CN105753984B - Enzyme-linked immunologic detecting kit and the application of a kind of early screening solid tumor or cancer - Google Patents
Enzyme-linked immunologic detecting kit and the application of a kind of early screening solid tumor or cancer Download PDFInfo
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- CN105753984B CN105753984B CN201610192049.0A CN201610192049A CN105753984B CN 105753984 B CN105753984 B CN 105753984B CN 201610192049 A CN201610192049 A CN 201610192049A CN 105753984 B CN105753984 B CN 105753984B
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Abstract
The invention discloses enzyme-linked immunologic detecting kit and the applications of a kind of early screening solid tumor or cancer.Kit of the invention is with SCNH2Polypeptide is as antigen, with rabbit-anti people SCNH2Monoclonal antibody specific association reaction, then the secondary antibody of rabbit with enzyme label is combined, and is developed the color using corresponding substrate, and addition colour developing sulfuric acid or phosphoric acid terminate liquid terminate reaction after colour developing, measures product in the case where absorbing light accordingly.Be experimentally confirmed: the present invention is for detecting SCNH2Anti-human SCNH2Monoclonal antibody specificity is high, high sensitivity；And error of the testing result because avoiding manual operation using enzyme linked immunological professional equipment；Can effectively, easily to the SCNH in liquid biopsy sample2Carry out accurate quantitative detection.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the enzyme linked immunosorbent detection of a kind of early screening solid tumor or cancer
Kit and application.
Background technique
Cancer has become the maximum healthy hidden danger of everyone at one's side." the global cancer that the World Health Organization announces
Disease report 2014 " display, China cancer patient newly-increased 3,070,000 in 2012, death about 2,200,000；Lung cancer, liver cancer, cancer of the esophagus, gastric cancer
Also it is much higher than world average with the new cases and the death rate of colorectal cancer；Even there is expert in Asia-Pacific anticancer conference in 2013
Declare, China increases 20% or more that cancer patient's quantity has accounted for the whole world newly every year, and the high-incidence impetus of cancer show no sign of it is inverse
Transfer toward gesture.Cancer also rejuvenation with the rapid development of society, the age of onset of cancer have advanceed to 40 years old or so, gradually at
For the first fatal disease of the mankind.Active prevention, early detection and early treatment are to tackle the most effective method of tumour.With people
Economic level raising, although many people are high to oneself health requirements, since operating pressure is bigger, majority
Do not recognize to be in sub-health state or be in do not have the Symptomatic early-stage cancer stage.Therefore blood (blood plasma and/
Or serum) and/or urine early screening to the discovery of the cancer or tumour of clinical early stage, determine by stages, therapeutic scheme, Yi Jiti
High therapeutic efficiency etc. has a very big significance.
The detection and diagnosis of cancer is all an intractable problem all the time.Currently, cancer detection and diagnosis are not yet
Some standardized universal means, the methods of the colonoscopy generallyd use, biopsy and PET-CT are invasive
By force, body wound is unavoidably caused, not only brings great pain to patient, it is also extremely inconvenient to operate, and expense
Valuableness, period are long, and general population is relatively difficult to undertake or bear, and is also only confined to certain cancer kinds.Such as colon cancer
Colonoscopy, the mammography for breast cancer, the PSA for prostate cancer check and cervix cancer cervix
Smear inspection etc..But other cancers, just without some good inspection methods, doctor can only go out in these cancer symptoms
Diagnosis can be just made after now, to lose best occasion for the treatment.In addition, is only there are certain diseases for influencing life in patient
Zhuan Shicaiqu hospital is checked, if if cancer, for most people, the cancer in this period has been in middle evening
Phase has affected opportunity adversely, and the treatment window for leaving oneself for is just very small.
The study found that tumour-specific target gene, tumour cell and tumor growth factor present in blood can be used as
A kind of marker is diagnosed, is treated and Prognosis scoveillance.Therefore, body fluid inspection is an important side of early-stage cancer diagnosis
Method can discriminate whether risk of cancer by the variation of each tumor markers index in detection blood or urine.Currently, such as
What easily early diagnoses early-stage cancer by testing liquid biopsy sample and tumour becomes hot fields for cancer diagnosis.
Carcinomebryonic antigen (CEA), cytokeratin 19 fragment (C-19), Serum Neuron Specific Enolase (NSE), sugar antigen 19 (CA19-9),
Carbohydrate antigen 125 (CA125), squamous cell carcinoma antigen (SCCAg), alpha-fetoprotein (AFP), prostate specific antigen (PSA) and
Human chorionic gonadotrophin (HCG) etc. is clinically more common tumor markers at present, but these markers are in cancer
Early diagnosis in value there is no at present final conclusion (Yang Dechang etc., 2001；Che Guowei etc., 2003).
SCNH2It is a member of Apelin family, but the other known member of its signal path and Apelin family
Signal path it is entirely different.SCNH2It is the peptide growth factor of 16 amino acid, amino acid sequence L-V-Q-P-
R-G-S-R-N-G-P-G-P-W-Q-G-NH2(Fang,et.al.,OJCD,2013(3):37-51).It is dynamic in most of lactation
Object or Marsupialia in the mammalian body, SCNH2It is a very conservative sequence, is disappeared in low vertebrate.
SCNH2It is high expression in the neoblast core of 17 days mice embryonics, once cell differentiation, such as skeletonization are thin
SCNH in born of the same parents2Level it is very high, but break up after osteocyte in SCNH2Just disappear (Fang, et.al., OJCD, 2013
(3):37-51).In addition, cancerous tissue chip immunohistochemical staining the results show that SCNH2Table in various solid tumors
Up to level be significantly higher than its it is normal organize, for example, compared with normal person, 90.2% breast cancer, 96.6% colorectal cancer,
SCNH in 87.5% lung cancer and 75.6% oophoroma2Level dramatically increase.In addition, the SCNH in cancer patient's blood plasma2's
Content is also considerably higher than the content (Fang, et.al., OJCD, 2013 (3): 37-51) of normal person.These evidences all show
SCNH2There is very strong association with cancer and embryonic development, is the extraordinary and associated specific biomarker of cancer.
SCNH2It includes the division growth of the tumour cell of separate sources that various cells, which can not only be promoted, is especially being promoted
The ability of angiogenesis is than it with family peptide growth factor Apelin-13 high.In the model of chicken embryo chorion revascularization,
The SCNH of 100 grams of molar concentrations (pM)2Promote blood vessel with the vascular endothelial growth factor (VEGF) of 100 nanomolar concentrations (nM)
The ability of generation is suitable, at 1 gram of molar concentration (pM), promotes the ability of angiogenesis to be significantly higher than the work of VEGF in body
With.Even in 0.1pM, still has the function of very strong Angiogensis.SCNH2It simultaneously can be to promote cancer thin significantly
The migration and infiltration of born of the same parents.SCNH2Rush cell Proliferation, angiogenesis, cancer cell migration and the effect of infiltration almost can be with
By SCN resistance H2Antibody inhibit (Fang, et.al., OJCD, 2013 (3): 37-51).It is above to demonstrate,prove the SCNH it was demonstrated that denier2
As 0.1pM can play the biology of other growth factors such as VEGF, Apelin-13 very high dose such as 1,000 multiple dose 100nM
Effect, and its expression in various solid tumors sharply increases (Fang, et.al., OJCD, 2013 (3): 37-51),
Therefore, the SCNH in blood is measured2Horizontal the early warning screening of cancer, early screening, checkout and diagnosis, chemotherapy effect are examined
Survey, the completeness detection of operation excision cancer and the recurrence of tumour monitoring etc. will have good clinical meaning.
Summary of the invention
It is an object of the present invention to provide SCN resistance H2Monoclonal antibody.
SCN resistance H provided by the invention2Monoclonal antibody by deposit number be CGMCC No.11599 hybridoma cell strain
It generates.
It is a further object to provide one plant of secretion SCN resistance H2Monoclonal antibody hybridoma cell strain.
The entitled SCNH-MoAb of hybridoma cell strain provided by the invention, classification naming are mouse hybridoma strain,
The cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 27th, 2015 (referred to as
CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), it protects
Hiding number is CGMCC No.11599.
It is a still further object of the present invention to provide said monoclonal antibody or the new applications of above-mentioned hybridoma cell strain.
The present invention provides said monoclonal antibody or above-mentioned hybridoma cell strains following 1) -6) at least one of in
Using:
1) detect or assist SCNH in detection sample to be tested2Content；
2) preparation detection or auxiliary detect SCNH in sample to be tested2The reagent or kit of content；
3) screening early stage solid tumor and/or cancer；
4) product of screening early stage solid tumor and/or cancer is prepared；
5) whether there is or not recurrences for monitoring treatment of cancer effect and/or monitoring cancer；
6) whether there is or not the products of recurrence for preparation monitoring treatment of cancer effect and/or monitoring cancer.
In above-mentioned application, the cancer is colorectal cancer, gastric cancer and/or cancer of pancreas.
Final object of the present invention be to provide it is a kind of detection or auxiliary sample to be tested in SCNH2The enzyme linked immunological of content
Kit.
SCNH in detection provided by the invention or auxiliary sample to be tested2The enzyme linked immunological kit of content includes independent packaging
Said monoclonal antibody, SCNH2The secondary antibody of polypeptide and enzyme label.
Mentioned reagent box further includes the working concentration and the SCNH for recording the monoclonal antibody2The working concentration of polypeptide
Carrier,
The working concentration of the monoclonal antibody is 0.1-100 μ g/ml；
The SCNH2The working concentration of polypeptide is 0.5-1 μ g/ml.
In mentioned reagent box,
The working concentration of the monoclonal antibody is 0.3125 μ g/ml；
The working concentration of the SCNH2 polypeptide is 1 μ g/ml.
In mentioned reagent box,
The enzyme, which is that horseradish peroxidase, alkaline phosphatase or glucose oxidase etc. are any, is suitable for label enzyme linked immunological
The enzyme of adsorption test；The enzyme is specially horseradish peroxidase.
Mentioned reagent box further includes substrate, and the substrate is o-phenylenediamine, dianisidine, 5-aminosalicylic acid, adjacent connection
Toluidines or 3,3 ', 5,5 '-methyl biphenyl amine；The substrate is specially 3,3 ', 5,5 '-methyl biphenyl amine.
Mentioned reagent box is following 1) -5) at least one of in application also belong to protection scope of the present invention:
1) detect or assist SCNH in detection sample to be tested2Content；
2) screening early stage solid tumor and/or cancer；
3) product of screening early stage solid tumor and/or cancer is prepared；
4) whether there is or not recurrences for monitoring treatment of cancer effect and/or monitoring cancer；
5) whether there is or not the products of recurrence for preparation monitoring treatment of cancer effect and/or monitoring cancer.
In above-mentioned application or mentioned reagent box, the sample to be tested is urine and/or serum and/or blood plasma.
The present invention utilizes SCN resistance H2Specific antibody detected using enzyme chain immunoabsorption in liquid biopsy sample
SCNH2Level, realize that early screening solid tumor or cancer, monitoring treatment of cancer effect, whether there is or not recurrences for monitoring cancer.Specific side
Method is as follows: with SCNH2Polypeptide is as antigen, with rabbit-anti people SCNH2Monoclonal antibody specific association reaction, then with enzyme mark
The secondary antibody of the rabbit of note is combined, and is developed the color using corresponding substrate, addition colour developing sulfuric acid or phosphoric acid terminate liquid after colour developing
Reaction is terminated, measures product in the case where absorbing light accordingly.Be experimentally confirmed: the present invention is for detecting SCNH2Anti-human SCNH2
Monoclonal antibody specificity is high, high sensitivity；And testing result is because avoiding artificial behaviour using enzyme linked immunological professional equipment
The error of work；Can effectively, easily to the SCNH in liquid biopsy sample2Carry out accurate quantitative detection.
Detailed description of the invention
Fig. 1 is rabbit-anti SCNH2Antibody titer testing result.
Fig. 2 is SCNH2Peridium concentration testing result.
Fig. 3 is SCNH in urine2Concentration Testing result.
Fig. 4 is SCNH in serum2Horizontal testing result.
Fig. 5 is SCNH in blood plasma2Horizontal testing result.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Sample diluent in following embodiments: contain 1% bovine serum albumin(BSA) (abbreviation BSA, Fisher, article No.
C79500 the phosphate buffer (PBS) of pH 7.4).
Cleaning solution in following embodiments: the phosphate buffer (PBS) of pH 7.4.
Zymolyte in following embodiments: 3,3 ', 5,5 '-methyl biphenyl amine (TBM, the super quick tetramethyl benzidine of a step
Tetramethylbenzidine) be Sigma company product, article No. T0440-1L.
Enzyme reaction terminate liquid in following embodiments: the sulfuric acid of 0.02M.
Cells frozen storing liquid in following embodiments: 50% calf serum；40% endless full nutrient solution (Sigma, article No.
R5886)；10%DMSO (dimethyl sulfoxide, Fisher, article No. MT25950CQC).
Complete culture solution in following embodiments is by FBS (Fisher, article No. MT35015CV) and RPMI1640 culture medium
The culture solution that (Fisher, article No. 32404-014) is uniformly mixed so as to obtain, volume fraction of the FBS in complete culture solution are 10%.
Embodiment 1, the acquisition of hybridoma cell strain and SCN resistance H2Monoclonal antibody preparation
One, the preparation of immunogene
SCNH2Polypeptide (amino acid sequence L-V-Q-P-R-G-S-R-N-G-P-G-P-W-Q-G-NH2) synthesize by the U.S.
GenScript Technology Co., Ltd. completes.Specific step is as follows: using Peptide synthesizer (TETRASTMPeptide
Synthesizer, Advanced ChemTech, Germany) synthesis in solid state SCNH2Polypeptide, after obtaining peptide carrier, by anti-
Phase high pressure liquid chromatograph (HPLC) after purification, analyze using HPLC and mass spectrograph purified, purity reaches
90% or more.Purified is freeze-dried further across dry ice into pulvis.
Two, animal immune
By the biologically active SCNH of synthesis2Polypeptide carries out immunity inoculation to rabbit as immunogene.Specific steps
It is as follows: SCNH prepared by step 12(solvent is the phosphate buffer of pH 7.4, SCNH to polypeptide2The concentration of polypeptide is 1 milli
Grams per milliliter) it is mixed with immunologic adjuvant freund adjuvant, progress dorsal sc multi-point injection, 0.1 milliliter/point.First immunisation injection is anti-
Commercial weight is 400 micrograms/rabbit, and the dosage of booster immunization is about 1/4 of initial dose or so.Every 2~3 weeks booster immunizations are primary.
The configuration of freund adjuvant: lanolin and paraffin oil being set in container in the ratio of 1:3, are allowed to mix with ultrasonic wave,
High pressure sterilization is set and is saved backup at 4 DEG C.Before first immunisation, BCG vaccine and the freund adjuvant of sterilizing are mixed into (BCG vaccine final concentration
For 4 mg/mls), emulsus is blended together with oscillator, fills antigen liquid (SCNH with a syringe2Polypeptide solution), another syringe dress
Equivalent adjuvant, the two are connected with vinyl tube, and then the two is aspirated back and forth, and approximate number ten minutes later can be completely newborn
Change.Injection is made with a wherein syringe after passed examination.When booster immunization, BCG vaccine is not added, directly according to above-mentioned step
Inoculation is carried out after the antigen and adjuvant of rapid equivalent volumes.2 weeks after the 2nd booster immunization, 2~3ml is taken from auricular vein
Blood prepares serum, detects the antibody titer of serum.Such as not up to expected potency, need to carry out booster immunization again, be when being satisfied with
Only.When antibody titer reaches expected horizontal, can bloodletting prepare antiserum.And the serum prepared is passed through into Protein G
(Fisher, article No. PI21359) is purified (by specification operation), and the IgG obtained after purification is polyclonal antibody.
Three, cell fusion and cloning
(1) cell fusion
(1) feeder layer is prepared
Select 6~10 weeks BALB/C mice peritoneal macrophages.It is operated in accordance with the following steps:
A, it draws neck to put to death, is immersed in 75% alcohol, 3~5 minutes；
B, skin, exposure peritonaeum are cut off with sterile scissors；
C, (forbidden with the culture solution RPMI1640 (Sigma, article No. R5886) that asepsis injector injects 5~6 milliliters of pre-coolings
Puncture intestinal tube)；
D, flushing liquor is sucked out in repeated flushing；
E, flushing liquor is put into 10 milliliters of centrifuge tubes, and 1200 revs/min from 5~6 minutes；
F, it is suspended with the culture solution of 20% calf serum (Fisher, article No. MT35015CV), adjustment cell number to 1 ×
105/ milliliter；
G, 96 orifice plates (Fisher, article No. 12565501) is added, 100 microlitres/hole；
H, 37 DEG C are put into, CO2Incubator culture.
(2) immune spleen cell is prepared
A, rabbit draws neck to put to death after last time booster immunization 3 days；
B, sterile to take spleen, culture solution is washed once；
C, spleen is ground, and crosses stainless steel mesh；
D, it is centrifuged, cell is washed 2 times with culture solution；
E, it counts；
F, 1 × 10 is taken8Splenic lymphocytes suspension is spare.
(3) myeloma cell is prepared
A, logarithmic growth myeloma cell is taken to be centrifuged；
B, it is washed 2 times with serum-free medium；
C, it counts, obtains 1 × 107Cell is spare.
(4) it merges
1. the splenocyte of the myeloma cell of step (3) preparation and step (2) preparation is mixed in the ratio of 1:10,50
It is washed 1 time, is centrifuged, 1200 revs/min, 8 minutes with the endless full nutrient solution of serum-free in milliliter centrifuge tube；Supernatant is abandoned, is inhaled with suction pipe
Net residual liquid, in order to avoid influence polyethylene glycol (abbreviation PEG, Sigma, article No. 1546569) concentration.Gently attack centrifuge tube bottom,
Keep cell precipitation slightly loose dynamic.
2. 1 milliliter of 45%PEG solution of 37 DEG C of pre-temperatures, side edged gentle agitation are added in 90 seconds.37 DEG C of water-bath effects 90
Second.
3. the endless full nutrient solution of 37 DEG C of pre-temperatures is added to terminate PEG effect, it is separately added into 1 milliliter, 2 millis at intervals of two minutes
It rises, 3 milliliters, 4 milliliters, 5 milliliters and 6 milliliters.
4. being centrifuged, 800 revs/min, 6 minutes.
5. filling with clearly, it is resuspended with the selection culture solution containing 20% serum HAT.
6. above-mentioned cell is added in 96 orifice plates of existing feeder layer, every hole adds 100 microlitres.One immune spleen can
It is inoculated with 4 piece of 96 orifice plate.
7. culture plate is set 37 DEG C, is cultivated in 5% carbon dioxide incubator.
Splenocyte and myeloma cell are after PEG is handled, the training in selection culture solution (HAT, Sigma, article No. H0262)
It supports, since myeloma cell lacks thymidine kinase or enzyme hypoxanthine guanine phosphoribosynltransferase, therefore is unable to growth and breeding, and hybridize
Oncocyte has above two enzyme, selects culture solution can be with growth and breeding in HAT.In with HAT selection culture 1~2 day, will have
A large amount of oncocytes are dead, 3~4 days posterior tuberosity vanished cells, and hybrid cell forms microcolony, and HAT selects culture solution to maintain 7~10 days
After should use HT culture solution (HT, Sigma, article No. H0137) instead, then maintain culture 2 weeks.During above-mentioned selection culture, every 2~3
It changes half culture solution.When hybridoma is covered with 1/10 area of bottom hole, it can start to detect specific antibody, needed for filtering out
The hybridoma cell line wanted.
(2) detection of antibody
The detection method of specific antibody uses enzyme-linked immunosorbent assay.
(3) clone of hybridoma
It is cloned using limiting dilution assay and carries out antibody positive wells clone.
(1) first 1 day preparation feeder layer (same to cell fusion) is cloned；
(2) hybridoma that will be cloned gently is dried up out of culture hole, is counted；
(3) adjustment cell is 3~10 cells/mls；
(4) tissue culture plate for the feeder layer for taking the previous day to prepare, every hole are added 100 microlitres of diluting cells.Be incubated in
37 DEG C, in 5% carbon dioxide incubator；
(5) liquid was changed at the 7th day, changed within every 2~3 days liquid 1 time later；
(6) 8~9 days visible cell Clone formations simultaneously detect antibody activity in time；
(7) cell in positive hole is moved to and expands culture in 24 orifice plates (Fisher, article No. 08-772-1H)；
(8) each clone promptly freezes.
The monoclonal hybridoma strain of above-mentioned antibody titer highest (1:250000) is named as SCNH-MoAb,
Classification naming is mouse hybridoma strain, and the cell strain is preserved in Chinese microorganism strain preservation on November 27th, 2015
Administration committee's common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of microbiology, institute, postcode 100101), deposit number is CGMCC No.11599.
Four, hybridoma freezes and recovers
1, hybridoma freezes
The cryopreservation methods of hybridoma are the same with the cryopreservation methods of other cell lines, every cryopreservation tube (Fisher, article No.
0339011) contain 1 × 106More than, but the hybridoma of archioporus can be changed because of culture environment difference, it is trained in 24 holes
It supports and is cultivated in plate, when covering with bottom hole, a hole fills a cryopreservation tube and freezes.
Frozen stock solution is preferably pre-chilled, and operational motion is soft, rapid.With cell cryopreservation device (Fisher, article No. when freezing
13900857) it is frozen.Freeze-stored cell will periodically recover, and the activity of cell and the stability of secretory antibody be checked, in liquid nitrogen
Middle cell can be reserved for several years or longer time.
2, cell recovery method
Glass ampule is carefully taken out from liquid nitrogen, is put in 37 DEG C of water-baths, the cell frozen is made to thaw in 1 minute, it will
Cell is washed twice with complete culture solution, is then moved into the culture bottle for the feeder cells that the previous day has prepared, set 37 DEG C,
It is cultivated in 5% carbon dioxide incubator, when cell forms colony, detects antibody activity.
With frozen stock solution (contain 10%DMSO, the culture solution of 20% serum) by hybridoma cell strain CGMCC No.11599 system
At 1 × 106The cell suspension of a/ml, saves for a long time in liquid nitrogen.When recovery, cryopreservation tube is taken out, is immediately placed in 37 DEG C of water-baths
Speed is melted, and moves into culture in culture bottle after centrifugation removal frozen stock solution.
Five, the preparation and purification of monoclonal antibody
(1) small lot prepares monoclonal antibody
By above-mentioned selected monoclonal cell strain, two days later using complete culture solution culture, the culture of half is collected by centrifugation
Liquid cooling is ensconced in 4 DEG C of refrigerators；In addition after half supplements fresh complete culture solution, suspension monoclonal cell strain continues to cultivate, such as
After this is repeated several times, the culture solution of all collections is purified (by specification operation) with Protein G, obtains SCN resistance H2List
Clonal antibody.
(2) large scale preparation monoclonal antibody
By preparing ascites in mouse Inoculation hybridoma to producing monoclonal antibody in large quantity.Ascites
Preparation: 0.5 milliliter of Pristane (norphytane) or atoleine is first injected intraperitoneally and is injected intraperitoneally after BALB/C mouse, 1~2 week
1×106A hybridoma, inoculating cell can produce ascites, the health status and sign of ascites of close observation animal after 7~10 days
As, it is as more as possible to ascites, and before mouse is frequency domain dead, mouse is put to death, is sucked ascites in test tube with dropper, general one
Mouse can obtain 5~10 milliliters of ascites.Ascites can also be extracted with syringe, can collected repeatedly for several times.Monoclonal Antibodies in Mice Ascites contains
Amount can reach 5~10 mg/mls, or also cell cryopreservation in ascites can get up, and transferred species mouse peritoneal then generates after recovery
A greater amount of ascites containing monoclonal antibody.The ascites of collection can be purified with Protein G (by specification operation), be obtained
A large amount of SCN resistance H2Monoclonal antibody.
Embodiment 2, SCN resistance H2Monoclonal antibody working concentration and SCNH2It is coated with the optimization of optimum concentration
One, SCN resistance H2The optimization of working concentration
1,96 orifice plate of antibody gradient dilution
The SCN resistance H for being prepared embodiment 1 with sample diluent2Monoclonal antibody solution be diluted to 1 mg/ml
Concentration prepares a SCN resistance H296 hole storage boards of antibody: first is classified as the 150 diluted antibody of μ l 1:100；Second is classified as
The 150 diluted antibody of μ l 1:200；And so on until the 12nd being classified as the 150 diluted antibody of μ l 1:204,800.Then,
This storage board is stored in spare in 2~8 degree of refrigerating box.
2、SCNH2Coated 96 orifice plate of polystyrene
SCNH prepared by embodiment 12Polypeptide (SCNH2) with PBS (PH 7.4) solution it is diluted to 4 different concentration: 2
μ g/ml, 1 μ g/ml, 0.5 μ g/ml and 0.25 μ g/ml；Then each hole of A, B row are added to the 2 μ g/ml's of 50 μ l
SCNH2, each hole of C, D row are added to the SCNH of the 1 μ g/ml of 50 μ l2, each hole of E, F row are added to 0.5 μ of 50 μ l
The SCNH of g/ml2, each hole of G, H row are added to the SCNH of the 0.25 μ g/ml of 50 μ l2.At room temperature, by SCNH2It is coated
96 orifice plates are placed on horizontal shaker and are incubated for 30 minutes.The liquid in 96 orifice plates is sopped up, it three times using cleaning solution washing, then will be every
300 μ l sample diluents are added in hole.This 96 orifice plate is placed on horizontal shaker, shakes 30 minutes, then, discards at room temperature
Sample diluent.
3, first antibody competitive binding antigen
Antibody in 96 orifice plate of antibody gradient dilution prepared by step 1 is added to 96 orifice plate of polystyrene being coated with
In corresponding hole, then every 50 μ l of hole discards antibody after this polystyrene board to be shaken to incubation 30 minutes at room temperature, uses
Cleaning solution washs three times.
4, secondary antibody marks first antibody
By HRP label anti-rabbit IgG secondary antibody (Fisher, article No. AP32PMI) with Sample dilution carry out 1:500 times it is dilute
It releases, 50 μ l of addition to each hole, is shaken after being incubated for 30 minutes at room temperature and discard secondary antibody, washed three times with cleaning solution.
5, substrate develops the color
The zymolyte (the super quick tetramethyl benzidine TBM of a step) of 100 μ l is added, the place of being protected from light is incubated for 30 minutes at room temperature
Afterwards, the enzyme reaction terminate liquid (sulfuric acid of 0.02M) of 50 μ l is added in every hole.
6, microplate reader is analyzed
The OD value at each hole 450nm is measured with microplate reader (TECAN microplate reader).And using antibody diluted concentration as abscissa,
Curve is drawn by ordinate of OD value.Curve is as shown in Figure 1, it can be seen from the figure that SCN resistance H2Monoclonal antibody be applicable in
Concentration is 0.1ng/ml-100ug/ml, optimal concentration 0.3125ng/ml.
Two, SCNH2The selection of coated optimum concentration
1, SCNH is prepared according to above-mentioned steps2Coated 96 orifice plate of polystyrene
2, the suitable first antibody working solution of preparation
The SCN resistance H for being prepared embodiment 1 with sample diluent2Monoclonal antibody carry out 1:1600 times and dilute, obtain the
One antibody-solutions.
3, the SCNH of gradient dilution is prepared2
With Sample dilution by SCNH2Carry out gradient dilution, concentration be respectively 400pg/ml, 2ng/ml, 4ng/ml,
20ng/ml, 40ng/ml, 200ng/ml, 400ng/ml, 2 μ g/ml, 4 μ g/ml, 20 μ g/ml and 40 μ g/ml, are placed on 2~8 degree
It is saved backup in refrigerating box.
4, competitive antigen-antibody reaction is prepared
1st column of 96 orifice plate of polystyrene in step 1 are added to the Sample dilution of 25 μ l, by gradient dilution in 3
SCNH2Successively it is added to the 2nd column, the 3rd column, until the 12nd column.Then the 25 μ l of first antibody solution prepared in step 2 is added to arrive
In each hole, is shaken after being incubated for 30 minutes at room temperature and discard liquid, washed three times with cleaning solution.
The step of repeating 5,6 in step 1.
As a result as shown in Fig. 2, SCNH2Coated applicable concentration is 0.5-1 μ g/ml, and optimal concentration is 1 μ g/ml.
Embodiment 3, SCN resistance H2The application of monoclonal antibody
One, SCN resistance H2SCNH of the monoclonal antibody in detection urine sample2Application in level
1, the collection of urine
(all normal persons and cancer patient sign the urine of collection normal person and cancer patient's (lung cancer and colorectal cancer)
Informed consent form is affixed one's name to, the ethics program of scientific research is all deferred in sample program and detection), before collecting urine, disinfecting paper need to be used
Then the processing that carries out disinfection of external genital and urethral orifice is collected intermediate urine with sterile vessel by towel.2 milliliters of urine is shifted
At room temperature into sterile centrifuge tube, the centrifugation of 1500 revolving speeds after ten minutes, takes supernatant to be divided in 3 different sterile 2
In the centrifuge tube of milliliter, and it is numbered registration.Wherein two pipes freeze in the refrigerator of -80 degree, and 1 pipe is stored in 2~8 degree
Refrigerating box in a short time for being detected.
2, the SCNH in urine sample is detected2It is horizontal
(1) SCNH for being 1 μ g/ml by concentration2It is coated with 96 orifice plate of polystyrene, 50 μ l is added in every hole, is shaking at room temperature
It is incubated for 30 minutes on bed.
(2) coating buffer is discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(3) cleaning solution is discarded.Per 100 μ l coating buffers of aerial addition, it is incubated for 30 minutes on shaking table at room temperature.
(4) coating buffer is discarded.First row is added to the Sample dilution of 25 μ l, the 1st, second Kong Zhi 12 of 2 rows
Add the SCNH of 25 μ l gradient dilutions in hole2, concentration is as follows: 400pg/ml, 2ng/ml, 4ng/ml, 20ng/ml, 40ng/ml,
200ng/ml, 400ng/ml, 2 μ g/ml, 4 μ g/ml, 20 μ g/ml and 40 μ g/ml.2nd drains into the secondary series of the 8th row to the 6th column
The different normal person's urine specimen of 25 μ l is added, totally 15 samples, two holes of each sample.Remaining hole is detection sample, altogether
18 samples, two holes of each sample.It is horizontal at room temperature to shake 5 minutes.Then, the concentration for adding 25 μ l is 312.5ng/ml's
The SCN resistance H prepared in embodiment 12Monoclonal antibody.It is incubated for 30 minutes on shaking table at room temperature.
(5) sample-antibody liquid is discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(6) cleaning solution is discarded.The secondary antibody that the anti-rabbit IgG of 50 μ l HRP label is added in every hole, is carried out with Sample dilution
1/500 times of dilution, shakes be incubated for 30 minutes at room temperature.
(7) two resistant to liquids are discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(8) cleaning solution is discarded.The zymolyte (the super quick tetramethyl benzidine TBM of a step) for adding 100 μ l, is protected from light at room temperature
After place is incubated for 30 minutes, the terminate liquid (sulfuric acid of 0.02M) of 50 μ l is added in every hole.
(9) microplate reader read plate is analyzed: measuring the OD value at each hole 450nm with microplate reader (TECAN microplate reader).With SCNH2
Concentration is abscissa, draws curve by ordinate of OD value.Dependence Results are shown in Fig. 3.It can be seen from the figure that in normal human urine
SCNH2Concentration is 0.05ng/ml, the SCNH in colorectal cancer patients urine2Concentration is 0.5ng/ml, in patients with lung cancer urine
SCNH2Concentration is 0.4ng/ml.SCNH in cancer patient's urine2Concentration is apparently higher than normal person, illustrates of the invention resist
SCNH2Antibody specificity is high, detection sensitivity is high, can effectively, easily to the SCNH in liquid biopsy sample2It is accurately fixed to carry out
Amount detection.
Two, SCN resistance H2SCNH of the monoclonal antibody in detection serum2Application in level
1, the acquisition of serum specimen
(all normal persons and cancer patient sign the serum of collection normal person and cancer patient's (cancer of pancreas and gastric cancer)
The ethics program of scientific research is all deferred in informed consent form, sample program and detection).Specific step is as follows: using ethylenediamine
After the vacuum drying pipe (anticoagulant tube, purple lid) of tetraacethyl (EDTA) processing collects 2 milliliters of new blood, at room temperature by it
Stand 30 minutes.At room temperature by sample, 1500 revolving speeds are centrifuged 10 minutes, and supernatant serum is then taken to be sub-packed in sterile 3
In 2 milliliters of different centrifuge tubes, and it is numbered registration.Two of them centrifuge tube sample freezes in the refrigerator of -80 degree,
1 pipe is stored in 2~8 degree of refrigerating box in a short time for detecting.
2, the SCNH in serum is detected2It is horizontal
(1) SCNH for being 1 μ g/ml by concentration2It is coated with 96 orifice plate of polystyrene, 50 μ l is added in every hole, is shaking at room temperature
It is incubated for 30 minutes on bed.
(2) coating buffer is discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(3) cleaning solution is discarded.100 μ l coating buffers are added in every hole, are incubated for 30 minutes on shaking table at room temperature.
(4) coating buffer is discarded.First row is added to the Sample dilution of 25 μ l, the 1st, second Kong Zhi 12 of 2 rows
Add the SCNH of 25 μ l gradient dilutions in hole2, concentration is as follows: 400pg/ml, 2ng/ml, 4ng/ml, 20ng/ml, 40ng/ml,
200ng/ml, 400ng/ml, 2 μ g/ml, 4 μ g/ml, 20 μ g/ml and 40 μ g/ml.3rd drains into the secondary series of the 8th row to the 6th column
The different normal human serum sample of 25 μ l is added, totally 15 samples, two holes of each sample.Remaining hole is detection serum sample
This, totally 18 samples, two holes of each sample.It is horizontal at room temperature to shake 5 minutes.Then, the concentration of 25 μ l of addition is
The SCN resistance H prepared in the embodiment 1 of 312.5ng/ml2Monoclonal antibody.It is incubated for 30 minutes on shaking table at room temperature.
(5) sample-antibody liquid is discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(6) cleaning solution is discarded.The secondary antibody that the anti-rabbit IgG of 50 μ lHRP label is added in every hole, carries out 1 with Sample dilution:
500 times of dilutions, shake be incubated for 30 minutes at room temperature.
(7) two resistant to liquids are discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(8) cleaning solution is discarded.The zymolyte (the super quick tetramethyl benzidine TBM of a step) for adding 100 μ l, is protected from light at room temperature
After place is incubated for 30 minutes, the terminate liquid (sulfuric acid of 0.02M) of 50 μ l is added in every hole.
(9) the OD value at each hole 450nm is measured with microplate reader (TECAN microplate reader).With SCNH2Concentration is abscissa, with
OD value is that ordinate draws curve.Dependence Results are shown in Fig. 4.It can be seen from the figure that the SCNH in normal human urine2Concentration is
0.9ng/ml, the SCNH in patients with gastric cancer urine2Concentration is 4ng/ml, the SCNH in Pancreas cancer patients urine2Concentration is 30ng/
ml.SCNH in cancer patient's urine2Concentration is apparently higher than normal person, illustrates SCN resistance H of the invention2Antibody specificity is high, examines
Survey high sensitivity, can effectively, easily to the SCNH in liquid biopsy sample2Carry out accurate quantitative detection.
Three, SCN resistance H2SCNH of the monoclonal antibody in detection blood plasma2Application in level
1, the acquisition of plasma specimen
After collecting 2 milliliters of new blood using vacuum drying pipe (non-anticoagulant tube, red cap), it is stood at room temperature
30 minutes.At room temperature by sample, 1500 revolving speeds are centrifuged 10 minutes, and supernatant blood plasma is then taken to be sub-packed in 3 sterile differences
2 milliliters of centrifuge tubes in, and it is numbered registration.Two of them centrifuge tube sample freezes in the refrigerator of -80 degree, 1 pipe
It is stored in 2~8 degree of refrigerating box in a short time for detecting.All normal persons and cancer patient endorsed informed consent
The ethics program of scientific research is all deferred in book, sample program and detection.
2, the SCNH in blood plasma is detected2It is horizontal
(1) use concentration for the SCNH of 1 μ g/ml2It is coated with 96 orifice plate of polystyrene, per 50 μ l of aerial addition, is existed at room temperature
It is incubated for 30 minutes on shaking table.
(2) coating buffer is discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(3) cleaning solution is discarded.100 μ l coating buffers are added in every hole, are incubated for 30 minutes on shaking table at room temperature.
(4) coating buffer is discarded.First row is added to the Sample dilution of 25 μ l, the 1st, second Kong Zhi 12 of 2 rows
Add the SCNH of 25 μ l gradient dilutions in hole2, concentration is as follows: 400pg/ml, 2ng/ml, 4ng/ml, 20ng/ml, 40ng/ml,
200ng/ml, 400ng/ml, 2 μ g/ml, 4 μ g/ml, 20 μ g/ml and 40 μ g/ml.Third drains into the secondary series of the 8th row to the 6th
Column add the different human normal plasma's sample of 25 μ l, totally 15 samples, two holes of each sample.Remaining hole is detection blood plasma sample
This, totally 18 samples, two holes of each sample.It is horizontal at room temperature to shake 5 minutes.Then, the concentration of 25 μ l of addition is
312.5ng/ml rabbit-anti SCNH2Antibody.It is incubated for 30 minutes on shaking table at room temperature.
(5) sample-antibody liquid is discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(6) cleaning solution is discarded.The secondary antibody that the anti-rabbit IgG of 50 μ l HRP label is added in every hole, is carried out with Sample dilution
1:500 times dilutes, and shakes be incubated for 30 minutes at room temperature.
(7) two resistant to liquids are discarded.It is washed three times using the cleaning solution of 300 μ l, 5 minutes every time.
(8) cleaning solution is discarded.The zymolyte (the super quick tetramethyl benzidine of a step, TBM) for adding 100 μ l, keeps away at room temperature
After being incubated for 30 minutes at light, the terminate liquid (sulfuric acid of 0.02M) of 50 μ l is added in every hole.
(9) the OD value at each hole 450nm is measured with microplate reader (TECAN microplate reader).With SCNH2Concentration is abscissa, with
OD value is that ordinate draws curve.Dependence Results are shown in Fig. 5.It can be seen from the figure that the SCNH in normal human urine2Concentration is
0.9ng/ml, the SCNH in Pancreas cancer patients urine2Concentration is 51ng/ml, the SCNH in stomach Urine in Patients2Concentration is 50ng/
ml.SCNH in cancer patient's urine2Concentration is apparently higher than normal person, illustrates SCN resistance H of the invention2Antibody specificity is high, examines
Survey high sensitivity, can effectively, easily to the SCNH in liquid biopsy sample2Carry out accurate quantitative detection.
Claims (11)
1. SCN resistance H2Monoclonal antibody, by deposit number be CGMCC No.11599 hybridoma cell strain generate.
2. one plant of secretion SCN resistance H2Monoclonal antibody hybridoma cell strain, deposit number be CGMCC No.11599.
3. monoclonal antibody described in claim 1 or hybridoma cell strain as claimed in claim 2 are following 1) -3) at least
A kind of application in:
1) preparation detection or auxiliary detect SCNH in sample to be tested2The reagent or kit of content；
2) product of screening early stage solid tumor and/or cancer is prepared；
3) whether there is or not the products of recurrence for preparation monitoring treatment of cancer effect and/or monitoring cancer.
4. application according to claim 3, it is characterised in that: the cancer is colorectal cancer, gastric cancer and/or cancer of pancreas.
5. SCNH in a kind of detection or auxiliary test sample2The enzyme linked immunological kit of content, the claim including independent packaging
Monoclonal antibody described in 1, SCNH2The secondary antibody of polypeptide and enzyme label.
6. kit described in 5 according to claim, it is characterised in that:
The kit further includes the working concentration and the SCNH for recording the monoclonal antibody2The load of the working concentration of polypeptide
Body,
The working concentration of the monoclonal antibody is 0.1-100 μ g/ml；
The SCNH2The working concentration of polypeptide is 0.5-1 μ g/ml.
7. the kit described in 5 or 6 according to claim, it is characterised in that:
The working concentration of the monoclonal antibody is 0.3125 μ g/ml；
The working concentration of the SCNH2 polypeptide is 1 μ g/ml.
8. the kit described in 5 or 6 according to claim, it is characterised in that:
The enzyme is horseradish peroxidase, alkaline phosphatase or glucose oxidase.
9. enzyme linked immunological kit according to claim 5 or 6, it is characterised in that: the sample to be tested be urine and/or
Serum and/or blood plasma.
10. in claim 5-8 any enzyme linked immunological kit it is following at least one of 1) or 2) in application:
1) product of screening early stage solid tumor and/or cancer is prepared；
2) whether there is or not the products of recurrence for preparation monitoring treatment of cancer effect and/or monitoring cancer.
11. application according to claim 3 or 4 or application described in any one of claim 10, it is characterised in that: described to test sample
Product are urine and/or serum and/or blood plasma.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102086228A (en) * | 2009-12-04 | 2011-06-08 | 北京维德维康生物技术有限公司 | ELISA (Enzyme Linked Immunosorbent Assay) Kit and application thereof |
| WO2014099984A1 (en) * | 2012-12-20 | 2014-06-26 | Amgen Inc. | Apj receptor agonists and uses thereof |
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2016
- 2016-03-30 CN CN201610192049.0A patent/CN105753984B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102086228A (en) * | 2009-12-04 | 2011-06-08 | 北京维德维康生物技术有限公司 | ELISA (Enzyme Linked Immunosorbent Assay) Kit and application thereof |
| WO2014099984A1 (en) * | 2012-12-20 | 2014-06-26 | Amgen Inc. | Apj receptor agonists and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| SCNH2 is a novel apelinergic family member acting as a potent mitogenic and chemotactic factor for both endothelial and epithelial cells;changge fang等;《Open J Clin Diagn.》;CIHR IRSC;20130630;第3卷(第2期);第37-51页 * |
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