antibody working concentration is 1: 4000.
Square matrix titration experiments are as follows：
According to square matrix titration experiments, antigen is done into gradient coating (5,4,2.5,2,1.25,1,0.75,0.5ug/ml), it will
The HPV antibody of label does the dilution of different dilutions (4000,6000,8000,10000), does non-blacked and blocks respectively
ELISA, experimental result are shown, when antigen protein package amount is 1ug/ml, and the anti-HPV antibody extension rate of label is 4000,
Block competition performance best.
One, the composition of ELISA kit is blocked
1. the ELISA Plate of pre-coated antigen：HPV16 E6 recombinant protein is coated with the amount of 1ug/ml with carbonate buffer solution
96 orifice plates, every 100 μ l of hole.4 DEG C of coatings overnight, routinely close and wash by ELISA method.
2. positive control standard items and negative control standard items.
The anti-HPV16 E6 monoclonal antibody of the invention of HRP label is carried out when in use with 1: 4000 dilution ratio dilute
It releases.
3. enzyme labelled antibody dilution：0.01M PBS, pH7.2.
4. cleaning solution：PBST
5. developing solution：TMB
6. terminate liquid：2M sulfuric acid.
Two, the preparation of ELISA kit is blocked
1. HPV16 E6 recombinant protein is coated with 96 orifice plates with the amount of 1ug/ml
With coating buffer by HPV16E6 antigen diluent at 1ug/ml, 100 μ l are added in every hole, and 4 DEG C of coatings are overnight.Next day inclines
Coating buffer, is washed 3 times with the cleaning solution diluted, is patted dry, and 200 μ l confining liquids are then added in every hole, and 37 DEG C of incubation 2h incline
Liquid in hole is removed, is sealed after dry with aluminium foil bag.
Coating buffer：PH9.6 carbonate buffer solution
Confining liquid：1%BSA
The preparation of the monoclonal antibody of the invention of 2.HRP label
Monoclonal antibody prepared by the present invention is marked with HRP, method is as follows：
For marking 5mg IgG：
1, it accurately weighs 5mg HRP and is dissolved in the distilled water of 0-5ml (brownish red)；
2, the NaIO4 solution that 0.5ml concentration is 0.06M is added, solution colour becomes grass green；
3,4 degree of placement 30min；
4, the ethylene glycol 0.5ml of 0.16mol/L is added to terminate oxidation reaction, room temperature avoid light place 30min, solution is at palm fibre
Yellow；
5, the IgG 1.0ml of 5mg/ml is added, solution brown color shoals；
6, bag filter, 4 degree of stirring dialysed overnights, the carbonate buffer solution (sodium carbonate of dialyzate 0.05M, PH9.5 are put into
3.18g, sodium bicarbonate 5.88g add distilled water constant volume 2L)；
7, dialysis is completed, and is sucked out, NaHB4 the solution 0.2ml, 4 degree of placement 2h of the Fresh of 5mg/ml is added；
8, isometric saturated ammonium sulfate (PH7.2), 4 degree of placement 30min are added；
9,10000rpm/min is centrifuged 10min, discards supernatant, with the PB of 20mM (Na2HPO4.12H2O7.16g,
NaH2PO4 3.12g adds dd water constant volume 2L) it is resuspended.Dialysed overnight.
10,10000rpm/min is centrifuged, and supernatant constant volume 2.50ml is added 2.5ml glycerol.- 20 degree save backup.
Three, the application of ELISA kit is blocked
It is specific to block ELISA kit detection operating procedure as follows：
1. when in use, all reagent constituents must all be restored to 18~25 DEG C of room temperature.It should be by each component before use
It is placed in room temperature at least one hour.
2. 50 μ l sample diluting liquids are added in each detection hole and control wells respectively.
3. the positive control of 50 μ l and negative control are added in corresponding control wells respectively, the suction nozzle of different controls is paid attention to
It replaces, with anti-cross-contamination.
4. the test sample of 50 μ l is added in remaining detection hole respectively, notice that the suction nozzle of different test samples will divide
It opens, to prevent pollution.
5. flicking ELISA Plate or being vibrated with oscillator, the solution in reaction plate is mixed.
6. ELISA Plate is closed, 37 degree of incubation 1h；
7. getting rid of the liquid in reacting hole, and washed 3 times with the cleaning solution diluted, pays attention to wash when washing every time
It washs liquid and fills it up with reacting hole, discard the cleaning solution in reacting hole and pat dry reaction plate.It can also be machine-washed and be washed 3 times with board-washing, it is each anti-
Ying Kongying adds the cleaning solution of 300 μ l or so.Pay attention to being careful when board-washing, in order to avoid the cross contamination between sample.
8. the anti-HPV16 ELIAS secondary antibody (take and use) of 100 μ l is added in reacting hole respectively, with strip of paper used for sealing capping plate
37 degree are incubated for 30 minutes.
9. after board-washing (see 7), the substrate solution of 100 μ l is added in reacting hole respectively, and in be protected from light, room temperature condition decentralization
It sets 10 minutes.Add behind the first hole i.e. Timing.
10. the terminate liquid that 50 μ l are added in each reacting hole terminates reaction.Paying attention to will be by adding the sequence of ELIAS secondary antibody to add
Terminate liquid.
11. measuring the absorbance value of sample and control at 450nm.
12. calculating the mean absorbance values of sample and control (see calculating).
Calculation method
Calculate average value the OD450 (=OD of tested sampleTEST), the average value (=OD of positive controlPOS), negative control
Average value (=ODNEG)。
The blocking rate of tested sample and positive control is calculated according to the following formula；(positive control blocking rate is in kind
It calculates)
Test validity
The average OD450 of negative control should be greater than 0.50.The blocking rate of positive control should be greater than 50%.
Result judgement
If the blocking rate of tested sample is greater than or equal to 40%, which can be judged to the positive, that is, have HPV16
Antibody exists, and immune effect is good.If the blocking rate of tested sample is less than or equal to 30%, which can be judged to yin
Property, i.e. nonreactive HPV16 antibody exists, it is proposed that renewed vaccination vaccine.If being detected the blocking rate of sample between 30~40%,
It is determined as doubtful, needs to detect again.
The preservation of biomaterial
Generate by above-described embodiment identification anti-HPV16 E6 albumen monoclonal antibody hybridoma cell strain in
It is preserved in China typical culture collection center (CCTCC, China, Wuhan, Wuhan University) on June 24th, 2018, deposit number is
CCTCC NO：C2018153, classification naming are HPV16 E6 (E7) hybridoma cell strain.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Beijing Suo Laibao Science and Technology Ltd
<120>It is a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 474
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
atgcaccaaa agagaactgc aatgtttcag gacccacagg agcgacccag aaagttacca 60
cagttatgca cagagctgca aacaactata catgatataa tattagaatg tgtgtactgc 120
aagcaacagt tactgcgacg tgaggtatat gactttgctt ttcgggattt atgcatagta 180
tatagagatg ggaatccata tgctgtatgt gataaatgtt taaagtttta ttctaaaatt 240
agtgagtata gacattattg ttatagtttg tatggaacaa cattagaaca gcaatacaac 300
aaaccgttgt gtgatttgtt aattaggtgt attaactgtc aaaagccact gtgtcctgaa 360
gaaaagcaaa gacatctgga caaaaagcaa agattccata atataagggg tcggtggacc 420
ggtcgatgta tgtcttgttg cagatcatca agaacacgta gagaaaccca gctg 474
<210> 2
<211> 474
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atgcatcaga aacgtaccgc aatgtttcag gatccgcagg aacgtccgcg taaactgccg 60
cagctgtgta ccgaactgca gaccaccatt catgatatta ttctggaatg tgtttattgc 120
aaacagcagc tgctgcgccg tgaagtttat gattttgcct ttcgtgatct gtgtattgtt 180
tatcgtgatg gtaatccgta tgccgtttgt gataaatgtc tgaaatttta tagcaaaatt 240
agcgaatatc gtcattattg ttattccctg tatggtacca ccctggaaca gcagtataat 300
aaaccgctgt gtgatttgct gattcgttgt attaattgtc agaaaccgct gtgtccggaa 360
gaaaaacagc gccatctaga taaaaaacag cgttttcata atattcgtgg tcgttggacc 420
ggtcgttgta tgagctgttg tcgtagcagc cgtaccagga gagagaccca gctg 474
<210> 3
<211> 29
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
tttgaattca tgcatcagaa acgtaccgc 29
<210> 4
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
tttctcgagc agctgggtct ctctcctggt acgg 34
Claims (10)
1. a kind of monoclonal antibody for specifically binding HPV16 E6 albumen, which is characterized in that it is by mouse hybridoma cell system
CCTCC NO：C2018153 secretion.
2. a kind of mouse hybridoma cell system CCTCC NO for secreting monoclonal antibody according to claim 1：
C2018153。
3. monoclonal antibody according to claim 1 is preparing the blocking ELISA for assessing HPV16 immune effect of vaccine
Application in kit.
4. a kind of for assessing the blocking ELISA kit of HPV16 immune effect of vaccine, which is characterized in that the kit packet
Containing monoclonal antibody according to claim 1, the monoclonal antibody resists for detecting in HPV16 vaccine recipient's blood
The content of HPV16 immune antiboidy.
5. blocking ELISA kit according to claim 4, which is characterized in that in the kit, for combining institute
The antigen for stating anti-HPV16 immune antiboidy in monoclonal antibody and HPV16 vaccine recipient's blood is by SEQ ID NO：Shown in 2
The HPV16 E6 recombinant protein of nucleic acid sequence encoding, peridium concentration 1ug/ml, the monoclonal antibody is marked with HRP
Monoclonal antibody, and be diluted when in use with 1: 4000 dilution ratio.
6. blocking ELISA kit according to claim 4 or 5, which is characterized in that when being detected with the kit,
The blocking rate of tested sample is greater than or equal to 40%, and the sample can be judged to the positive, that is, with the presence of HPV16 antibody, exempt from
Epidemic disease works well；If the blocking rate of tested sample is less than or equal to 30%, the sample can be judged to feminine gender, i.e. nonreactive
HPV16 antibody exists, it is proposed that renewed vaccination vaccine；If the blocking rate of tested sample is judged to doubting between 30~40%
Seemingly, it needs to detect again.
7. a kind of prepare the method according to claim 4 for blocking ELISA kit, the described method comprises the following steps：
(1) HPV16 E6 recombinant protein is prepared, by SEQ ID NO：Nucleic acid sequence encoding shown in 2；
(2) using the recombinant protein of step (1) acquisition as immunogene, monoclonal antibody is prepared by hybridoma technology；
(3) the recombinant protein coated elisa plate for obtaining step (1)；
(4) monoclonal antibody obtained with HRP markers step (2)；
To which preparation is to block ELISA as the blocking ELISA reagent of HPV16 immune antiboidy in principle detection vaccine immunity person serum
Box.
8. the method according to the description of claim 7 is characterized in that in step (1), when expanding E6 protein gene, to close
Numeral optimizes, in favor of obtaining immunogene.
9. the method according to the description of claim 7 is characterized in that the peridium concentration of recombinant protein is 1ug/ in step (3)
ml。
10. method according to any one of claims 7 to 9, which is characterized in that existed with the monoclonal antibody that HRP is marked
It is diluted when use with 1: 4000 dilution ratio.
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