CN104830805B - Anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application - Google Patents
Anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application Download PDFInfo
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Abstract
The invention belongs to cell engineering fields, specifically disclose anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application.Hybridoma cell strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 20th, 2014, and deposit number is CGMCC No.9812；Monoclonal antibody of the present invention can be used for the Immunohistochemical detection of ELISA, chemiluminescence, western blot, immunofluorescence and tissue, and verifying work as the clinical samples of colorectal cancer tumour hepatic metastases marker with it for the functional study of people's Clonorchiasis Sinensis provides support.When kit containing said monoclonal antibody of the present invention is detected, high sensitivity, high specificity, detection range is wide, easy to operate, and "dead" pollution, kit is at low cost, clinical adaptable, is particularly suited for China's clinic clinical detection screening.
Description
Technical field
The present invention relates to cell engineering fields, anti-more particularly, to anti-human Clonorchiasis Sinensis monoclonal
Body hybridoma and its monoclonal antibody and application.
Background technology
Colorectal cancer (colorectal cancer, CRC) is the most common solid tumor of western countries, accounts for about malignant tumour
The 10% of the death rate.In the U.S., colorectal cancer occupies the second of the malignant tumour cause of the death, and every year about 52,000 colorectal cancer
Death.In recent years, China's colorectal cancer incidence rate is also in rising trend, occupies the 5th pernicious cause of the death.Colorectal cancer patients
In lysis, there are about half can progress to hepatic metastases, wherein 30% patient's liver is only metastasis site, 15~35%
Colorectal cancer patients first visit when find simultaneity hepatic metastases, then there is heterochronia hepatic metastases in 20~25% patient.Liver turns
Shifting is the common cause of the death of colorectal cancer, more than half nearly patient can die of hepatic metastases.If Liver Metastases from Colorectal Cancer is abandoned controlling
Then poor prognosis is treated, median survival time is 6~12 months, although by systemic chemotherapy, diagnosis of hepatic metastases can not be cut off, patient
Median survival time there was only 12~24 months, life span is more than that 5 years persons are rare, and colorectal cancer simultaneity or heterochronia liver turn
It is always the problem for perplexing clinician to move, and how to find colorectal cancer hepatic metastases as early as possible, or after radical resection for colorectal cancer it is right
It is assessed in hepatic metastases danger level, it is the significant challenge faced at present to and guide aftertreatment.Post operative colo-rectal cancer liver is turned
Shifting, which is caused danger, to be predicted, contributes to postoperative progress targetedly auxiliary treatment, to improve overall survival.
Colorectal cancer hepatic metastases is a complicated and continuous process, and cancer cell first is detached from from primary carcinoma stove, and degradation is extracellular
Matrix is detached from primary tumor and enters portal vein, then by signal transduction, reaches liver under the action of associated receptor and the factor, wears
Go out blood vessel to adhere to again, so liver formed new vessels, be proliferated again, finally formed hepatic metastases stove.It is each in the process
Kind adhesion molecule, angiogenic factor, extracellular stromatin enzyme etc. all act.It is clinically tied at present to detect
The method of carcinoma of the rectum hepatic metastases stove mainly passes through the iconographies means such as B ultrasound, CT, MR and DSA, but to the inspection rate of hepatic metastases stove
Only 50~90%, and also may be used though being detected in art it is difficult to make specific diagnosis to diameter 1cm micro metastasis below
It was found that some early stage colorectal cancer hepatic metastases lesions, but when metastatic lesion diameter is less than 4mm, detects discovery rate and be greatly lowered.
Early stage, the tumor markers such as CEA, CA 19-9 lacked specificity again because of atypical symptoms, diagnosed relative difficulty.Either
The hepatic metastases or the recurrent hepatic metastases of root value criterion that i.e. oneself has found when colorectal cancer is made a definite diagnosis for the first time, only l/4 when making a definite diagnosis~
1/3 hepatic metastases stove is confined to a leaf liver, most of to have involved two leaves, and is multiple, it is difficult to operation excision.Therefore,
The early stage of metastatic liver cancer, correctly it is extremely important to improve prognosis to guiding treatment for diagnosis.Find early diagnosis knot that is effective and stablizing
The biological markers of carcinoma of the rectum hepatic metastases will generate far-reaching influence to the clinical diagnosis of colorectal cancer hepatic metastases and treatment.
Clonorchiasis Sinensis (TCTP), also known as P21 (mouse TCTP), P23 (people TCTP), Q23 are that one kind is deposited extensively
It is protein family that is highly conserved in sequence and having very high homology in animal, plant and yeast, is initially considered that TCTP is
A kind of growth associated protein, research prompt TCTP in recent years has very important biological function, including adjusts the cell cycle
Process and pernicious transfer, calbindin, extracellular histamine release albumen, anti-apoptotic and anti-malarial effect etc.；TCTP can
Enhance the transfer ability of cell, the content in hepatic metastasis of colonic carcinoma patients serum significantly increases, and TCTP is in serum for detection
Expression quantity is significant for the transfer for monitoring colorectal cancer.
Invention content
The technical problem to be solved by the present invention is to overcome present in existing colorectal cancer hepatic metastases detection technique cannot
The defect correctly diagnosed in time, provides a kind of anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma cell strain MQ-ANTI-
TCTP-2, the cell strain can secrete anti-human Clonorchiasis Sinensis monoclonal antibody.
Second object of the present invention is to provide the anti-human Clonorchiasis Sinensis list of above-mentioned hybridoma cell strain secretion
Clonal antibody.
Third object of the present invention is to provide the applications of above-mentioned anti-human Clonorchiasis Sinensis monoclonal antibody.
Fourth object of the present invention is to provide immune containing above-mentioned anti-human Clonorchiasis Sinensis monoclonal antibody
Detection kit.
The purpose of the present invention is give realization by the following technical programs：
A kind of anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma cell strain MQ-ANTI-TCTP-2, classification life
Name is people's Clonorchiasis Sinensis (Hu-TCTP) monoclonal antibody hybridoma cell, and the cell strain was October 20 in 2014
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center day, deposit number is CGMCC No.9812, is protected
Hiding address is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The preparation method of above-mentioned hybridoma cell strain is as follows：Serum titer is taken to be more than 1：104BALB/c mouse splenocyte
It is merged according to a conventional method with 50%PEG-4000 with SP2/0 myeloma cell；With the HAT RPMI- containing 20% calf serum
1640 Screening of Media fused cells carry out ELISA sieves with people's Clonorchiasis Sinensis of recombinant expression coating elisa plate
Choosing；By multiple limiting dilution, the hybridoma cell strain of the anti-human Clonorchiasis Sinensis of stably excreting is finally obtained.
The anti-human Clonorchiasis Sinensis monoclonal antibody obtained by the secretion of above-mentioned hybridoma cell strain；Preferably, institute
It is IgG1 types to state monoclonal antibody, and light chain is κ chains.
The preparation method of the anti-human Clonorchiasis Sinensis monoclonal antibody：It is CGMCC using deposit number
The strain of hybridoma of No.9812 is with 1 × 106/ only amount injection pretreated 8~10 week old of paraffin BALB/c female mices
Ascites is extracted in abdominal cavity when mouse web portion expands after breeding observing 10~14 days.Using affinity chromatography Protein G
Sepharose Fast Flow monoclonal antibody purifications, and by Sepharose S-200 molecular sieve desalinations, PBS is eluted, with
SDS-PAGE measures the purity of monoclonal antibody, and purity reaches 90% or more.
Application of the anti-human Clonorchiasis Sinensis monoclonal antibody of any one of the above in preparing tumour diagnostic reagent；
Preferably, the tumour is colorectal cancer.
Immunity detection reagent containing above-mentioned anti-human Clonorchiasis Sinensis monoclonal antibody, the immune detection examination
Agent box also contains marker, and the marker is fluorescent marker, radioactively labelled substance or enzyme marker.
Compared with prior art, the invention has the advantages that：
The present invention provides a kind of anti-human Clonorchiasis Sinensis (TCTP) monoclonal antibody, the monoclonal antibody by
Hybridoma cell strain MQ-ANTI-TCTP-2 secretions obtain, and at present there has been no the antibody of the TCTP of production domesticization, foreign countries can detect
The antibody product of TCTP there are expensive, potency is low, with natural TCTP albumen reaction effect it is bad the shortcomings of, it is of the present invention
After monoclonal antibody-purified, it is swollen to prove that it can identify that natural people translates control by immunofluorescence dyeing and immunohistochemical staining
Tumor albumen；In addition, carrying out Western-blot to tying straight SW480 cancer cell and Lovo by monoclonal antibody purification and demonstrating this
Monoclonal antibody can identify people's Clonorchiasis Sinensis of denaturation；Monoclonal antibody of the present invention can be used for ELISA, change
Luminous, western-blot, immunofluorescence and tissue Immunohistochemical detection is learned, is people's Clonorchiasis Sinensis work(
It can research and its clinical samples verification work offer support as colorectal cancer tumour hepatic metastases marker.
When kit containing said monoclonal antibody of the present invention is detected, high sensitivity (0.1ng/mL), specificity
By force, detection range is wide, easy to operate, and "dead" pollution, kit is at low cost, clinical adaptable, is particularly suited for China
Clinical clinical detection screening.
Description of the drawings
Fig. 1 be monoclonal antibody A2 of the present invention to tie straight carcinoma cell immunization trace as a result, it is 23kDa that it, which combines the molecular weight of band,
Near.
Fig. 2 is results of the monoclonal antibody A2 of the present invention to the straight cancer cell Lovo immunofluorescence dyeings of knot.
Fig. 3 is the standard curve that monoclonal antibody A2 of the present invention carries out chemiluminescence detection.
Fig. 4 is result (microscope magnifications 100 of the monoclonal antibody A2 of the present invention to liver cancer tissue immunohistochemical staining
Times).
Specific implementation mode
The content further illustrated the present invention with reference to the accompanying drawings of the specification with specific embodiment, but should not be construed as to this
The limitation of invention.Without departing from the spirit and substance of the case in the present invention, to simple made by the method for the present invention, step or condition
Modifications or substitutions all belong to the scope of the present invention；Unless otherwise specified, technological means used in embodiment is art technology
Conventional means known to personnel.
The preparation of 1 hybridoma cell strain of embodiment
One, prepared by recombined human Clonorchiasis Sinensis antigen
Tctp gene is transferred from colorectal cancer cell Lovo, builds pronuclear recombination expression vector pET22b (+)/TCTP,
It expresses in Escherichia coli Escherichia coli BL21, is purified using Ni Sepharose affinitive layer purification columns,
Purity is obtained up to 90% or more rhTC TP albumen (amino acid sequence such as SEQ ID NO：Shown in 1).
Wherein, the primer sequence of amplification people's Clonorchiasis Sinensis (TCTP) gene is：
Sense primer：5'-GTCGGATCCATGATTATCTACCGG-3'(SEQ ID NO：2)；
Downstream primer：5'-TTGCGGCCGCTTAACATTTTTCCATTTCTAAACCA-3’(SEQ ID NO：3)；Wherein,
Upstream introduces BamHI restriction enzyme sites, and downstream introduces Not I restriction enzyme sites.
Two, mouse immune
The TCTP recombinant proteins of purifying are taken to be mixed with 250 μ l Freund's adjuvant adjuvants, with the amount subcutaneous abdomen of 100 μ g/500 μ l
Multi-point injection BALB/c mouse, the three weeks amount subcutaneous abdomen multi-point injection BALB/c mouses with 1100 μ g/500 μ l in interval, from the
It is immunized and starts three times, the second week tail vein after being immunized every time acquires mouse blood, and indirect ELISA method detection serum titer reaches
1：Prepare cell fusion after 50000 or more, merges first 3 days, tail vein injection booster immunization is primary, 100 μ g of antigen dose.Freund
Freund's complete adjuvant and the equal commodity in use product of incomplete Freund's adjuvant.
Three, immune serum titration
Immune serum potency is measured using indirect elisa method.30 μ g TCTP recombinant proteins are taken to be dissolved in 10ml 0.05M,
The carbonate buffer solution of pH9.6 is coated with micro- 96 orifice plate of polystyrene, and 100 holes μ l/, 4 DEG C overnight.Next day (is contained using PBS
0.1% (V/V) Tween-20) board-washing three times, with 200 holes μ l/ containing 10% newborn bovine serum confining liquid 10mM PBS, 37 DEG C of closings
1h, three times using PBS (containing 0.1% (V/V) Tween-20) board-washing, mouse 15 days tail vein bloods after third time is immune,
Mouse immune serum is used containing 2% newborn bovine serum, 10mM PBS with 10-1~10-8It dilutes again, 96 orifice plates of addition, 100 holes μ l/, 37
DEG C 1h, PBS (containing 0.1% (V/V) Tween-20) board-washing three times after, be added 1：10000 times of dilution horseradish peroxidase marks
Remember goat anti-mouse igg (Sigma, INC.), 100 holes μ l/, 37 DEG C of incubation 1h, ibid after board-washing, TMB develops the color, 100 holes μ l/, room
Temperature is protected from light 20min, adds 50 holes μ l/ 2M H2SO4Termination reaction, surveys 450nm absorption values, right using mice serum before immune as feminine gender
According to judging the potency of immune serum so that ratio >=2.1 of measured value and control value are positive.
Four, the preparation of hybridoma
Serum titer is taken to be more than 1：104Mouse, merge preceding 3 days, after taking recombinant antigen and isometric PBS mixings, with
The amount intraperitoneal injection BALB/c mouse to be fused of every 100 μ g/500 μ L carry out booster immunization.It is sterile to take mouse spleen, spleen is made
The murine myeloma cell strain SP20 of cell suspension and exponential phase presses 10：1 ratio mixing, the centrifugation of 500 × g room temperatures
5min abandons supernatant, flicks centrifugation bottom of the tube with finger, keeps precipitation loose, and centrifuge tube is placed in 37 DEG C of water-baths, will be in 37 DEG C of water-baths
In 50% polyethylene glycol (PEG, MW4000, Sigma) of the heat preservation one after another drop of addition centrifuge tube of dropper, centrifugation is shaken in drop
Pipe drips off in 1min, 2min is stood after dripping off, every 1 minute 1640 culture medium 1ml, 2ml of serum-free that 37 DEG C of preheatings are added,
3ml, 4ml, 5ml and 10ml terminate the effect of polyethylene glycol, and cell mixture 500 × g room temperatures centrifuge 5min, abandon supernatant, add
Enter HAT culture solutions (hypoxanthine (H), aminopterin-induced syndrome (A) and thymidine (T) (HAT, Sigma)) and cell be gently resuspended,
Cell is divided into 96 orifice plates, per 200 μ l of hole.After culture three days, cell fusion situation is observed, replaces half HAT culture solutions, even
The continuous a few days replaces HT culture solutions (hypoxanthine (H) and thymidine (T) (HT, Sigma)) and trains until there is Clone formation
Support three days, by indirect ELISA method filter out the hybridoma that can be reacted with TCTP recombinant proteins (with negative serum
The ratio of A450 is judged to the positive more than 2.1), replace 1640 complete mediums.
Five, the hybridoma of anti-human TCTP monoclonal antibodies is secreted in screening
Indirect elisa method screens cells and supernatant, and the higher positive colony hybridoma of potency is selected to carry out sub- gram
The continuous cloning of limiting dilution assay 2~3 times is used in combination in Longhua, until to 100% cell positive rate, it is anti-finally to obtain stably excreting
15 plants of TCTP cell strain of monoclonal antibody send to after by cloning positive rate up to liquid nitrogen cryopreservation after 100% cell amplification cultivation.
And will wherein one plant be named as MQ-ANTI-TCTP-2, which was deposited in Chinese microorganism strain on October 20th, 2014
Preservation administration committee common micro-organisms center, deposit number are CGMCC No.9812.
Six, the volume analysis of hybridoma cell strain MQ-ANTI-TCTP-2 secretory antibodies：By hybridoma cell strain MQ-
ANTI-TCTP-2 is cultivated on 96 microwell plates, the yield of antibody is generated in order to analyze it, with 10~12 days supernatants of cell fusion
It is analyzed.Two kinds of fusion proteins GST, GST-TCTP are used for ELISA, hybridoma cell strain MQ-ANTI-TCTP-2 secretions
The OD of antibody A 2 and GST-TCTP450It is 1.62, the OD of antibody A 2 and GST450It is 0.13.
The specific method of ELISA is：30ug fusion proteins are coated on 96 hole polystyrene plates, 4 DEG C overnight.Use elution buffer
Liquid (PBS for containing 0.05% (V/V) Tween-20) elutes plate 3 times.The PBS containing 10% calf serum is added into micropore,
37 DEG C of closing 2h.The culture supernatant of hybridoma cell strain MQ-ANTI-TCTP-1 is added in micropore (holes 100uL/), 37 DEG C
It is incubated 1h.It washes after version and the sheep anti-mouse igg polyclonal antibody of horseradish peroxidase-labeled is added, 37 DEG C of incubation 1h.Elution buffer
100 μ lTMB colour developings are added in liquid board-washing three times, per hole, and after 15~30 minutes, 2M H are added2SO4Solution terminates reaction, and microplate reader exists
It is detected at absorbance value 450nm.
The preparation of 2 anti-TCTP monoclonal antibodies of embodiment and property analysis
1, the preparation of antibody：Positive rate hybridizes up to 100% MQ-ANTI-TCTP-2 after the cloning that embodiment 1 is obtained
Tumor cell strain is with 1 × 106/ only amount injection 8~10 pretreated week old of atoleine BALB/c female mices abdominal cavity, raise
It supports and extracts ascites when mouse web portion expands after observing 6~7 days.Mouse ascites are acquired after 7~10 days, 12000rpm centrifuges 10min
Supernatant is collected, is purified with ProteinGharose (being purchased from GE companies)：ProteinG fillers are loaded in purification column, it will be pure
Change column to connect with Akta purifying instrument, with equilibration buffer (method provided to specifications is prepared) 5 column volumes of balance, wait for pure
The ascites of change dilute 10 times with equilibration buffer after with 1.5ml/min speed loadings, be washed till baseline with equilibration buffer after loading,
With elution buffer antibody elution, antibody peak is collected, the antibody of elution uses Tri-Hcl (pH9.0) to neutralize immediately；After purification anti-
Body A2 is named as antibody A 2 in -20 DEG C of preservations, the antibody of MQ-ANTI-TCTP-2 secretions.
2, the immunological properties of antibody A 2
(1) measurement of antibody concentration：People Clonorchiasis Sinensis TCTP monoclonal antibody A2 are obtained after ascites is purified,
It is measured using the Smart Spec plus nucleic acid-protein analyzers that BIO-RAD companies produce, a concentration of 1.15mg/ml.
(2) antibody subtype is identified：The Asia of hybridoma cell strain is identified using the mouse monoclonal antibody subtype identification kit of Hbt companies
The hypotype of type, MQ-ANTI-TCTP-2 secretory antibodies is IgG1 types, and light chain is κ chains.
(3) the Western blot identifications of antibody：It collects and ties straight cancer cell (Lovo, HT29, SW620 and SW480), with 1
× SDS lysis buffers cracking after loading, after 12%SDS-PAGE with Bio-Rad reactance body transfer devices by protein delivery extremely
On pvdf membrane, the closing of 5% skimmed milk power overnight, wash by the Tris-HCl buffer solutions (containing 0.1% (V/V) Tween-20) of pH7.4
Film 3 times, each 10min, 1：800 are added purified anti-human TCTP monoclonal antibodies A2, after being incubated at room temperature 1h, pH7.4's
Tris-HCl buffer solutions (containing 0.1% (V/V) Tween-20) wash film 3 times, each 10min, are added 1：5000 diluted goat-antis
Mouse IgG polyclonal antibodies (Sigma) are secondary antibody, are incubated at room temperature 1h, and TTBS washes film 3 times, extra solution is sucked with filter paper, are tiled
In on clean tin foil, 1.4ml is addedSerial Western chemiluminescent substrates reaction solution (A：B=1：
1), make film complete wetting in reaction solution, it is rapid to take out, surplus liquid is sucked with filter paper, is laid on another tin foil, with guarantor
Fresh paper wraps film, is put into X-ray magazine, develops in dark place.There is single spy in anti-TCTP monoclonal antibodies A2
Anisotropic band, the results are shown in Figure 1.
(4) the confocal immunofluorescence microscopy identification of antibody：It collects and ties straight cancer cell Lovo, be laid on sheet glass and grow overnight,
PBS washs cell, fixes cell using the paraformaldehyde of precooling, anti-TCTP monoclonal antibodies A2 (1 is added：500 dilutions), 37 DEG C
It is incubated 1h, using PBS as negative control.PBS develop a film after 1：1000 are added fluorescent marker sheep anti mouse secondary antibody (Sigma), 37 DEG C of incubations
After 1h, PBS develop a film, under the microscope, the cell dyed through anti-TCTP monoclonal antibodies A2 observes green fluorescence to fluorescence microscopy
(wavelength 488nm), as shown in Figure 2.As a result prove that anti-TCTP monoclonal antibodies A2 prepared by hybridoma can recognize that naturally
People's TCTP albumen.
(5) the immunohistochemistry identification of antibody：The liver cancer tissue of paraffin embedding is collected from Nanfang Hospital, utilization resists after purification
TCTP monoclonal antibodies A2 (1：500 dilutions) immunohistochemical staining is carried out, the immunohistochemical kit of new company is stepped using Fujian,
Colouring method is with reference to novel agent box specification is stepped, and DAB colour developings, negative control group replaces primary antibody with PBS, as a result, it has been found that through anti-TCTP
Brown particle is found in cytoplasm on the tissue specimen of monoclonal antibody A2 dyeing, as shown in Figure 4.In colorectal cancer tumour
The immunohistochemical staining result of cell proves that anti-TCTP monoclonal antibodies A2 prepared by hybridoma can know in liver metastasis model
Not natural people's TCTP albumen.
The preparation of immunity detection reagent of the embodiment 3 containing anti-TCTP monoclonal antibodies
One, applied to the development of chemiluminescence detection kit
S1. acridinium ester label antibody method
S11. by 0.1mg antibody (ABCAM.Catalog Number:Ab58362 it) is added in ultra-filtration centrifuge tube (50KD),
8000r/min centrifuges 5~6min and is concentrated.200uL PB buffer solutions, 8000~9000r/min is added in antibody after concentration
5~6min is centrifuged, filtrate is discarded, adds 200uL PB buffer solutions, centrifuge as stated above, this step repeats 5~6 times.
S12. will several times centrifuge after centrifuge tube take out, discard filtrate, centrifugal column inverted, 2000~3000r/min from
Heart 1min, collects filtrate, and whole process collects about 200 μ L of antibody.
S13. 100 are pressed with pretreated antibody and acridinium salt：1 mass ratio mixes well, and shakes 16~20 hours.
S14. the antibody diluted of acridinium salt will be marked to required concentration, deposits in 2~8 DEG C.
S2. magnetic bead coated antibody flow
S21. mix well suspension magnetic bead with eddy mixer, take 1mL (10mg) suspension (the preservation liquid of magnetic bead) arrive from
In heart pipe；
S22. centrifuge tube is placed on magnetic separator 1 minute (as needed can be for more time) and carefully removes supernatant.
S23. 1ml connections buffer solution (0.1M MES) is added, suspension magnetic bead is mixed well with eddy mixer.
S24. 40ul coupling agents (1- ethyls -3- (3- DimethylAminopropyls) carbodiimide hydrochloride) are added.It is added real
Apply the antibody that example 2 is prepared.
S25. suspension magnetic bead is mixed well with eddy mixer, room temperature rotation is overnight.
S26. 4mg magnetic beads/ml, 2~8 DEG C of preservations are diluted to TBST elution buffers.
The preparation of S3.TCTP standard items：Configure calibration object with TCTP sterlings, concentration is respectively 0,0.39,1.56,6.25,
25.00、100.00ng/mL。
S4. chemiluminescence starts agent A：
S5. chemiluminescence starts agent B
S6. concentrated cleaning solution
PH value is adjusted to 7.0, with 20 times of dilutions of distilled water when use.
The TCTP chemistry of above-mentioned preparation give out light immune analytic reagent kit concrete operations it is as follows：
A.4 DEG C refrigerator takes out kit, equilibrium at room temperature 15 minutes；
B. corresponding plastic tube is added in the standard items of each concentration point and 50 μ L of sample to be tested respectively, then adds the anti-of magnetic bead
50 μ L of body；37 DEG C incubate 30 minutes；
D. it is washed 5 times with the cleaning solution after dilution, fills cleaning solution, impregnated 20 seconds every time；
F. plastic tube is added in 50 μ L of acridinium ester label, 37 DEG C incubate 30 minutes；
G. it is washed 5 times with the cleaning solution after dilution, fills cleaning solution, impregnated 20 seconds every time；
H. chemiluminescence is added and starts agent each 50 μ L of A and B, directly measures its luminous intensity (RLU), time of measuring 0.5 second/
Pipe；
I. with a concentration of abscissa of calibration object, RLU values are that ordinate draws standard curve (Fig. 3), are existed with each RLU values to be measured
The TCTP antigen concentrations of the sample are investigated and prosecuted on standard curve.
Above-mentioned detection kit is examined and determine according to manufacture conventional in the art and identification regulation, the results are shown in Table 1.
The technical indicator testing result of 1 kit of table
| Inspection project | Test stone | Inspection result |
| Accuracy | Average recovery rate is in 90%-100% | 95.8% |
| Specificity | With cross reacting rate≤0.01% of its analog | ＜ 0.01% |
| Accuracy | ≤ 15% (n=10) | 5.6% |
| Sensitivity | ≤0.5ng/mL | 0.1ng/mL |
| Stability | Each reagent set splits 37 DEG C at least 7 days | Meet the above standard |
The above result shows that the above-mentioned accuracy for preparing obtained TCTP chemical luminescence immune assay determination reagent kits, spy
The opposite sex, accuracy, sensitivity and stability comply fully with requirement.
The ratio of kit that 4 embodiment 3 of embodiment is prepared and commercially available enzyme linked immunological kit to patient's blood sample measured value
It is marked compared with 50 parts of patients of kit and commercially available enzyme linked immunological protein detection kit (EIAB companies) while detection of embodiment 3 are used
This, the comparison result of two kinds of kits is shown in Table 2.
2 enzyme linked immunological kit comparison result of table
From listed testing result data analysis, kit of the present invention detects 40 parts of positives in 50 parts of positive samples, positive
Rate is 80%, and enzyme exempts from kit and detects 35 parts of positives, positive rate 70%.As a result it shows the testing result of this kit and faces
Bed accessory has higher coincidence rate, is more convenient for promoting and applying, securely and reliably.
SEQUENCE LISTING
<110>Nanfang Medical Univ
<120>Anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application
<130>
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 172
<212> PRT
<213>TCTP albumen
<400> 1
Met Ile Ile Tyr Arg Asp Leu Ile Ser His Asp Glu Met Phe Ser Asp
1 5 10 15
Ile Tyr Lys Ile Arg Glu Ile Ala Asp Gly Leu Cys Leu Glu Val Glu
20 25 30
Gly Lys Met Val Ser Arg Thr Glu Gly Asn Ile Asp Asp Ser Leu Ile
35 40 45
Gly Gly Asn Ala Ser Ala Glu Gly Pro Glu Gly Glu Gly Thr Glu Ser
50 55 60
Thr Val Ile Thr Gly Val Asp Ile Val Met Asn His His Leu Gln Glu
65 70 75 80
Thr Ser Phe Thr Lys Glu Ala Tyr Lys Lys Tyr Ile Lys Asp Tyr Met
85 90 95
Lys Ser Ile Lys Gly Lys Leu Glu Glu Gln Arg Pro Glu Arg Val Lys
100 105 110
Pro Phe Met Thr Gly Ala Ala Glu Gln Ile Lys His Ile Leu Ala Asn
115 120 125
Phe Lys Asn Tyr Gln Phe Phe Ile Gly Glu Asn Met Asn Pro Asp Gly
130 135 140
Met Val Ala Leu Leu Asp Tyr Arg Glu Asp Gly Val Thr Pro Tyr Met
145 150 155 160
Ile Phe Phe Lys Asp Gly Leu Glu Met Glu Lys Cys
165 170
<210> 2
<211> 24
<212> DNA
<213>Expand the sense primer of people's tctp gene
<400> 2
gtcggatcca tgattatcta ccgg 24
<210> 3
<211> 35
<212> DNA
<213>Expand the downstream primer of people's tctp gene
<400> 3
ttgcggccgc ttaacatttt tccatttcta aacca 35
Claims (1)
1. one kind containing anti-human Clonorchiasis Sinensis monoclonal antibody immunity detection kit, the anti-human translation controls tumour
Protein monoclonal antibody is secreted by hybridoma cell strain MQ-ANTI-TCTP-2；The cell strain was on October 20th, 2014
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9812；
Anti-human Clonorchiasis Sinensis monoclonal antibody contained in the immunity detection reagent includes by acridinium ester label
Anti-human Clonorchiasis Sinensis monoclonal antibody and the coated anti-human Clonorchiasis Sinensis monoclonal antibody of magnetic bead；
Further include in the immunity detection reagent：
Chemiluminescence starts agent A：
65%HNO3 10.0ml
Urea peroxide 2.0g
Prochin300 1mL
Distilled water 1000mL；
Chemiluminescence starts agent B：
NaOH 10.0g
Triton X-100 20mL
Prochin300 1mL
Distilled water 1000mL；
Concentrated cleaning solution：
Tris 24g
NaCl 160g
KCl 4g
HCl 15mL
Deionized water 100mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510129941.XA CN104830805B (en) | 2015-03-24 | 2015-03-24 | Anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510129941.XA CN104830805B (en) | 2015-03-24 | 2015-03-24 | Anti-human Clonorchiasis Sinensis monoclonal antibody hybridoma and its monoclonal antibody and application |
Publications (2)
| Publication Number | Publication Date |
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| CN104830805A CN104830805A (en) | 2015-08-12 |
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| ACCESSION：NP_003286.1;MIAO X ET AL.;《GENBANK》;20140316;1-2 * |
| PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODY AGAINST TRANSLATIONALLY CONTROLLED TUMOR PROTEIN;QIANG M ET AL;《HYBRIDOMA》;20110228;第30卷(第1期);摘要、第82页左栏材料和方法、第83页右栏第1段 * |
| 翻译控制肿瘤蛋白(TCTP)研究进展;吕素芳 等;《科学技术与工程》;20060228;第6卷(第04期);424-428 * |
| 翻译控制肿瘤蛋白TPT1的原核表达、纯化、抗体制备和初步应用;赵一璠 等;《细胞与分子免疫学杂志》;20100331;第26卷(第03期);摘要、第247页左栏1.2方法 * |
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