CN108872596A - A kind of ELISA detection kit of HPV16 L1 antibody - Google Patents
A kind of ELISA detection kit of HPV16 L1 antibody Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of ELISA detection kit of HPV16L1 antibody.ELISA kit of the invention includes：The secondary antibody of ELISA Plate, horseradish peroxidase label；Wherein ELISA Plate is coated with HPV16L1 recombinant protein；And ELISA Plate includes the ELISA Plate main body with graphene conductive layer, the temperature regulating device for controlling the graphene conductive layer temperature and the power supply device being electrically connected with temperature regulating device.When kit of the invention is used for the bioactivity of antibody, graphene conductive layer heats reagent in ELISA Plate and plate, can save insulating box and be incubated for process, simplify the experimental implementation of ELISA.Simultaneously, it is coated with HPV16L1 recombinant protein in the reacting hole of ELISA Plate, is suitable for detection HPV16L1 antibody, has the advantages that take and use, without in addition preparing HPV16L1 recombinant protein, the time required to greatly reducing testing cost, shortening detection HPV16L1 antibody.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of ELISA detection kit of HPV16 L1 antibody.
Background technique
Cervical carcinoma accounts for the second of female gynecological malignant tumour, has become the important illness for threatening women's health, China
The number nearly 50,000 of cervical carcinoma is died of every year, and the etiological study of cervical carcinoma is paid attention to by domestic and foreign scholars always, and research finds to draw
The key for playing cervical carcinoma is the infection of HPV.HPV belongs to Papillomaviridae Papillomavirus, is spherical, double-stranded cyclic DNA
Virus.HPV mainly passes through direct or indirect contact stain article or by the Sex transmitted pathogen mankind.HPV's is special infected with host
Anisotropic and tissue specificity can only infect the skin of the mankind and the basal cell that mucous membrane is impaired.
Cervical mucosa of the HPV through damaging infects basal cell layer first, and virus is hidden with a small amount of dissociative DNA state in substrate
Into the cell, to realize immunologic escape.When basal cell constantly breaks up, it is mature, migrated to mucosal surface, HPV viruse is just a large amount of to be increased
It grows.With the apoptosis of mucosal surface epithelial cell, virion is largely discharged into epithelial surface, can be into one as the new source of infection
Step aggravates infection.Long-term HPV persistent infection causes host cell related gene expression to change, and can gradually develop as uterine neck
Cancer.
HPV has identified specific hypotype a about more than 100 so far, causes a disease different with prognosis according to it and can be divided into high-risk
Type HPV (HR-HPV) and low risk HPV (LR-HPV), wherein 15 kinds of HR-HPV, including HR-HPV16,18,31,33,35,
39,45, type of 5l, 52,56,53,58 etc., can cause precancerous lesion and cervical carcinoma after infection.Wherein HPV16 and HPV18 is most main
The epidemic strain wanted.
HPV L1 glutelin is the major structural protein of human papilloma virus, is virus infected cell and cell immune response
The principal target of challenge virus, and the albumen is more conservative.Therefore have some for the exploitation preventing/treating HPV sense of L1 albumen
The research of the antibody of dye.
It after obtaining HPV16 L1 antibody, needs to detect its potency by ELISA, and is there is no at present for HPV16
The ELISA detection kit of L1 antibody.It needs to use antigen in detection process, and when some HPV16 L1 Antibody preparations, it uses
The HPV16 L1 antibody that nucleic acid vaccine immunity obtains causes ELISA also to need in addition to prepare HPV16 L1 when detecting antibody anti-
Original, time-consuming, complicated for operation, at high cost, causes significant wastage.
Therefore a kind of ELISA detection kit for HPV16 L1 antibody is developed, has the advantages that take and use, is not necessarily to
In addition HPV16 L1 recombinant protein is prepared, the time required to greatly reducing testing cost, shortening detection HPV16 L1 antibody.
Summary of the invention
HPV16 L1 glutelin is the major structural protein of human papilloma virus, is that virus infected cell and cellular immunity are anti-
The principal target of challenge virus is answered, and the albumen is more conservative.Therefore have some for L1 albumen exploitation preventing/treating HPV
The research of the antibody of infection.
It after obtaining HPV16 L1 antibody, needs to detect its potency by ELISA, and is there is no at present for HPV16
The ELISA detection kit of L1 antibody.
In view of this, the purpose of the present invention is to provide a kind of ELISA kit for HPV16 L1 antibody test,
Can self-heating, be repeatedly incubated for without insulating box, it is easy to operate, and have the advantages that take i.e. use, save the time, reduce at
This.
To achieve the above object, the technical scheme is that：
A kind of ELISA kit for HPV16 L1 antibody test comprising：ELISA Plate, horseradish peroxidase (HRP)
The secondary antibody of label；The ELISA Plate is coated with HPV16 L1 recombinant protein；
The ELISA Plate includes the ELISA Plate main body with graphene conductive layer, for controlling the graphene conductive layer temperature
The temperature regulating device of degree and the power supply device being electrically connected with temperature regulating device；
The temperature regulating device includes temperature detecting module and control module；
The temperature detecting module is used to detect the real time temperature data of graphene layer, and the real time temperature data are passed
It is handed to the control module；
The control module is used to be arranged the control instruction to temperature, and receives the real-time temperature of the temperature detecting module
Degree evidence, and corresponding control command is extracted according to the control instruction and the real time temperature data, the control command is used
In controlling the graphene conductive layer heating or stopping heating, so that the real time temperature data are corresponding with the control instruction
Temperature data match.
HPV16 L1 glutelin is very conservative, and the L1 albumen of different strains has more identical epitope, based on
Upper consideration, HPV16 L1 protein sequence of the recombination HPV L1 albumen containing overall length of the invention.
As a preferred option, above-mentioned HPV16 L1 recombinant protein is the HPV16 L1 recombinant protein of Bacillus coli expression,
Its amino acid sequence includes the full length amino acid sequence of HPV16 L1 albumen.
The working principle of ELISA Plate can be heated：The control module of temperature regulating device issues instruction to graphene after temperature is arranged
Conductive layer heating, graphene conductive layer is heating up, and conducts heat to reagent box main body, makes the main body temperature liter of ELISA Plate
Height further transfers heat to ELISA Plate reacting hole, heats to it.
As a preferred option, the temperature detecting module includes temperature sensor, and the temperature sensor is at least arranged
In the inner wall of ELISA Plate main body one at.
As a preferred option, the bottom hole of the ELISA Plate and the wall bottom of the ELISA Plate are in contact.
When the two is in contact, directly heats and existed simultaneously with air heat transfer：The graphene conductive layer liter of ELISA Plate inner wall
Wen Houke heats ELISA Plate bottom hole, and then transfers heat to reagent in hole, and reagent heats in device to hole；Meanwhile graphene conductive layer adds
In hot plate after air, the part that ELISA Plate reacting hole is contacted with air is heated.
As a preferred option, the ELISA Plate also has plate lid.
As a preferred option, above-mentioned ELISA Plate is detachable ELISA Plate, is coated with the HPV16 L1 weight comprising multiple
The enzyme mark strip of histone.
Further, above-mentioned ELISA Plate is 96 hole elisa Plates, the above-mentioned enzyme mark strip for being coated with the HPV16 L1 recombinant protein
For 12 hole enzyme mark strips or 8 hole enzyme mark strips.
As a preferred option, mentioned reagent box also includes sample dilution buffer, preparation：BSA 1g,NaCl
0.8g, KH2PO40.02g, Na2HPO4.12H2300 0.01g of O 0.29g, KCl 0.02g, Proclin, adds distilled water extremely
100ml is adjusted to pH 7.4.
The present invention changes by the Proclin 300 of novel non-toxic as preservative instead of traditional hypertoxic Sodium azide.
As a preferred option, mentioned reagent box also includes tmb substrate liquid and terminate liquid；
The tmb substrate liquid is one-component developing solution；
The substrate solution：TMB 20mg, dehydrated alcohol 10ml, adds distilled water to 100ml；
The terminate liquid is 2mol/L H2SO4Aqueous solution.
Kit of the invention uses one-component developing solution, and when use no longer needs to using two-component (i.e.：A liquid, B liquid, behaviour
Make more cumbersome), the developing solution of one-component need to only add once, and sensitivity is higher, specific more preferable, colour developing later period (termination
The result retention time is longer later), convenient for having the sufficient time to record and analysis result.
As a preferred option, mentioned reagent box also includes the PBST solution of 5 times of concentration, 10 times of concentration or higher concentration.
As a preferred option, above-mentioned graphene conductive layer is located at the inner surface of the ELISA Plate main body.
As a preferred option, when above-mentioned ELISA kit is used for chicken HPV L1 antibody test, the horseradish peroxidase
The secondary antibody of label is the anti-chicken antibody of horseradish peroxidase label.For example, the goat-anti chicken antibody of horseradish peroxidase label.
As a preferred option, mentioned reagent box also includes negative control and positive control, and the negative control is distillation
Water or PBS solution, the positive control are HPV16 L1 antibody.
The secondary antibody of above-mentioned horseradish peroxidase label is concentrate, and when use needs first to dilute.And half after dilution is small
When interior use.
Kit of the invention can save 6 months in 2-8 degrees Celsius, and -20 degrees Celsius save 12 months.Wherein horseradish peroxide
The concentrate for changing the secondary antibody of enzyme label is only stored in 4 degrees Celsius.
The second object of the present invention is to provide the side that a kind of above-mentioned ELISA kit is used to detect HPV16 L1 antibody
Method saves the multiple incubation process of insulating box, easy to operate.
To achieve the above object, the technical scheme is that：
The method that the ELISA kit of purpose one is used to detect HPV16 L1 antibody, includes the following steps：
1) ELISA kit is powered on, be arranged the temperature regulating device controlled at 37 DEG C；Blank is set
Group, negative control group and experimental group；
2) product are loaded：Reasonable concentration gradient is set, measuring samples are diluted with PBS solution, by the measuring samples after dilution
It is added in the experimental group reacting hole of the ELISA Plate, negative control group adds PBS solution, and after insulation reaction 60-90min, PBST is molten
Liquid washing；
3) add secondary antibody：To being added what the horseradish peroxidase marked in the reacting hole of the negative control group and experimental group
Secondary antibody, after insulation reaction 30-60min, the washing of PBST solution；
4) it develops the color：TMB- hydrogen peroxide urea solution is added in every reacting hole, is protected from light insulation reaction 5-10min, rear to be added
2mol/L H2SO4Aqueous solution；
5) result detects：2mol/L H is added2SO4Aqueous solution 15min in, with 450nm wavelength detecting as a result, and counting
Calculation obtains the potency of HPV16 L1 antibody.
As a preferred option, the HPV16 L1 antibody of kit of the invention detection is chicken antibody, at this time horseradish peroxide
The secondary antibody for changing enzyme label is the anti-chicken antibody of horseradish peroxidase label.For example, the goat-anti chicken antibody of horseradish peroxidase label.
Further, it can be used for detecting the HPV16 L1 antibody in the raw sample of chicken serum, Egg-white and other chicken pot pie.
The beneficial effects of the present invention are：ELISA detection kit provided by the invention for HPV16 L1 antibody：
1) have the advantages that take i.e. use, without in addition preparation HPV16 L1 recombinant protein, greatly reduce testing cost,
The time required to shortening detection HPV16 L1 antibody.
2) enzyme mark hole has been coated with HPV16 L1 recombinant protein, and completes to close, subsequent only to need to be incubated for antibody to be checked, incubation
Secondary antibody, colour developing, simplify experimental implementation.
3) when being used for the bioactivity of antibody, graphene conductive layer heats reagent in ELISA Plate and plate, can save insulating box
Incubation process simplifies the experimental implementation of ELISA.
Detailed description of the invention
Fig. 1 is the ELISA Plate schematic diagram of ELISA kit.Wherein, 1：ELISA Plate main body；11：Plate lid；2：Temperature regulating device；
3：Attaching plug.
Fig. 2 is ELISA Plate main body schematic top plan view.Wherein, 1：ELISA Plate main body；12：ELISA Plate reacting hole；13：ELISA Plate
Partition.
Specific embodiment
The preferred embodiment of the present invention will be described in detail (referring to attached drawing) below.Tool is not specified in preferred embodiment
The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition, illustrated embodiment are to preferably say to the contents of the present invention
It is bright, but be not that the contents of the present invention are only limitted to illustrated embodiment.So those skilled in the art are according to foregoing invention
Content carries out nonessential modifications and adaptations to embodiment, still falls within protection scope of the present invention.
1 ELISA kit of embodiment
The present invention develops ELISA kit comprising：Secondary antibody, the sample of ELISA Plate, horseradish peroxidase (HRP) label
Dilution buffer, TMD substrate solution and terminate liquid, positive control, negative control；The ELISA Plate is coated with HPV16 L1 recombination egg
It is white.
1. Elisa plate structure
ELISA Plate includes comprising ELISA Plate main body 1, temperature regulating device 2, power supply device 3.Specially：With graphene conductive layer
ELISA Plate main body 1, with graphene conductive layer be electrically connected, for control the graphene conductive layer temperature temperature regulating device 2,
The power supply device 3 being electrically connected with temperature regulating device；
Wherein the graphene conductive layer is fixedly connected with the temperature regulating device.
1) ELISA Plate main body 1
Preferably, the graphene conductive layer is located at the inner surface of the ELISA Plate main body.
Reagent box main body 1 further preferably includes box cover.
The outer layer of reagent box main body 1 may include heat-barrier material, such as foam, glass fibre, asbestos.
The graphene conductive layer of reagent box main body 1 plays heat effect, and reagent box wall plays heat preservation, supporting role.
ELISA Plate main body as shown in Figure 1 and Figure 2, is detachable ELISA Plate, and include multiple 12 holes enzyme mark strip.
2) it is electrically connected with graphene conductive layer, for controlling the temperature regulating device 2 of the graphene conductive layer temperature, institute
Stating temperature regulating device includes temperature detecting module and control module.
Wherein temperature detecting module is used to detect the real time temperature data of graphene layer, and the real time temperature data are passed
It is handed to the control module；Control module is used to be arranged the control instruction to temperature, and receives the temperature detecting module
Real time temperature data, and corresponding control command, the control are extracted according to the control instruction and the real time temperature data
Order is for controlling the graphene conductive layer heating or stopping heating, so that the real time temperature data refer to the control
Corresponding temperature data is enabled to match.
Preferably, above-mentioned temperature detecting module includes temperature sensor, and the temperature sensor is at least set to the enzyme
In the inner wall of target main body one at.
1. the control module of temperature regulating device issues instruction and heats to graphene conductive layer after temperature is arranged, graphene conductive
Layer is heating up, and conducts heat to ELISA Plate main body, increases the main body temperature of ELISA Plate, it is anti-further to transfer heat to enzyme mark
Ying Kong heats it.
2. temperature sensor detects temperature change and is transferred to control module in the inner wall of ELISA Plate main body；Until inner wall
The temperature of middle temperature sensor detection reaches setting value, and such as 37 degrees Celsius, control module is extracted and issued after receiving temperature data
Stop call for heat, graphene conductive layer stops heating.
3) power supply device 3 being electrically connected with temperature regulating device
It is chosen as battery or attaching plug etc..If selecting attaching plug, when using this kit, plug connects power supply.
Preferably, the reacting hole bottom of above-mentioned ELISA Plate and the wall bottom of ELISA Plate are in contact.When the two is in contact, directly
Heating and air heat transfer exist simultaneously：ELISA Plate bottom hole can be heated after the graphene conductive layer heating of ELISA Plate inner wall, in turn
Reagent in hole is transferred heat to, reagent heats in device to hole；Meanwhile in graphene conductive layer heating ELISA Plate main body after air, ELISA Plate
The part that reacting hole is contacted with air is heated.
If the two is not in contact, in graphene conductive layer heating ELISA Plate main body after air, heat is passed by air
It is handed to the hole material and further, reagent in hole of ELISA Plate.
ELISA Plate is preferably 96 hole elisa Plates, is further preferably detachable ELISA Plate, includes multiple 12 hole enzyme marks
Item.
2.HPV16 L1 recombinant protein
ELISA Plate reacting hole of the invention is coated with HPV16 L1 recombinant protein.
Preferably, the preparation process of the HPV16 L1 recombinant protein includes：
1) the nucleotide sequence overall length of HPV16 L1 is cloned；
2) it is connected to carrier, obtains recombinant vector；
3) recombinant vector converts Escherichia coli, obtains recombination bacillus coli；
4) expression of recombinant e. coli is induced, purifying obtains recombinant protein, i.e. HPV16 L1 recombinant protein.
3. other
1) secondary antibody of horseradish peroxidase (HRP) label
The secondary antibody of horseradish peroxidase (HRP) label is five times of concentrates, is stored in 4 DEG C.
When the anti-chicken antibody that the secondary antibody of horseradish peroxidase label is horseradish peroxidase label, for example, horseradish peroxidating
The goat-anti chicken antibody of enzyme label, kit of the invention can be used for the chicken HPV L1 antibody detected.
2) sample dilution buffer, preparation：BSA 1g, NaCl 0.8g, KH2PO40.02g, Na2HPO4.12H2O
300 0.01g of 0.29g, KCl 0.02g, Proclin, adds distilled water to 100ml, is adjusted to pH 7.4.
3) tmb substrate liquid and terminate liquid；
The tmb substrate liquid is one-component developing solution；
The substrate solution：TMB 20mg, dehydrated alcohol 10ml, adds distilled water to 100ml；
The terminate liquid is 2mol/L H2SO4Aqueous solution.
Terminate liquid is 2mol/L H2SO4Aqueous solution.
Further, ELISA kit of the invention also includes the PBST solution of 5 times of concentration, 10 times of concentration or higher concentration,
As cleaning solution.
2 ELISA kit application method of embodiment
1) before carrying out ELISA experiment, ELISA kit is assembled according to Fig. 1 Fig. 2.
Plug connects power supply, and temperature regulating device 2 is arranged in advance, is configured to the temperature of kit, and such as 37 DEG C.When setting temperature
The control module of temperature regulating device issues instruction and heats to graphene conductive layer afterwards, and graphene conductive layer is heating up.Such as graphene
Conductive layer is not in contact with ELISA Plate bottom hole, is heated in reagent box main body after air, and air conducts heat to ELISA Plate,
Further to reagent in hole；If the two contacts, directly heats bottom hole and air heating ELISA Plate exists simultaneously.
Temperature regulating device of the invention ensure that the stabilization of temperature.
2) ELISA experimental implementation is carried out by step, during insulation reaction, splice lid 11, in favor of heat preservation.
1. blank group, negative control group and experimental group is arranged；
2. being loaded product：Reasonable concentration gradient is set, measuring samples are diluted with PBS solution, by the measuring samples after dilution
It is added in the experimental group reacting hole of the ELISA Plate, negative control group adds PBS solution, and after insulation reaction 60-90min, PBST is molten
Liquid washing；
3. plus secondary antibody：To being added what the horseradish peroxidase marked in the reacting hole of the negative control group and experimental group
Secondary antibody, after insulation reaction 30-60min, the washing of PBST solution；
4. developing the color：TMB- hydrogen peroxide urea solution is added in every reacting hole, is protected from light insulation reaction 5-10min, rear to be added
2mol/L H2SO4Aqueous solution；
5. result detects：2mol/L H is added2SO4Aqueous solution 15min in, with 450nm wavelength detecting result.
3) enzyme mark strip after the reaction was completed, is removed, is detected for result.
When the anti-chicken antibody that the secondary antibody of horseradish peroxidase label is horseradish peroxidase label, for example, horseradish peroxidating
The goat-anti chicken antibody of enzyme label, kit of the invention can be used for the chicken HPV L1 antibody detected.Further, can be used for detecting chicken
HPV L1 antibody in the raw sample of serum, Egg-white and other chicken pot pie.
ELISA kit of the invention is reusable.When experiment next time, other HPV16 L1 recombinant protein is taken to be coated with
Enzyme mark strip, and be assembled into ELISA kit shown in Fig. 1 Fig. 2 and tested.
ELISA detection kit for HPV16 L1 antibody of the invention, has been coated with HPV16 L1 recombinant protein, tool
Have and take the advantages of using, without in addition preparation HPV16 L1 recombinant protein, greatly reduces testing cost, shortens detection
The time required to HPV16 L1 antibody.And can self-heating, exempt from insulating box be incubated for process, it is easy to operate.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (10)
1. a kind of ELISA kit for HPV16 L1 antibody test, which is characterized in that the kit includes：ELISA Plate,
The secondary antibody of horseradish peroxidase label；The reacting hole of the ELISA Plate is coated with HPV16 L1 recombinant protein；
The ELISA Plate includes the ELISA Plate main body with graphene conductive layer, for controlling the graphene conductive layer temperature
Temperature regulating device and the power supply device being electrically connected with temperature regulating device；
The temperature regulating device includes temperature detecting module and control module；
The temperature detecting module is used to detect the real time temperature data of graphene layer, and the real time temperature data are transferred to
The control module；
The control module is used to be arranged the control instruction to temperature, and receives the real time temperature number of the temperature detecting module
According to, and corresponding control command is extracted according to the control instruction and the real time temperature data, the control command is for controlling
It makes the graphene conductive layer heating or stops heating, so that real time temperature data temperature corresponding with the control instruction
Degree evidence matches；
The HPV16 L1 recombinant protein includes the full length sequence of HPV16 L1 albumen.
2. ELISA kit according to claim 1, which is characterized in that the HPV16 L1 recombinant protein is large intestine bar
The HPV16 L1 recombinant protein of bacterium expression.
3. ELISA kit according to claim 1, which is characterized in that the ELISA Plate is detachable ELISA Plate, includes
Multiple enzyme mark strips for being coated with the HPV16 L1 recombinant protein.
4. ELISA kit according to claim 1, which is characterized in that the ELISA Plate is 96 hole elisa Plates.
5. ELISA kit according to claim 1, which is characterized in that the kit also includes sample dilution buffer
Liquid；
The preparation of the samples dilution buffer：BSA 1g, NaCl 0.8g, KH2PO40.02g, Na2HPO4.12H2O
300 0.01g of 0.29g, KCl 0.02g, Proclin, adds distilled water to 100ml, is adjusted to pH 7.4.
6. ELISA kit according to claim 1, which is characterized in that the kit also includes tmb substrate liquid and end
Only liquid；
The tmb substrate liquid is one-component developing solution；
The substrate solution：TMB 20mg, dehydrated alcohol 10ml, adds distilled water to 100ml；
The terminate liquid is 2mol/L H2SO4Aqueous solution.
7. ELISA kit according to claim 1, which is characterized in that the graphene conductive layer is located at the enzyme mark
The inner surface of plate main body.
8. ELISA kit according to claim 1, which is characterized in that the ELISA kit is anti-for chicken HPV L1
When physical examination is surveyed, the secondary antibody of the horseradish peroxidase label is the anti-chicken antibody of horseradish peroxidase label.
9. ELISA kit according to claim 1, which is characterized in that the kit also includes negative control and sun
Property control, the negative control be distilled water or PBS solution, the positive control be HPV L1 antibody.
10. the method that ELISA kit described in claim 1 is used to detect HPV L1 antibody, which is characterized in that including following step
Suddenly：
1) ELISA kit is powered on, be arranged the temperature regulating device controlled at 37 DEG C；And blank is set
Group, negative control group and experimental group；
2) product are loaded：Reasonable concentration gradient is set, dilutes measuring samples with PBS solution, the measuring samples after dilution is added
In the experimental group reacting hole of the ELISA Plate, negative control group adds PBS solution, after insulation reaction, the washing of PBST solution；
3) add secondary antibody：To be added that the horseradish peroxidase marks in the reacting hole of the negative control group and experimental group two
It is anti-, after insulation reaction, the washing of PBST solution；
4) it develops the color：TMB- hydrogen peroxide urea solution is added in every reacting hole, is protected from light insulation reaction, rear that 2mol/L H is added2SO4's
Aqueous solution；
5) result detects：2mol/L H is added2SO4Aqueous solution 15min in, with 450nm wavelength detecting as a result, calculate HPV
The potency of L1 antibody.
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