CN109580959A - A kind of ELISA kit detecting heparin-binding epidermal growth factor - Google Patents
A kind of ELISA kit detecting heparin-binding epidermal growth factor Download PDFInfo
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Abstract
The invention belongs to technical field of immunoassay, and in particular to a kind of ELISA kit for detecting heparin-binding epidermal growth factor.The kit includes: HB-EGF albumen rabbit polyclonal antibody, dilution, cleaning solution, developing solution, terminate liquid and the HB-EGF protein standard substance of 96 hole elisa Plates of pre-coated anti-HB-EGF protein monoclonal antibody, horseradish peroxidase (HRP) label.Double antibody sandwich method detection kit prepared by the present invention has the characteristics that high sensitivity, high specificity, quantitative accurate, easy to use, favorable reproducibility, HB-EGF content in detectable cell, serum, blood plasma and animal tissue's sample, for the qualitative and quantitative detection of HB-EGF albumen in basic research and clinical diagnosis, and high-volume sample can be quickly detected simultaneously, application prospect is very wide.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of detection heparin-binding epidermal growth factor
ELISA kit.
Background technique
Heparin-binding epidermal growth factors (Heparin-binding Epidermal Growth Factor, HB-EGF),
That is HB-EGF albumen.
In recent years, the experimental results prompt, HB-EGF or can become a new tumor markers, some researches show that,
The HB-EGF expression of partial breast cancer and patients with prostate cancer and lysophosphatidic acid -1 (LPA-1, a kind of rush tumour growth turn
Move the factor) level be positively correlated, prompt people HB-EGF or can be used as the kinds cancers such as breast cancer, oophoroma and prostate cancer
Diagnosis and clinical monitoring molecular marker.
Important biomarker of the HB-EGF as kinds of tumors occurrence and development accurately and rapidly detects it to swollen
Tumor early diagnosis, the research of tumor development mechanism develop tracking by stages and the meaning of clinical treatment and the judgement of prognosis situation
Justice is very crucial, thus be badly in need of establishing it is a kind of it is easy, efficiently, can be used for all kinds of common samples such as cell suspension, serum, blood plasma etc.
The reagent that quickly detects of high throughput and method.
Commercially available HB-EGF protein ELISA kit domestic at present is there are poor specificity, the disadvantages of low output, therefore needs out
High specific, the HB-EGF antibody of high-affinity and the development of related kit are sent out, supports every base of country's HB-EGF albumen
Plinth research, and further develop clinical in vitro diagnosis in vitro kit and critical material is provided, accelerate the exploitation of product, fills up technology neck
The blank in domain.
Summary of the invention
The object of the present invention is to provide a kind of low cost, detection sensitivity is high, testing result is stable, reproducible heparin
The Double antibody sandwich ELISA kit of associativity epidermal growth factor (HB-EGF).
The technical solution adopted by the present invention are as follows:
It is a kind of for detecting the ELISA kit of heparin-binding epidermal growth factor, the kit is using double
Antibody sandwich, the kit include the HB-EGF protein monoclonal antibody of specificity, horseradish peroxidase (HRP) label
Polyclonal antibody.
The monoclonal antibody includes light chain variable region, and sequence amino acid sequence is as shown in SEQ.ID.NO.2；Further include
Heavy chain variable region, amino acid sequence is as shown in SEQ.ID.NO.1.
Wherein, the HB-EGF protein monoclonal antibody of the specificity is for being coated with；The horseradish peroxidase
(HRP) polyclonal antibody marked is used for HB-EGF Protein Detection.
Wherein the HB-EGF monoclonal antibody is mouse monoclonal antibody；The polyclonal antibody of HRP label is rabbit polyclonal
Antibody.
The ELISA kit for detecting heparin-binding epidermal growth factor further includes sample diluting liquid, washing
Liquid, substrate developing solution, reaction terminating liquid and HB-EGF albumen standard items；
The sample diluting liquid is the phosphate buffer (PBS) containing 0.5% bovine serum albumin(BSA) (BSA)；It is described to wash
Washing liquid is the 0.1mol/L phosphate buffer (PBS) containing 0.1% Tween-20 (Tween-20)；The substrate show liquid by
Developing solution A and developing solution B composition, developing solution A be hydrogen peroxide, developing solution B be tetramethyl benzidine, developing solution using when take
A, B liquid of volume mix；The terminate liquid is the sulfuric acid of 2mol/L, and the HB-EGF protein standard substance is recombination HB-EGF egg
It is white.
After the mouse monoclonal antibody is by recombination HB-EGF protein immunization BALB/c mouse, obtained using hybridoma technology preparation
?；The rabbit polyclonal antibody of the HRP label purifies skill by ammonium sulfate method by recombinating HB-EGF protein immunization New Zealand White Rabbit
Art obtains；It is obtained according to a conventional method using horseradish peroxidase (Horseradish peroxidase, HRP) labelled antibody
The rabbit polyclonal antibody of HRP label, is used for HB-EGF Protein Detection.
The preferred high specific of the present invention, the monoclonal antibody of highly sensitive anti-HB-EGF and polyclonal antibody match
Combination, mouse monoclonal antibody is coated on solid phase carrier, it may specifically bind in recombination HB-EGF albumen and sample
It is compound to form solid matrix antibody-antigen-enzyme mark detection antibody when the rabbit polyclonal antibody that HRP label is added for HB-EGF albumen
Object reads each sample absorbance value, with standard curve using color developing agent to terminating after the colour developing of corresponding substrate under 450nm wavelength
The content of HB-EGF albumen in the sample that compares that you can get it.
Beneficial effects of the present invention:
For the present invention using the HB-EGF protein content in double antibody sandwich method quantitative detection sample, detection method is easy easily
Row, detection sensitivity and accuracy height, high specificity, it is reproducible and can and meanwhile quick detection high-volume sample.
The present invention selects HB-EGF albumen for standard items, and HB-EGF contains the Heparin-binding of 21 amino acid residue
Area, thus with heparin and the like heparan sulfate proteoglycan (heparan sulfate proteoglycan,
HSPG) there is stronger affinity.
Solid-phase enzyme-linked immune method (ELISA method) have technology maturation, high sensitivity, specificity it is good, easy to operate, detection at
Sheet is low, is suitable for the advantages such as high-volume pattern detection.
Coated antibody is mouse monoclonal antibody in kit of the invention, only one epitope, specificity is very well；Detect antibody
It is mostly anti-for rabbit, it is detected after being coupled HRP.The coating and detection antibody of the chief component of kit are that independent research produces
Product, therefore cost is greatly reduced relative to import external product, it will be played in the relevant rudimentary and clinical research of HB-EGF
Important function has a vast market foreground.
Detailed description of the invention
Fig. 1 is the sample-adding schematic diagram of each sample and control group in 96 hole elisa Plates.
Fig. 2 is the examination criteria curve graph of HB-EGF enzyme linked immunological double antibody sandwich method kit.
Specific embodiment
It is explained further the present invention combined with specific embodiments below, but is not intended to limit protection scope of the present invention.Below
Method, equipment in case study on implementation, material are conventional method in that art, equipment and material if do not illustrated.
It is prepared by the component of embodiment 1:HB-EGF enzyme linked immunological kit
(1) preparation of HB-EGF protein standard substance is recombinated:
Standard items in the reagent are recombination HB-EGF albumen, with pET-30a-HB-EGF (Invitrogen company) matter
Grain is transformed into Escherichia coli and the albumen is induced to be expressed in vitro, then the HB- of high-purity is obtained by affinity chromatography method
EGF albumen.
(2) preparation of anti-HB-EGF mouse monoclonal antibody:
A. animal immune:
Using female BAl BIc/c mouse inbred lines (being purchased from Nanjing University's zootype research institute) as immune animal, with above-mentioned
Recombination HB-EGF albumen is that immunogene is immunized, and immunizing dose is the recombination HB-EGF that 100 μ g are immunized in every mouse every time.It is first
When secondary immune, immunogene and isometric complete Freund's adjuvant are made emulsifier, subcutaneous abdomen multi-point injection, interval is after 3 weeks,
It takes same dose immunogene and isometric incomplete Freund's adjuvant that emulsifier is made, carries out second of booster immunization, secondary immunity
Tail vein blood afterwards measures serum titer using indirect elisa method, and serum titer reaches 1:105After above, two potency are selected
Highest mouse carries out third time booster immunization (method is with second of booster immunization), and interval carried out last time impact after 3 weeks
Immune, impact is immunized to take 200 μ g HB-EGF albumen, and addition PBS is diluted to 200 μ L, is injected intraperitoneally, and completion impact immune 3~
Mouse spleen cells are taken to carry out cell fusion after 4 days.
Wherein, the specific method is as follows for the indirect elisa method: recombination HB-EGF albumen is taken out, with pH 9.6,
HB-EGF albumen is diluted to 1 μ g/mL and 96 hole elisa Plates, every 100 μ L of hole, 4 DEG C of packets is added by the carbonate buffer solution of 0.05mol/L
It is stayed overnight, takes out and be coated with overnight ELISA Plate, after TBS-T washing 3 times, ELISA Plate is patted dry, 1 week after being immunized for the second time, from mouse
Tail vein is taken a blood sample in right amount, and 5000g is centrifuged 15min and separates serum, with the Sample dilution (phosphorus containing 0.5% bovine serum albumin(BSA)
Phthalate buffer) serum is diluted by 1:100,1:1000,1:10000,1:100000,1:1000000 gradient, every hole
ELISA Plate to be detected is added in 100 μ L, 37 DEG C, after being incubated for 1h, washs 3 times through TBS-T, pats dry ELISA Plate, every hole is added 100 μ L's
1:5000 dilutes the goat anti-mouse secondary antibody (being purchased from Jackson Immuno Research company) of HRP label, 37 DEG C of incubations
30min.ELISA Plate is taken out, after TBS-T is washed 5 times, every hole is added 100 μ L and substrate display liquid is added, and 37 DEG C are protected from light colour developing 10
~15min is then added 50 μ L terminate liquids and terminates reaction, reads light absorption value under microplate reader 450nm wavelength.Choose serum titer
Reach 1:105It is immune to carry out third time for the above mouse.
B. cell fusion and cloning
The mouse spleen for taking immune completion obtains splenocyte suspension through 80 mesh mesh screens grinding filter centrifugation, after cell count, leads to
Cross ratio of the cell-fusion techniques by splenocyte in 5:1 and the SP2/0 murine myeloma cell (in being purchased from logarithmic growth phase
The Kunming Ke Yuan cell bank) mixing, hybridoma is screened using HAT special culture medium, myeloma cell and splenocyte etc. are not melted
It closes or the cell of fusion not yet in effect will be unable to grow, and the hybridoma of effective integration will grow in culture hole, be proliferated, simultaneously
Secretory antibody.Cells and supernatant is taken within the 9th~12 day after fusion, to recombinate HB-EGF albumen as envelope antigen, using indirect
ELISA method measures antibody-secreting amount in supernatant, screens positive hole, and carry out cloning to positive cell by limiting dilution assay
Culture, until obtaining the monoclonal hybridoma strain of stably excreting anti-HB-EGF specific antibody.
The above cell fusion and cloning process are common classical way in immunology monoclonal antibody technique.
C. the production and purifying of anti-HB-EGF monoclonal antibody
Positive hybridoma cell is chosen, by 5 × 105A cell number/only inject the BALB/c for using atoleine sensitization in advance
Mouse peritoneal, 7~14d observation mouse web portion obviously expand, and can extract ascites.12000g centrifugation 10min is gone after ascites acquisition
Except grease and precipitating, supernatant, as anti-HB-EGF monoclonal antibody are collected, using Protein G method antibody purification, acquisition resists
Body dispenses after SDS-PAGE electrophoresis and indirect elisa method identification antibody purity and specificity, saves backup in -20 DEG C.
The specific method is as follows for Protein G method antibody purification: 1:3 is mixed well antibody by volume with acetate buffer
After be added dropwise caprylic acid, after 4 DEG C of standing 1h, 4 DEG C of centrifugation 30min of 2500g abandon precipitating, collect supernatant, are then added 0.1
10 × PBS of times volume adjusts pH to 7.4, is fitted into counter-balanced Protein G-chromatographic column, washes away foreign protein using PBS, most
Antibody protein is eluted by elution buffer (glycine solution for the 0.1mol/L that pH value is 2~3).
Antibody purity is verified through SDS-PAGE electrophoresis, and antibody can obtain higher purity, anti-HB- through second of elution
EGF monoclonal antibody IgG (H+L) molecular weight is about 160KD, and wherein IgG heavy chain is about 55KD, and IgG light chain is about 25KD.
The measurement of d.HB-EGF monoclonal cell strain sequence
In the present embodiment, heavy chain and light chain area gene are carried out to positive monoclonal cell strain using Protocols in Molecular Biology
Amplification, and carry out sequencing.
Antibody gene extracts amplification, and the specific method is as follows: collecting the good hybridoma of growth conditions, utilizes Thermo
The Trizol of company extracts hybridoma total serum IgE, by the HiScript Q RT SuperMix for of Nanjing Nuo Weizan company
The operation scheme of qPCR (+gDNA wiper) specification by mRNA reverse transcription be cDNA, -20 DEG C freeze it is spare.Reverse transcription reaction
System is 5 μ LRNA (2500ng), and 10 μ L 4 × gDNA, 10 5 × supermix of μ L II add dd H2O complements to 50 μ L, total anti-
Answering volume is 50 μ L.
The area source of mouse light chain FR1 and constant region gene sequences (NC000072.6) are searched by ncbi database, according to sequence
Design light chain PCR primer, upstream sequence are as follows: GCGGAGCTCGATRTTGTGATGACCCARAC, downstream sequence are as follows:
GCGTCTAGACTCATTCCTGTTGAAGCTCT；
The area source of mouse heavy chain FR1 and hinge area gene order (NC000078.6) are searched by ncbi database, according to sequence
Design heavy chain PCR primer, upstream sequence are as follows: GCGCTCGAGCAGKTCCAGCTGAAGCAGTC, downstream sequence are as follows:
GCGACTAGTGCATTTGCATGGAGGACAG。
Using the cDNA of hybridoma cell strain as template, PCR amplification obtains the light chain and heavy chain fragment of antibody.By Nanjing promise
The operation scheme of the Phanta Max Super-Fidelity DNA Polymerase specification of Wei Zan company carries out PCR, PCR
Reaction system are as follows: 25 μ L 2 × Phanta, 1 μ L dNTP, 4 10 μM of μ L primer pairs, 4 μ L hybridoma cDNA, 1 μ L DNA are poly-
Synthase, total reaction volume are 50 μ L.Amplification condition are as follows: 94 DEG C of initial denaturation, 3min；94 DEG C of denaturation, 30s；64 DEG C of annealing, 30s；Prolong
72 DEG C are stretched, 5min.Glue recycling and sequencing analysis are carried out to PCR product according to OMEGA company plastic recovery kit specification, obtained
The heavy chain variable amino acid sequence of hybridoma is SEQ.ID.NO.1, and light-chain variable sequence amino acid sequence is
SEQ.ID.NO.2。
Further, the potency of the monoclonal antibody in the verified present invention after purification reaches 1 × 108。
(3) preparation of HB-EGF rabbit polyclonal antibody
New zealand rabbit (being purchased from Nanjing University's zootype research institute) is chosen as immune animal, to recombinate HB-EGF albumen
It is immunized for immunogene, immunizing dose is the recombination HB-EGF albumen that 500 μ g are immunized in every rabbit every time.First immunisation will be exempted from
Emulsifier, the subcutaneous multi-point injection of the nape of the neck is made in epidemic focus and the complete Freund's adjuvant of equivalent, and interval takes same dose to exempt from 2~3 weeks
Emulsifier is made in epidemic focus and equivalent incomplete Freund's adjuvant, and booster immunization is immunized 4~5 times altogether, and indirect elisa method measures serum
Potency reaches 1:105After, arteria carotis communis takes blood, and by ammonium sulfate method purified polyclonal antibodies, packing is protected in -20 DEG C of low temperature
Deposit the preparation for enzyme labelled antibody.Wherein specific step is as follows for ammonium sulfate method antibody purification:
A. 1:1 is mixed antibody serum by volume with PBS, and saturated ammonium sulfate solution is then added dropwise, and is sufficiently mixed
It is even make 20% ammonium sulfate, stand 30min, in 4 DEG C, 2500g is centrifuged 30min, abandons precipitating, receives supernatant；
B. continuous dropwise addition saturated ammonium sulfate solution is relayed toward supernatant, makes 50% ammonium sulfate, is sufficiently mixed,
30min is stood, in 4 DEG C, 2500g is centrifuged 30min, abandons supernatant, collects precipitating；
C. the PBS of 1 times of volume is added into precipitating, sufficiently continuously adds ammonium sulfate after dissolution precipitating, makes
40% ammonium sulfate, is sufficiently mixed, and stands 30min, and in 4 DEG C, 2500g is centrifuged 30min, abandons supernatant, receives and precipitates, this step
It is rapid to repeat 2~3 times；
D. it is dissolved and is precipitated with PBS, as HB-EGF rabbit polyclonal antibody slightly purifies product.
(4) Antibody preparation of horseradish peroxidase-labeled
A. it weighs HRP 5mg to be dissolved in 1mL0.2mol/L pH5.6 acetate buffer, the nothing containing 1%DNFB is added
Hydrous ethanol solution 0.1mL, at room temperature gentle agitation 1h；
B. the 0.1mol/L NaIO of the fresh configuration of 0.5mL is added4Then step (3) are added in solution, 4 DEG C of placement 30min
5~the 10mg of polyclonal antibody to be marked being prepared adjusts pH value to 9.0~9.5 using carbonate buffer solution, sufficiently mixes
Even, 4 DEG C stand overnight；
C. the 4mg/ml NaBH of 0.1mL Fresh is added4Solution mixes, in 4 DEG C of placement 3h；
D. aforesaid liquid is fitted into bag filter, is dialysed in pH 7.4, the PBS solution of 0.01mol/L, 4 DEG C overnight；
E. liquid in bag filter is collected, 3000g is centrifuged 30min, removes sediment, supernatant is enzymic-labelled antibody.
Embodiment 2: the establishment of the enzyme linked immunological kit of detection HB-EGF albumen
The enzyme linked immunological kit of detection HB-EGF albumen is set up, includes following reagent:
A. anti-HB-EGF albumen mouse monoclonal antibody；
The anti-HB-EGF albumen rabbit polyclonal antibody of b.HRP label；
C. HB-EGF protein standard substance is recombinated；
D. it is coated with buffer: pH value 9.6, the carbonate buffer solution of 0.05mol/L；
E. confining liquid: contain the phosphate buffer of 0.5% (volumn concentration) bovine serum albumin(BSA)；
F. sample diluting liquid: contain the phosphate buffer of 0.5% (volumn concentration) bovine serum albumin(BSA)；
G. cleaning solution: contain the phosphate buffer of 0.1% (volumn concentration) tween；
H. substrate developing solution: being made of developing solution A and developing solution B, and developing solution A is hydrogen peroxide, and developing solution B is tetramethyl
Benzidine, developing solution using when take isometric A, B liquid to mix；
I. terminate liquid: the sulfuric acid of 2mol/L.
The preparation of embodiment 3:HB-EGF enzyme linked immunological kit
(1) orthogonal test gropes optimum antibody combination and the working concentration of enzyme linked immunological kit
Optimum antibody combination is groped using orthogonal test method and optimum antibody uses concentration, anti-HB-EGF albumen is small
The anti-HB-EGF albumen rabbit that mouse monoclonal antibody is diluted to 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL and 0.25 μ g/mL, HRP label is more
Clonal antibody is diluted to 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL and 0.25 μ g/mL, standard concentration is 100pg/ μ L, 10pg/ μ L,
1pg/ μ L and 0.1pg/ μ L.According to Orthogonal experiment results (table 1), in the standard protein group of various concentration, HB-EGF mouse Dan Ke
There is obvious reducing tendency in the absorbance value that grand antibody concentration detects after 1 μ g/mL, and HB-EGF rabbit polyclonal antibody concentration exists
There is obvious reducing tendency in the absorbance value detected after 0.5 μ g/mL, it is determined that HB-EGF mouse monoclonal antibody is as coated antibody
Best effort concentration be 1-2 μ g/mL, it is 0.5-2 μ that preferably 1 μ g/mL, HRP, which mark the best effort concentration of rabbit polyclonal antibody,
G/mL, preferably 0.5 μ g/mL.
1. orthogonal test of table gropes the optimum antibody combination and working concentration result of enzyme linked immunological kit
(2) the batch preparation of kit
A. a large amount of preparations of ELISA Plate:
Coated elisa plate: with coating buffer, anti-HB-EGF monoclonal antibody is diluted to concentration 1 μ g/mL, 100 μ L/
Hole is coated with 96 hole elisa Plates, 4 DEG C of overnight incubations；With 200 hole μ L/ of cleaning solution, board-washing 3 times；Closing: 100 μ L closing is added in every hole
Nonspecific binding site is closed in fluid-tight, is incubated at room temperature 1h；Then use 200 hole μ L/ of cleaning solution, board-washing 3 times；Vacuum packet is used after patting dry
Installation packaging, 4 DEG C save backup.
B. a large amount of preparations of protein standard substance: diluting HB-EGF albumen with sample diluting liquid, be lyophilized after packing, -20 DEG C of guarantors
It deposits spare.
C. a large amount of preparations of enzyme labelled antibody: anti-HB-EGF albumen rabbit polyclonal after purification is marked with HRP, is added 50%
Glycerol saves backup for -20 DEG C after packing.
The detecting step of embodiment 4:HB-EGF enzyme linked immunological kit
Involved cell in the present embodiment, blood, source of mouse or source of people used in tissue equipotential scientific research, do not there is special theory
It is bright, it is conventional purchase gained.
(1) collection, processing and preservation of sample
A. cell culture supernatant: 1000 × g is centrifuged 10min removal particle and polymer, collects supernatant, -20 DEG C of preservations
It is spare.
B. serum: venous blood collection, after collecting blood without heat source and endotoxic test tube, 4 DEG C of 1000 × g centrifugations
15min avoids haemolysis, collects serum, -20 DEG C save backup.
C. blood plasma: venous blood collection collects blood using EDTA test tube, and 1000 × g is centrifuged 15min at 4 DEG C, removal cell and
Particle, -20 DEG C save backup.
D. tissue sample: after cutting sample, weighing weight, and liquid nitrogen quick freeze saves backup, and sample still needs to protect after thawing
The PBS of appropriate volume is added in the temperature for holding 2~8 DEG C, is homogenized sample sufficiently with scissors or homogenizer, 1000 × g centrifugation
15min collects supernatant for detecting.
(2) it is loaded
A. the standard items of enzyme mark coating plate and freeze-drying are taken out, standard items are 1ng HB-EGF albumen, and the dilution of 1mL sample is added
Liquid dilution standard product, standard concentration 1pg/ μ L are placed 20 minutes at room temperature, by standard items from 1pg/ μ L, by 2 times times
Than carrying out gradient dilution, 7 points are diluted, dilution are respectively taken 100 μ L be added in 96 hole elisa Plates according to the position of Fig. 1, blank
Hole is the sample diluting liquid that negative control is equal volume, and 1,2,3 three hole of A row is set as blank control wells, HB- in Fig. 1
EGF albumen is the preferable range of linearity in the section μ L 0.001~1pg/.
B. test serum or plasma sample are taken, is added in reacting hole, 100 holes μ L/, 37 DEG C of incubation 1h；
C. cleaning solution board-washing 3 times, 200 holes μ L/, pat dry ELISA Plate.
(3) detection antibody is added
A. HRP labelled antibody is diluted to 0.5 μ g/mL with sample diluting liquid, be added in reacting hole, 100 holes μ L/, room temperature
Lower incubation 1h；
B. cleaning solution board-washing 3 times, 200 holes μ L/, pat dry ELISA Plate.
(4) it develops the color
A. 100 μ L substrate developing solutions (1:1 is mixed A, B liquid by volume) is added, reacts at room temperature 15min, 50 μ is then added
L terminate liquid terminates reaction；
B. light absorption value is read under microplate reader 450mm wavelength.
(5) using the concentration of HB-EGF protein standard substance as abscissa, light absorption value is ordinate foundation for the foundation of standard curve
Standard curve (such as Fig. 2), equation y=0.8049x+0.0189, regression coefficient R2>=0.99, sample is calculated according to standard curve
In HB-EGF protein content.
The measurement of embodiment 5:HB-EGF enzyme linked immunological kit major parameter
The major parameter of kit is measured, specificity, accuracy and precision including kit, specific method
It is as follows with result:
(1) specific detection of kit
Recombinant protein EGFR, TNF-α, IL-1 β, HGF are diluted to 100pg/ μ L, detected with HB-EGF enzyme linked immunological
Kit is detected, experiment results proved, and the EGFR of the kit and 100pg/ μ L, TNF-α, IL-1 β, HGF are without intersecting
Reaction shows kit specificity preferably, can identify HB-EGF albumen in specific manner.
(2) accuracy determination of kit
By addition recovery experiment come the accuracy of detection kit, to human serum sample (by Affiliated Hospital of Jiangsu University
Clinical laboratory provide) in addition recombination HB-EGF albumen, added HB-EGF protein concentration be 1pg/ μ L, 0.1pg/ μ L,
0.05pg/ μ L and 0pg/ μ L, then detects sample after addition with prepared kit, is calculated according to standard curve
HB-EGF protein content in each sample, the HB-EGF protein content subtracted in blank serum obtain measured HB-EGF
Protein content, the protein content divided by theory addition is TIANZHU XINGNAO Capsul.
It the results are shown in Table 2, TIANZHU XINGNAO Capsul is 95%-110% as the result is shown, illustrates that the accuracy of prepared kit is good
Good, serotonin confrontation detection nothing significantly interferes with.
Table 2.HB-EGF protein reagent box accuracy determination
| The recombination HB-EGF concentration (pg/ μ L) of addition | Measured value | The rate of recovery |
| 0 | 0.032 | - |
| 0.05 | 0.059 | 117.0% |
| 0.1 | 0.111 | 111.6% |
| 1 | 0.975 | 97.50% |
(3) the precision measurement of kit
A. repetitive test
5 parts of the selection blood serum samples containing different HB-EGF protein concentrations (derive from clinical laboratory, Affiliated Hospital of Jiangsu University
There is provided), it is detected in parallel in 3 pieces of ELISA Plates, standard control curve is respectively set in 3 repetitions of each sample on every block of plate, each plate,
The coefficient of variation C.V, C.V=(standard deviation S D/ average value Mean) × 100% for calculating separately each testing result, are shown in Table 3,
Average coefficient of variation was 7.631% (works as C.V > 15% and show that otherness is larger between different groups), illustrated prepared kit
There is good repeatability.
Batch interior repetitive test of table 3.HB-EGF protein reagent box
B. differences between batches detect
5 parts of blood serum samples containing different HB-EGF protein concentrations of selection repeat detection 5 times, each each sample in batches
If 3 repetitions, standard control curve is set in each plate.It calculates with the coefficient of variation between 5 testing results of a sample
(C.V), the coefficient of variation average out to 12.64% for being shown in Table 4,5 parts of samples, 5 testing results (works as C.V > 15% and shows different groups
Between otherness it is larger), illustrate that prepared kit differences between batches are smaller.
The detection of table 4.HB-EGF protein reagent box differences between batches
Embodiment 6: kit detects the application of HB-EGF protein content in human serum
Human serum HB-EGF protein content is measured using prepared HB-EGF enzyme linked immunological detection kit, is examined altogether
20 human serum samples (volunteer from health) is surveyed, the results are shown in Table 5, the HB-EGF protein content model in the human serum of detection
It encloses for 0.1~5pg/ μ L, meets the range of normal value of HB-EGF protein content in human serum.The above result shows that utilizing this hair
Bright kit energy quantitative detection goes out the content of HB-EGF albumen in human serum sample, therefore, ELISA kit of the present invention
Sensitivity is fully able to meet basic research and clinical diagnosis needs, carries out accurate quantitative analysis measurement to people's HB-EGF protein content.
HB-EGF determining the protein quantity in 5. human serum of table
| Serum sample | HB-EGF protein content (pg/ μ L) |
| Serum 1 | 0.674 |
| Serum 2 | 0.389 |
| Serum 3 | 0.687 |
| Serum 4 | 0.578 |
| Serum 5 | 2.174 |
| Serum 6 | 0.879 |
| Serum 7 | 1.369 |
| Serum 8 | 0.753 |
| Serum 9 | 0.645 |
| Serum 10 | 0.414 |
| Serum 11 | 0.941 |
| Serum 12 | 0.587 |
| Serum 13 | 0.844 |
| Serum 14 | 0.524 |
| Serum 15 | 0.247 |
| Serum 16 | 0.574 |
| Serum 17 | 2.178 |
| Serum 18 | 0.894 |
| Serum 19 | 1.017 |
| Serum 20 | 0.249 |
Embodiment 7: kit detects the application of HB-EGF protein content in cell conditioned medium
Using prepared HB-EGF enzyme linked immunological detection kit measurement human breast cancer cell line MDA-MB-231,
Abortion syndrome SKOV3, bronchial epithelial cell system BEAS-2B cell (being purchased from Chinese Academy of Sciences Kunming cell bank) culture supernatant
HB-EGF protein content in liquid the results are shown in Table 6, and testing result meets the normal value model of HB-EGF protein content in cell supernatant
It enclosing, this shows-, the content of HB-EGF albumen in cell conditioned medium is gone out using kit energy quantitative detection of the present invention.
HB-EGF determining the protein quantity in 6. cell conditioned medium of table
Embodiment 8: kit detects the application of HB-EGF protein content in human plasma
The blood of 20 healthy volunteers is collected using the anticoagulant test tube of EDTA, 4 DEG C of 1000 × g are centrifuged 15min, and utilization is made
HB-EGF protein content in standby HB-EGF enzyme linked immunological detection kit detection human plasma, the results are shown in Table 7, meets people's blood
The range of normal value of HB-EGF protein content in slurry, the above result shows that, go out people's blood using kit energy quantitative detection of the present invention
Starch the content of HB-EGF albumen in sample.
HB-EGF determining the protein quantity in 7. human plasma of table
Embodiment 9: kit detects the application of HB-EGF protein content in tissue samples
The ovary tissue of 10 female adult mouse (purchased from Nanjing University's zootype research institute) is collected as detection mark
This weighs weight after cutting sample, and PBS is added by the concentration ratio of 1 μ g/ μ L, is homogenized sample sufficiently using homogenizer,
1000 × g is centrifuged 15min, collects supernatant for detecting, the results are shown in Table 8, meet HB-EGF protein content in animal tissue
Range of normal value, the above result shows that, go out HB-EGF in Mouse Ovary Tissues sample using kit energy quantitative detection of the present invention
The content of albumen.
HB-EGF determining the protein quantity in the tissue of table 8.
| Tissue samples | HB-EGF protein content (pg/ μ L) |
| 1 | 0.0102 |
| 2 | 0.0361 |
| 3 | 0.0305 |
| 4 | 0.0285 |
| 5 | 0.0235 |
| 6 | 0.0656 |
| 7 | 0.0451 |
| 8 | 0.0230 |
| 9 | 0.0556 |
| 10 | 0.0237 |
Sequence table
<110>Co., Ltd, Jiangsu Lai Sen biotechnology research institute
<120>a kind of ELISA kit for detecting heparin-binding epidermal growth factor
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Claims (10)
1. a kind of for detecting the ELISA kit of heparin-binding epidermal growth factor, the kit includes specificity
HB-EGF protein monoclonal antibody, the monoclonal antibody light chain variable region is as shown in SEQ ID.NO. 2, heavy chain variable region
As shown in SEQ .ID.NO. 1.
2. kit according to claim 1, which is characterized in that the kit further includes polyclonal antibody, described more
Clonal antibody is the polyclonal antibody of horseradish peroxidase-labeled.
3. kit according to claim 1, which is characterized in that the HB-EGF monoclonal antibody is anti-for murine monoclonal
Body.
4. kit according to claim 2, which is characterized in that the polyclonal antibody of the horseradish peroxidase-labeled
For rabbit polyclonal antibody.
5. kit according to claim 1, which is characterized in that the kit further include sample diluting liquid, cleaning solution,
The standard items of substrate developing solution, reaction terminating liquid and HB-EGF albumen.
6. kit according to claim 5, which is characterized in that
The sample diluting liquid is the phosphate buffer containing 0.5% (percent by volume) bovine serum albumin(BSA)；
The cleaning solution is the 0.1 mol/L phosphate buffer containing 0.1% (percent by volume) Tween-20.
7. kit according to claim 5, which is characterized in that
The substrate shows that liquid is made of developing solution A and developing solution B, and developing solution A includes hydrogen peroxide, and developing solution B includes tetramethyl
Base benzidine, developing solution using when take isometric A, B liquid to mix；
The terminate liquid is the sulfuric acid of 2 mol/L.
8. kit according to claim 1, which is characterized in that the test object of the kit is serum, on cell
Clearly, blood plasma or tissue.
9. a kind of method for obtaining heparin-binding epidermal growth factor content, which is characterized in that any using claim 1-7
Kit described in is detected.
10. a kind of system for obtaining heparin-binding epidermal growth factor content, which is characterized in that the system comprises rights to want
Seek kit described in any one of 1-7 claim.
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