CN106554418A - 2 monoclonal antibody of anthrax toxin acceptor and preparation method and application - Google Patents
2 monoclonal antibody of anthrax toxin acceptor and preparation method and application Download PDFInfo
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- CN106554418A CN106554418A CN201610919910.9A CN201610919910A CN106554418A CN 106554418 A CN106554418 A CN 106554418A CN 201610919910 A CN201610919910 A CN 201610919910A CN 106554418 A CN106554418 A CN 106554418A
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Abstract
The invention belongs to technical field of pharmaceutical biotechnology, specifically, the present invention is with regard to a kind of 2 monoclonal antibody of anthrax toxin acceptor and preparation method and application.The monoclonal antibody is CCTCC by preserving number：It is prepared by the hybridoma cell strain of C201628.CMG2 monoclonal antibodies of the present invention have the advantages that specificity is good, affinity is high, solve the problems, such as prepared by CMG2 high-affinity antibodies.And 2 monoclonal antibody of anthrax toxin acceptor can be used for developing tumour diagnostic reagent and staging, the diagnostic reagent of typing, while the medicine or pharmaceutical carrier for CMG2 positive tumors can be developed for.
Description
Technical field
The present invention relates to molecular biology and biological technical field, relate more specifically to a kind of 2 Dan Ke of anthrax toxin acceptor
Grand antibody and preparation method and application.
Background technology
2 (anthrax toxin receptor 2, ANTXR2 of anthrax toxin acceptor；Also known as capillary
Morphogenesis gene 2, CMG2).CMG2 be single pass transmembrane receptor, encoding gene be located at No. four chromosome 4q21.21,
Molecular weight 45kDa.It is proved that CMG2 can mediate anthrax toxin to enter host cell, it is one of two receptors of anthrax toxin,
And be the receptor most strong with anthrax toxin affinity.There is experiment to show, the internal native ligand of CMG2 is laminin，LN
And IV Collagen Type VIs, but the physiological function of CMG2 is not yet illustrated (laminin).
So far, tumor cell CMG2 expression and its meaning lack report, and our previous experiments are pointed out, CMG2 with
The dryness of GCSCs, epithelial-mesenchymal convert (epithelial-mesenchymal transition, EMT) phenotype and invade
Attack transfer ability closely related；The Preliminary detection result of clinical gastric cancer specimen is also pointed out, high expression of the CMG2 on stomach organization
It is proportionate with TNM stage, with the survival of patients time in negative correlation.Therefore, CMG2 is probably the new feature marks of GCSCs
Thing, also points out CMG2 to be probably new thinking and target spot (Enrichment of cancer in the diagnosis of tumor and treatment
stem cells based on heterogeneity of invasiveness.Stem Cell Rev.2009Mar；5(1)：
66-71)。
1) CMG2 is probably the dryness mark of stomach cancer stem cell
Find that CMG2 (ANTXR2) is expressed stomach cancer stem cell is high first, and with stomach cancer stem cell dryness, EMT phenotypes
And invasive ability is closely related.CMG2 can be interacted with LRP6, and the latter can activate Wnt signal paths, it was found that CMG2
Relevant with Src-Erk Pathway Activations, this two paths can induce EMT, and EMT is the critical event of Invasion and Metastasis process.Cause
This speculates the functional markers as stomach cancer stem cell, and CMG2 may activate Wnt paths energy by interacting with LRP6
Activation Src-Erk paths, induce EMT and promote gastric cancer invasion transfer (The LDL receptor-related protein
LRP6 mediates internalization and lethality of anthrax toxin.Cell.2006Mar 24；
124(6):1141-54), as shown in Figure 1.
2) invasive ability of the obvious stomach cancer stem cell of expression energy of silence or downward CMG2
After the expression of silence CMG2, p-Src 416 (positive regulator phosphorylation site) phosphorylation is reduced and p-Src527 (negative regulators
Phosphorylation site) phosphorylation increases, and points out CMG2 to have positive regulating and controlling effect to Src.Therefore, effects of the CMG2 in GCSCs can
Classical Wnt signal path (Wnt/ β-catenin) and non-classical Wnt/Jun kinase pathways can be regulated and controled by LRP6, in addition
It is likely present the regulatory pathway for activating Src-Erk.The activation of these signal paths may raise the EMT such as Snail, Twist
The expression of associated transcription factor, so as to promote the generation of EMT and the enhancing of invasive ability.
3) in CMG2 monoclonal antibodies and CMG2 be effective oncotherapy means
By silence and strike it is low CMG2's it is demonstrated experimentally that suppress CMG2 can reduce EMT generation and suppress tumor
Invasive ability.In current Biotherapeutics means, Antybody therapy is most effective and most ripe means.It is prepared by design
Monoclonal anti physical ability with CMG2 as target spot reaches the effect for the treatment of tumor, and this point foreign countries have some correlational studyes to report.
Amit chaudhary can suppress pathologic blood by preparing TEM8 monoclonal antibodies (another receptor of anthrax toxin)
Pipe is generated so as to suppress propagation (the TEM8/ANTXR1Blockade Inhibits of tumor
PathologicalAngiogenesis and Potentiates Tumoricidal Responses against
Multiple Cancer Types.Cancer Cell 21,212–226,February 14,2012)；Lorna M report target
Effect (the Targeting the for the treatment of tumor can be reached to TEM8 and CMG2 by suppressing tumor-blood-vessel growth
anthrax receptors,TEM-8and CMG-2,for antiangiogenic therapy.Front Biosci.；16:
1574–1588.).By antibodypadia (https://www.antibodypedia.com/) online lookup, CMG2's
Antibody is mainly based on polyclonal antibody, and the monoclonal antibody report and effect of only only a few are failed to understand, in above-mentioned Amit
Also mention in chaudhary articles and start the monoclonal antibody effect on driving birds is not good for preparing TEM8 and failure, finally adopt antibody library skill
Art obtains its antibody.Therefore, for the monoclonal antibody applied research of target spot, current CMG2 not yet has been reported that its main cause is
Be not successfully prepared high-affinity has the CMG2 monoclonal antibodies for preferably combining effect with natural CMG2.
The content of the invention
For above-mentioned problems of the prior art, it is an object of the invention to provide a kind of anthrax toxin acceptor 2 is single
Clonal antibody and preparation method and application, solve the problems, such as prepared by CMG2 high-affinity antibodies, and anthrax toxin acceptor 2 is single
Clonal antibody can be used for developing tumour diagnostic reagent and staging, the diagnostic reagent of typing, while pin can be developed for
Medicine or pharmaceutical carrier to CMG2 positive tumors.
In order to realize foregoing invention purpose, the technical solution used in the present invention is as follows：
2 monoclonal antibody of anthrax toxin acceptor, is CCTCC by preserving number：It is prepared by the hybridoma cell strain of C201628.
The hybridoma cell strain is named as 8-F3, culture title：Hybridoma cell strain 8-F3 (IgG1), by Chinese allusion quotation
The center preservation of type culture, address：Wuchang, wuhan Luo Jia Shan Wuhan University, preservation date：On 2 25th, 2016.
2 monoclonal antibody of the anthrax toxin acceptor is the monoclonal antibody of CMG2 specificitys.
The preparation method of 2 monoclonal antibody of anthrax toxin acceptor, comprises the steps of：
1) extracellular region protein is recombinated as immunogen immune Balb/c mice with the CMG2 of purification；
2) by hybridoma technology by many times of the inguinal lymph node cells of Balb/c mices and myeloma cell SP2/0 Jing
Cell fusion, using indirect elisa method (enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay,
ELISA screening positive clone) is carried out, cloning filters out the hybridoma cell strain of energy stably excreting CMG2 monoclonal antibodies.
Further, the Balb/c mices in the step 1 are 8 week old, respectively with Freund's complete adjuvant and Freund
The vola of Freund's incomplete adjuvant, point first immunisation and the 9th day Balb/c mice immune twice, the Freund's complete adjuvant and institute
The antigen dose for stating incomplete Freund's adjuvant is 100 micrograms.
Further, step 2) include：Inguinal lymph nodes separation cell is mixed with myeloma cell, with poly- second two
Alcohol 1450 is fusion agent, forms fused cell, and fused cell is suspended from the HAT containing calf serum, and (H-Hypoxanthine time is yellow
Purine, A-Aminopterin methotrexates, T --- Thymidine thymidines) in culture fluid, juxtaposition CO2In 37
DEG C culture；Screened with indirect elisa method, during screening, screened with CMG2 restructuring extracellular region protein coatings, to detection
Positive colony hole carries out Cell-cloned using limiting dilution assay, is placed in CO2In cultivate in 37 DEG C, until all cell growths
Till the culture fluid in hole is positive.
A kind of screening side of anthrax toxin acceptor 2 monoclonal antibody optimal with 2 protein binding of natural anthrax toxin acceptor
Method, comprises the steps of：
1) 10 stomach organization paraffin sections are taken, paraffin section is routinely dewaxed to water：Paraffin section is dipped in into dimethylbenzene
Middle 10min, three times；Take out paraffin section and be placed in 10min in 100% dehydrated alcohol, it is secondary；It is at different levels that 90%-70% is inserted successively
The each 10min of ethanol；Taking-up is placed in 10min in distilled water；
2) incubate dry paraffin section for 37 DEG C, paraffin is drawn a circle；
3) antigen retrieval：3M carbamide room temperature acts on 30min；Distilled water shakes washes 5min, three times；0.01MpH7.4PBS shakes and washes
5min, three times；
4) Deca 3%H2O2Room temperature acts on 10min；Distilled water shakes washes 5min, three times；PBS shakes and washes 10min, secondary；
5) 1% bovine serum albumin of Deca, 37 DEG C of effect 30min, inclines, does not wash；
6) Deca adopts the different CMG2 monoclonal antibodies of identical dilution factor and solubility, while right as feminine gender with Mus Ig supernatants
According to 4 DEG C overnight；PBS shakes and washes 10min, three times, gets rid of liquid (tissue is sure not drying), lies against in wet box；
7) Deca be marked with HRP sheep anti mouse two resist, room temperature effect 30min；PBS shakes and washes 10min, three times, gets rid of liquid
(tissue is sure not drying), lies against in wet box；
8) Deca developer DAB working solutions, control colour developing under room temperature effect 5-30min, or light microscopic；After colour developing completely, steam
Distilled water bath color development stopping；
9) ethanol (70%-100%) dehydrations at different levels, every grade of 10min；Take out paraffin section and insert 10min in dimethylbenzene, three
It is secondary；
10) gummy mounting.
Further, step 9) in dehydration before if necessary haematoxylin redye.
Further, anthrax toxin acceptor 2 monoclonal optimal with 2 protein binding of natural anthrax toxin acceptor for filtering out
Antibody.
2 monoclonal antibody of anthrax toxin acceptor is in tumour diagnostic reagent and staging, the diagnostic reagent of typing is prepared
Application, the reagent is independent tumor markerses.
2 monoclonal antibody of anthrax toxin acceptor is in the medicine or pharmaceutical carrier for preparing treatment CMG2 positive tumors
Using.
Further, the tumor includes gastric cancer, colon and rectum carcinoma, the esophageal carcinoma, hepatocarcinoma, pulmonary carcinoma, renal carcinoma and mammary gland
Cancer.
The preparation method of 2 monoclonal antibody of anthrax toxin acceptor of the present invention, by efficient immune programme for children, is drenched by separating
The sensitization bone-marrow-derived lymphocyte fawned on is merged with SP2/0 cells, is successfully prepared the CMG2 monoclonals that can be combined with natural CMG2
Antibody, the CMG2 monoclonal antibodies have the advantages that specificity is good, affinity is high, solve the preparation of CMG2 high-affinity antibodies
Problem；2 monoclonal antibody of anthrax toxin acceptor of the present invention shows that in group change detection which has different detections effects to tumor
Really：CMG2 monoclonal antibodies are that part gastric cancer sample and partial breast cancer sample are distinguished in the histochemical staining situation of same tumor
In strong positive expression, positive expression, do not express substantially, and positive strong and weak classification and classification with tumor is without obvious relation；
Histochemical staining situation of the CMG2 monoclonal antibodies in other tumors is that the expression of Partial tumors strong positive, Partial tumors express one
As, while there is situation about not expressing, at identical conditions, in pulmonary carcinoma, renal carcinoma, breast carcinoma, the esophageal carcinoma, there is the sample to be in
Strong positive is expressed；CMG2 monoclonal antibodies histochemical staining situation in the normal tissue shows CMG2 Dan Ke to be negative substantially
Specificity of the grand antibody in lesion detection, 2 monoclonal antibody of anthrax toxin acceptor are combined almost without target protein, point out CMG2 mono-
Clonal antibody can have as the antitumor drug or pharmaceutical carrier for cmg2 positive tumors, i.e. CMG2 monoclonal antibodies
The expression popularity of tumor and specificity.CMG2 monoclonal antibodies histochemical staining situation in different tumors is similar to simultaneously, shows
CMG2 monoclonal antibodies have potential diagnosis and classify, classification diagnosis value, and as diagnosis and classification, classification diagnosis
Reagent is independent tumor markerses.
Description of the drawings
Fig. 1 is Gene chip analysis ANTXR2 (CMG2) in the high expression of stomach cancer stem cell；
Fig. 2 is part positive findingses in 10 gastric cancer samples that the 8-F3 of the present invention contaminates；
Fig. 3 is that commercial antibodies identify CMG2 recombiant proteins；Wherein, Fig. 3 A are the rabbit polyclonal antibodies of Protech companies
With the reaction of CMG2 recombiant proteins；Fig. 3 B are the reaction of the rabbit multi-resistance with CMG2 recombiant proteins of Sigma companies；Fig. 3 C are
The rabbit polyclonal antibody of Protech companies and the reaction of CYCS recombiant proteins；Fig. 3 D are the rabbit multi-resistance and CYCS weights of Sigma companies
The reaction of histone；
Fig. 4 is the specificity of two standby strain antibody 3B11 and 8-F3 of ELISA systems of identification；Wherein, Fig. 4 A are 3B11 and TEM8
The reaction of recombiant protein；Fig. 4 B are the reactions of 3B11 and CMG2 recombiant proteins；Fig. 4 C are the corresponding CMG2 monoclonal antibodies of 8-F3
With the reaction of TEM8 recombiant proteins；Fig. 4 D are the reactions of the corresponding CMG2 monoclonal antibodies of 8-F3 and CMG2 recombiant proteins；
Fig. 5 is the comparing results of the 8-F3 with Protech companies antibody of the present invention；
Fig. 6 is partial results in the gastric cancer that the 8-F3 of the present invention contaminates and breast carcinoma sample；
Fig. 7 is part positive findingses in the kinds of tumors organization chip that the 8-F3 of the present invention contaminates；
Fig. 8 is part positive findingses in various normal structure chips that the 8-F3 of the present invention contaminates.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, it is with reference to embodiment and accompanying drawing, right
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only to explain the present invention, not
For limiting the present invention.
The preparation method of 2 monoclonal antibody of anthrax toxin acceptor that the present invention is provided, mainly comprising step：
1) extracellular region protein is recombinated as immunogen immune Balb/c mice with the CMG2 of purification,
Comprising：Use Freund's complete adjuvant and incomplete Freund's adjuvant respectively, immunity 8 weeks twice of point first immunisation and the 9th day
The vola of the Balb/c mices in age, Freund's complete adjuvant and incomplete Freund's adjuvant dosage are 100 micrograms；
2) many cells of the inguinal lymph node cells of mice and myeloma cell SP2/0 Jing are melted by hybridoma technology
Closing, screening positive clone being carried out using indirect elisa method, cloning filters out the hybridization of energy stably excreting CMG2 monoclonal antibodies
Tumor cell strain,
Comprising：Inguinal lymph nodes separation cell is mixed with myeloma cell, with Macrogol 1450 as fusion agent, shape
Into fused cell, fused cell is suspended from the HAT culture fluid containing calf serum, juxtaposition CO2In 37 DEG C cultivate；With indirect
ELISA method is screened, and during screening, is screened with CMG2 restructuring extracellular region protein coatings, and the positive colony hole to detecting is adopted
Cell-cloned is carried out with limiting dilution assay, CO is placed in2In in 37 DEG C cultivate, until all cell growth holes culture fluid it is equal
Till being positive.
The preparation of 1 CMG2 monoclonal antibodies of embodiment
1) immunogen prepares
(aminoacid sequence is SEQ ID NO to adopt the CMG2 albumen of protokaryon escherichia coli expression：2) do immunogen.Specifically
Method is：The DNA sequence of synthesis CMG2 extracellular regions, (coded sequence is SEQ ID NO to introduce Nde I and Hind III：1) enzyme action
The DNA sequence of the CMG2 extracellular regions of synthesis is connected Pet22b prokaryotic expressions after Nde I and Hind III double digestions by site
Carrier, converts DH5a escherichia coli amplification plasmid and verifies whether successfully to expanding plasmid double digestion after checking successful connection；Will
Plasmid conversion BL21 (DE3) of successful connection carries out abduction delivering, as the albumen expressed is with the His labels being pre-designed,
With the corresponding recombiant protein of Ni column purifications, CMG2 prokaryotic recombinant proteins are obtained as immunogen.
2) immunogen immune Balb/c mice
CMG2 prokaryotic recombinant proteins are taken out from refrigerator, protein concentration are determined with Lowry methods (Folin- phenol reagent process), are made
1.0mg/ml is diluted to the 10mmol/L PBS (phosphate buffered saline(PBS)) of pH7.4.Choose 5-10 only 8 week old, body weight
The female Balb/c mices of 25g.Antigen emulsifying adopts antigen emulsator emulsifying.During first immunisation, by immunogen protein and grade body
Long-pending Freund's complete adjuvant emulsifying mixing, every mice carry out extremity foot-pad immunization by the immunogenic dosage of 100 μ g.Female
Balb/c mices carry out respectively within the 9th day second it is immune, adjuvant uses incomplete Freund's adjuvant, amount of antigen, volume injected and way instead
Footpath is constant, and indirect elisa method determines potency within the 21st day.Selecting 5 best mices of immune effect carries out cell fusion.
3) by immune Balb/c mice serums titration
12 days after second immunity, blood examination is taken from mouse tail vein and survey serum antibody titer；Use 0.1M NaCO3(PH9.6)
CMG2 prokaryotic recombinant proteins are diluted to best effort concentration 3-5 μ g/ml by coating buffer, add 100 μ l protein liquids, preservative film bag per hole
In 4 DEG C overnight, board-washing machine washing plate 5 times is finally upside down in Sptting plate in absorbent paper plate, and in making hole, cleaning mixture is drained.Blood sampling and
Dilute serum：Rat-tail is pinched, after 75% alcohol disinfecting, a breach is cut in tail vein with shears, 20 μ l of blood, 2000rpm centrifugations is taken
30min, takes 1 μ l of supernatant and adds 99 μ l antibody diluents to mix, and carry out doubling dilution, from 1:100 to 1:3200, by dilution
Tested serum adds 100 μ l per hole, while taking serum 1 before mouse immune:Negative control is done in 100 dilutions, and antibody diluent does sky
It is white to compare.37 DEG C are incubated 1 hour, with board-washing machine cyclic washing more than 5 times；Horseradish peroxidase goat anti-mouse igg is used
0.1% PBST (phosphate buffer of the pH7.4 containing 0.05% tween 20) is diluted to 1:3000, add 100 μ l per hole, 37
DEG C incubation 1 hour, wash 5 times；Plus 100 μ l/ holes of TMB (3,3', 5,5'- tetramethyl benzidine) nitrite ion, room temperature dark place 1-3 point
Clock, adds 50 μ l of terminate liquid observation results, terminates displaing yellow after TMB colour developings per hole, is read with enzyme-linked immunosorbent assay instrument record 450nm
Number, it is so that 2.1 times of each hole OD values more than negative control OD value are surveyed after blank control wells zeroing, as positive.Serum titer reaches
1:3200, can be used for cell fusion.
4) preparation of antigen sensibilization B cell and SP2/0 cell suspension
5 best Balb/c mices of immune effect are taken, eyeball of mouse, sacrificed by exsanguination, eye blood collected after centrifugation blood is extractd
Make clearly the positive control of ELISA, sterile working removes skin, be placed on aseptic operating platform, separate the lymph node of extremity enlargement, put
Enter to fill in the glass dish of 10ml incomplete culture mediums, washing is carefully peelled off the connective tissue and fatty tissue of surrounding, changes a glass
Glass ware, lymph node is pulled out, is placed in 200 mesh stainless (steel) wires, is ground with the inner core of syringe, with full culture of often cannoing be used up
Base is rinsed, and primed lymphocyte is entered in solution through mesh, lymphocyte is moved in 10ml glass centrifuge tubes,
1500rpm horizontal centrifugal 10min, remove supernatant.Same method, cannot be used up full culture medium 10ml washed cell 1 time, and precipitation is collected by centrifugation
Cell, by the cell resuspended mixing of 10ml incomplete culture mediums, cell counting about 1 × 108Individual cell.
SP2/0 cells are taken out from liquid nitrogen, is put in 37 DEG C of water-baths rapidly, is constantly rocked, until cell solution is complete
Dissolving, cell is transferred in 10ml centrifuge tubes, and 1500rpm horizontal centrifugal 10min abandon supernatant, and 10ml complete culture solutions are resuspended
Precipitation, is transferred to cell suspension in 50ml culture bottles, puts 37 DEG C, 5%CO2Cultivate in incubator.After cell growth is good
Cell is screened one week with the Selective agar medium containing 8-AG；Merge first 2 days, 1 bottle of cell is reached into 4 bottles, then just merging same day cell
In exponential phase, just, cell size is uniform, round and bright for vigor, merges the same day, with connector bend dropping tube by SP2/0 cells
Gently blow down from tube wall, be collected in centrifuge tube, be centrifuged, abandon supernatant, precipitation is cannotd be used up after full culture medium washing, and 10ml is not complete
Full culture medium is resuspended, cell counting, and about 5 × 107。
5) preparation of feeder cells
A non-immune Balb/c mice is taken, eyeball is plucked, sacrificed by exsanguination, 70% ethanol soaking disinfection 5-10min are cut off
Mouse skin, lifts peritoneum with tweezers, cuts an osculum with shears, and connector bend dropping tube is drawn ice-cold incomplete culture medium and rinses abdomen
Chamber, washing liquid is drawn in 50ml centrifuge tubes.Same method, full culture medium flushing abdominal cavity of cannoing be used up 3 times, collects washing liquid, under room temperature
1000rpm horizontal centrifugal 10min, go supernatant, 10ml incomplete culture mediums re-suspended cell simultaneously to count.
6) myeloma cell and primed lymphocyte fusion
The PEG 1450 (Macrogol 1450) of sigma companies of the U.S. is placed in into pre-temperature in 37 DEG C of incubators before fusion, is inhaled
Take 1 × 107Individual myeloma cell's suspension and 1 × 108Individual primed lymphocyte suspension (cell number 1：10) to 50ml it is aseptic from
In heart pipe, 30ml RPMI-1640 (Roswell Park Memorial Institute 1640) culture medium is added, it is fully mixed
Even, 1500rpm centrifugation 10min abandon supernatant, flick ttom of pipe, make cell mass loosely into pasty state, by 37 DEG C of water-baths of centrifuge tube, with drop
Pipe draws 1450 solution of 50%PEG of 0.8ml pre-temperatures, is being slowly added in cell along tube wall, side edged away from ttom of pipe about 2cm
Centrifuge tube is rotated, is added in 1min or so, then stood 90 seconds, be added dropwise over RPMI-1640 culture medium 30ml of 37 DEG C of pre-temperatures
Terminate fusion, add within 3min, fast after speed is first slow, action is soft, centrifuge tube is stood 5min in 37 DEG C of incubators, is taken
Go out centrifuge tube, 1500rpm centrifugation 5min, supernatant discarded add 10ml HAT culture medium re-suspended cells, gently blow and beat, mix, will
Fused cell is seeded to 96 porocyte culture plates for being covered with feeder cells, and by 100 μ l/ holes, every piece of culture plate stays 6 holes to be inoculated with
SP2/0 cells, as the negative control that HAT is selected, put 37 DEG C, 5%CO2Cultivate in incubator.
After fusion the 4-5 days can under inverted microscope observation of cell growing state, and add 100 μ of HAT culture medium
L, the 10-12 days available indirect elisa methods survey hybridoma potency, and the changes HT culture medium culturings for 14-15 days.
7) screening of specific hybrid oncocyte
12-15 days after fusion, when cell grows to the 1/4-1/2 bottom holes of full culture hole, trained using indirect elisa method detection
Foster supernatant, screening positive clone；With CMG2 albumen coated elisa plates (0.3 μ g/ holes), 4 DEG C of coatings overnight, are machine-washed using front board-washing
Wash once, pat dry liquid, add the 0.2% of 50 μ l cells and supernatants and 50 μ l PBST, positive control to select the immunity of mice
Serum, negative control select SP2/0 training supernatants, blank cleaning mixture, 37 DEG C of incubation 1h；Washing ELISA Plate：100 are added per hole
μl 1：The goat anti-mouse ig antibody of the HRP labellings of 6000 0.1% PBST dilutions, 37 DEG C are incubated 45 minutes；Washing, pats dry liquid
Body, plus 100 μ l/ holes of freshly prepared TMB solution, room temperature dark place reaction 2-3 minutes, plus terminate liquid is per 50 μ l terminating reactions of hole,
Microplate reader examines 450nm absorbances.
As a result it is：With CMG2 as antigen, successively immunity Balb/c mices 20, co-melting to close 2 times, successively screens altogether and obtains high
16 plants of the CMG2 antibody of affinity, Jing after 4 time cloningizations and repeatedly ELISA screenings, obtains secreting the miscellaneous of CMG2 monoclonal antibodies
Tumor cell strain is handed over, Jing is frozen for several times for these hybridomies, subculture in vitro separately culture more than 3 months can stably excreting CMG2 Dan Ke
Grand antibody, it is final frozen in liquid nitrogen container.
8) prepared by CMG2 monoclonal antibodies ascites
After filtering out positive colony, positive hybridoma cell is carried out by colonized culture using limiting dilution assay immediately, made
Standby feeder cells, it is resuspended with 10ml incomplete culture mediums, to collect positive colony cell and simultaneously count, full culture medium of cannoing be used up is by the positive
Clone cell is diluted to 100/20ml, takes one piece of 96 porocyte culture plates added with feeder cells in advance, adds 200 μ l thin
Remaining positive colony cell is transferred to amplification culture in 24 orifice plates by born of the same parents' suspension, collects cell liquid nitrogen cryopreservation, while will culture
Plate is in 37 DEG C, 5%CO2Cultivate in incubator, cell is progressively expanded basis of microscopic observation cell growth status after the 3rd day training
Support.The mice of 8 week old or Jing are produced into the paraffin oil of mouse peritoneal injection sterilizing, after 10 days by 108Individual hybridoma note
Mouse peritoneal is incident upon, and mouse ascites is obtained for purification CMG2 Dan Ke using extraction or by the way of killing cell in 10-14 days
Grand antibody.
9) identification of CMG2 monoclonal antibodies subclass
Using Sigma Co., USA Mouse Monoclonal Antibody Isotyping Reagents (ELISA/
Ouchterlony Double Diffusion, Stock No.ISO-2 LOT 114k4817), operation by specification is carried out.
(1) coated elisa plate：CMG2 albumen is diluted to into best effort concentration 3ug/ml with coating buffer, adds 100 μ l per hole
Antigen liquid, per plant plus 12 holes in 4 DEG C overnight, are washed 5 times, empty dry.
(2) close：Add 350 μ l of 5%BSA albumen (bovine serum albumin) confining liquid, 37 DEG C of incubation 1h-1.5h per hole, wash
Wash 5 times, it is empty dry.
(3) the Hybridoma Cell Culture supernatant for adding 100 μ l fresh per hole, is stored at room temperature 1h, shakes and wash 3min, totally 5 times.
(4) with antibody diluent with 1：2000 dilution IgG1, IgG2a, IgG2b, IgG3, IgM, IgA.
(5) culture supernatant for adding 100 μ l to dilute per hole, every kind of supernatant add 2 holes, are stored at room temperature 30min, shake and wash
3min, totally 5 times.
(6) 100 μ l 1 are added per hole：The rabbit-anti sheep IgG antibodies of 5000 dilutions, are stored at room temperature 15min, shake and wash 3min, and totally 5
It is secondary.
(7) 100 μ l substrate nitrite ions, room temperature lucifuge reaction 10min-15min are added per hole.
(8) 50 μ l terminate liquids are added per hole, observes result, determine Ig subclass.
10) purification CMG2 monoclonal antibodies in ascites
The ascites of fresh collection, 3000r/min 15 minutes, remove cell component (or it is frozen during the solidss that formed
Matter) etc.；The limpid ascites in upper strata is taken, equivalent adds PH7.2 veronal buffered saline (VBS；0.004mol/L barbital,
0.15mol/L NaCl, 0.8mmol/L Mg2+, 0.3mmol/L Ca2+) dilution；Then diluted in ascites with every 10ml and added
150mg SiO 2 powders, mix, and suspension is shaken frequently in incubation at room temperature 30 minutes；3000g is centrifuged 20 minutes, and lipid etc. leads to
Cross the method to remove, you can the CMG2 monoclonal antibody ascites that must be clarified.
(1) the monoclonal antibody-purified required buffer of CMG2 are configured：
Binding Buffer (A liquid):100mmol/L sodium phosphates (sodium phosphate), 100mmol/L citric acids
Sodium (sodium citrate), pH7.0.
Elution Buffer (B liquid):100mmol/L hydrochloric acid, pH2.7.
Assemble Buffer (C liquid):1mol/L tri- (methylol) aminomethane (Tris-HCl), pH9.0.
(2) by CMG2 monoclonal antibodies ascites and A liquid with 1：10 ratio mixing, 0.45 μm of membrane filtration wait loading.
(3) AKTA Explorer are accessed from HiTrap rProtein A HP posts, A, B liquid fully balances respective pipeline.
(4) sample for preparing (2) balances pillar with A liquid after loading, then with the eluting post of B liquid, receives from A pipe loadings
Collection eluting peak is into the collecting pipe for adding a small amount of C liquid in advance.
(5) pH to 7.0-8.0 of wash-out protein is adjusted, by CMG2 monoclonal antibody subpackage freezen protectives.
The screening of the CMG2 monoclonal antibody optimal with natural CMG2 protein binding of embodiment 2
A kind of technical scheme of SABC, screen from the one group of CMG2 monoclonal antibody for obtaining it is optimal can with it is natural
The optimal CMG2 monoclonal antibodies of CMG2 protein binding.
Specifically, (China is purchased from to the preservation of the First Affiliated Hospital of Third Military Medical University of PLA Pathology Deparment
PLA Hospital No.1 Attached to Military Medical Univ. No. 3) 10 stomach organization paraffin sections be used to detect prepared CMG2
Immunoreactivity of the monoclonal antibody to natural CMG2, filters out the CMG2 monoclonal antibodies of the same terms the following group best results
Strain, comprises the steps of：
1) stomach organization paraffin section is taken, paraffin section is routinely dewaxed to water：Paraffin section is dipped in dimethylbenzene
10min, three times；Take out paraffin section and be placed in 10min in 100% dehydrated alcohol, it is secondary；90%-70% wine at different levels is inserted successively
The each 10min of essence；Taking-up is placed in 10min in distilled water.
2) incubate dry paraffin section for 37 DEG C, paraffin is drawn a circle.
3) antigen retrieval：3M carbamide room temperature acts on 30min；Distilled water shakes washes 5min, three times；0.01M pH7.4PBS shake and wash
5min, three times.
4) Deca 3%H2O2Room temperature acts on 10min；Distilled water shakes washes 5min, three times；PBS shakes and washes 10min, secondary.
5) 1% bovine serum albumin of Deca, 37 DEG C of effect 30min, inclines, does not wash.
6) Deca adopts the different CMG2 monoclonal antibodies of identical dilution factor and solubility, while right as feminine gender with Mus Ig supernatants
According to 4 DEG C overnight；PBS shakes and washes 10min, three times, gets rid of liquid (tissue is sure not drying), lies against in wet box.
7) Deca be marked with HRP sheep anti mouse two resist, room temperature effect 30min；PBS shakes and washes 10min, three times, gets rid of liquid
(tissue is sure not drying), lies against in wet box.
8) Deca developer DAB working solutions, control colour developing under room temperature effect 5-30min, or light microscopic；After colour developing completely, steam
Distilled water bath color development stopping.
9) haematoxylin is redyed if necessary；Ethanol (70%-100%) dehydrations at different levels, every grade of 10min；Take out paraffin section to put
Enter 10min in dimethylbenzene, three times.
10) gummy mounting.
One group of CMG2 monoclonal antibody to preparing is dyeed to 10 parts of stomach organization paraffin sections respectively, is contrasted identical
Under concentration and the most obvious CMG2 monoclonal antibodies of dilution factor effect are considered that the CMG2 optimal with natural CMG2 protein binding is mono-
Clonal antibody.
The hybridoma cell strain of the optimal CMG2 monoclonal antibodies of present invention screening is named as 8-F3, and its preserving number is
CCTCC NO：201628, culture title：Hybridoma cell strain 8-F3 (IgG1), depositary institution are protected for Chinese Typical Representative culture
Tibetan center abbreviation CCTCC, address：Wuhan, China Wuhan University (wuchang, wuhan Luo Jia Shan Wuhan University), preservation date is
2 months 2016 No. 25.
Fig. 2 shows, identical concentration is diluted to prepared CMG2 monoclonal antibodies and 10 gastric cancer groups are contaminated respectively
Paraffin section is knitted, Color is optimal at identical conditions for the corresponding CMG2 monoclonal antibodies of 8-F3, positive rate highest.
The specificity identification of 3 CMG2 monoclonal antibodies of embodiment
1) commercial antibodies checking CMG2 recombiant proteins
The rabbit polyclonal antibody (article No. 16723-1-AP) and the rabbit multi-resistance of sigma companies of the Protech companies of purchase
The effect of CMG2 antigens prepared by (article No. SAB1400784) checking.Coating CMG2 recombiant proteins and same expression way and pure
The unrelated CYCS recombiant proteins changed：By CMG2 recombiant proteins and CYCS recombiant proteins respectively with coating diluted to 3 μ g/
Ml, ELISA batten adds 100 μ l per hole, and 4 DEG C of coatings are overnight.Next day takes out plank and abandons antigen, board-washing.Respectively by purchase
The rabbit polyclonal antibody of Protech companies and the rabbit multi-resistance of sigma companies make 1：1000,1：2000,1：4000,1：8000,1：
16000 ..., add in the bar hole of correspondence CMG2 recombiant proteins and CYCS recombiant proteins by 100 μ l/ holes, separately make blank and negative and positive
Control wells.37 DEG C of incubation 1h, board-washing add two to resist, and 1：3000HRP labelling goat anti-rabbit iggs, add correspondence bar by 100 μ l/ holes
Kong Zhong, 37 DEG C of incubation 40min.Liquid is abandoned, board-washing is patted dry, add 100 μ l/ holes of tmb substrate liquid, lucifuge colour developing 10min to add eventually
Only 50 μ l/ holes of liquid.As a result as shown in figs. 3 a-3d, Fig. 3 A are the rabbit polyclonal antibodies and CMG2 recombiant proteins of Protech companies
Reaction, bearing reaction are preferable；Fig. 3 B are the reactions of rabbit multi-resistance and the CMG2 recombiant proteins of Sigma companies, and bearing reaction is preferable；Figure
3C is the reaction of the rabbit polyclonal antibody with CYCS recombiant proteins of Protech companies, as a result reactionless；Fig. 3 D are Sigma companies
Rabbit multi-resistance and CYCS recombiant proteins reaction, it is as a result reactionless.That is, the rabbit polyclonal antibody of Protech companies and
The rabbit multi-resistance of sigma companies can preferably recognize CMG2 recombiant proteins, and not react with CYCS recombiant proteins.
2) ELISA identifies the specificity of CMG2 monoclonal antibodies
TEM8 recombiant proteins are another receptor proteins of anthrax toxin, have similar function and higher with CMG2
Albumen homology, TEM8 albumen be prepared by recombinant by the expression of identical expression way identified and whether can recombinate egg with TEM8
It is white to combine.By TEM8 recombiant proteins and CMG2 recombiant proteins respectively with coating diluted to 3 μ g/ml, the every hole of ELISA battens
100 μ l are added, 4 DEG C of coatings are overnight.Next day takes out plank and abandons antigen, board-washing.Monoclonal antibody 3B11,8-F3 is made into 1：1000,
1：2000,1：4000,1：8000,1：16000 ..., add in correspondence bar hole by 100 μ l/ holes, separately make the control of blank and negative and positive
Hole.37 DEG C of incubation 1h, board-washing add two to resist, and 1：3000HRP labelling goat anti-mouse IgGs, add correspondence bar hole by 100 μ l/ holes
In, 37 DEG C of incubation 40min.Liquid is abandoned, board-washing is patted dry, add 100 μ l/ holes of tmb substrate liquid, lucifuge colour developing 10min to add and terminate
50 μ l/ holes of liquid.As a result as shown in figs. 4 a-4d, Fig. 4 A are the reactions of 3B11 and TEM8 recombiant proteins, as a result reactionless；Fig. 4 B are
3B11 and the reaction of CMG2 recombiant proteins, as a result have and react and have dilution factor；Fig. 4 C are the corresponding CMG2 monoclonal antis of 8-F3
The reaction of body and TEM8 recombiant proteins, it is as a result reactionless；Fig. 4 D are the corresponding CMG2 monoclonal antibodies of 8-F3 and CMG2 restructuring eggs
White reaction, as a result has and reacts and have dilution factor.That is, the corresponding CMG2 monoclonal antibodies of 8-F3 can preferably recognize CMG2
Recombiant protein, and nonrecognition is with the TEM8 recombiant proteins of belt transect His labels, with preferable specificity.
3) SABC contrasts the specificity of CMG2 monoclonal antibodies
The CMG2 polyclonal antibodies of the Protech companies for selecting the external Evaluated effect of purchase optimal, using description condition
And the optimum condition that 8-F3 gropes, identical tumor tissue section is contaminated respectively, whether counterstaining situation determines its specificity
Unanimously.Concrete operations are described as follows：
(1) two parts of routines of paraffin section of gastric cancer, colon and rectum carcinoma are dewaxed to water：Paraffin section is dipped in into diformazan
10min in benzene, three times；Take out paraffin section and be placed in 10min in 100% dehydrated alcohol, it is secondary；It is each that 90%-70% is inserted successively
The each 10min of level ethanol；Taking-up is placed in 10min in distilled water.
(2) 37 DEG C incubate dry paraffin section, and paraffin is drawn a circle.
(3) antigen retrieval：3M carbamide room temperature acts on 30min；Distilled water shakes washes 5min, three times；0.01M pH7.4PBS shake
Wash 5min, three times.
(4) Deca 3%H2O2Room temperature acts on 10min；Distilled water shakes washes 5min, three times；PBS shakes and washes 10min, secondary.
(5) 1% bovine serum albumin of Deca, 37 DEG C of effect 30min, inclines, does not wash.
(6) CMG2 polyclonal antibody of the Deca using 8-F3 the and protech companies of the present invention, while with corresponding kind
IgG is negative control, and 4 DEG C overnight；PBS shakes and washes 10min, three times, gets rid of liquid (tissue is sure not drying), lies against in wet box.
(7) corresponding two anti-, the room temperature effect 30min for having HRP labellings of Deca labelling；PBS shakes and washes 10min, three times, gets rid of
Liquid (tissue is sure not drying), lies against in wet box.
(8) Deca developer DAB working solutions, control colour developing under room temperature effect 5-30min, or light microscopic；After colour developing completely, steam
Distilled water bath color development stopping.
(9) haematoxylin is redyed if necessary；Ethanol (70%-100%) dehydrations at different levels, every grade of 10min；Take out paraffin section to put
Enter 10min in dimethylbenzene, three times.
(10) gummy mounting.
As a result as indicated by figures 5 a-5b, Fig. 5 A are the negative right of the corresponding CMG2 monoclonal antibodies of 8-F3 from left to right respectively
According to and its dye gastric cancer, colon and rectum carcinoma positive findingses；Fig. 5 B are Protech companies CMG2 polyclones from left to right respectively
The negative control and its dye gastric cancer, the positive findingses of colon and rectum carcinoma of antibody；As a result show, the 8-F3 of the present invention is corresponding
CMG2 monoclonal antibodies with the Color close with the CMG2 polyclonal antibodies of Protech companies, to gastric cancer, colon cancer,
Rectal cancer histochemical staining is almost consistent, and in pigmented section and dyeing positioning, result is consistent, therefore the 8-F3 of the present invention is corresponding
CMG2 monoclonal antibodies can substitute the CMG2 polyclonal antibodies of Protech companies.Prove the CMG2 monoclonal antibodies of the present invention
With the preferably effect and specificity of identification CMG2, it is the monoclonal antibody of CMG2 specificitys.
The diagnosing tumor application of 4 CMG2 monoclonal antibodies of embodiment
The research of CMG2 early stages is primarily directed to its effect in the invasion and attack of anthrax toxin, only has pin by patent consulting
Reduce to preparing its receptor acting of monoclonal antibodies block anthrax toxin to it is artificial into damage.We pass through stem-cell research
Experiment find differential expression of the CMG2 monoclonal antibodies in the differential expression of tumor stem cell and non-tumor stem cell very
Substantially, point out which there be important effect in tumor, suppress swollen by blocking or striking low CMG2 molecules and be likely to be breached
The effect of tumor, also points out, and CMG2 monoclonal antibodies are probably the target spot of important antineoplaston.Existed by the 8-F3 of the present invention
Find in the histochemical staining of a series of tumor sample that CMG2 monoclonal antibodies have the expression popularity and specificity of tumor.It is main
Applied research and result is wanted to have following：
1) histochemical staining situation of the CMG2 monoclonal antibodies in same tumor
The First Affiliated Hospital of Third Military Medical University of PLA Pathology Deparment is preserved with the 8-F3 of the present invention and (be purchased from
The First Affiliated Hospital of Third Military Medical University of PLA) 200 gastric cancer and breast cancer tissue's paraffin section adopt phase
Same testing conditions carry out histochemical staining, analyze the expression of the CMG2 monoclonal antibodies in same tumor.8-F3 is diluted to
Optimal concentration, contaminates 200 gastric cancer and breast carcinoma sample respectively, and as a result as shown in Fig. 6 A-6F, wherein Fig. 6 A-6C are 8-F3 pair
There are different expression types in the CMG2 monoclonal antibodies answered in gastric cancer, Fig. 6 D-6F are the corresponding CMG2 monoclonal antis of 8-F3
There are different expression types in breast carcinoma in body.Fig. 6 A and Fig. 6 D show
Sample is expressed in strong positive, and Fig. 6 B and Fig. 6 E show part gastric cancer and breast carcinoma sample positive expression, and Fig. 6 C and Fig. 6 F shows
It is shown with part gastric cancer and breast carcinoma sample is not expressed substantially.As a result display that positive strong and weak classification and classification with tumor without substantially
Relation, points out CMG2 monoclonal antibodies to be probably independent tumor markerses.
2) histochemical staining situation of the CMG2 monoclonal antibodies in other tumors
With the tumor tissue array (multiple organ, OD-CT-Com04-001) of the Xin Chao companies of purchase, comprising multiple organ cancer 7
Plant, wherein the esophageal carcinoma 5, gastric cancer 6, colon cancer 5, hepatocarcinoma 6, pulmonary carcinoma 6, breast carcinoma 6, renal carcinoma 6, and it is various
7 by cancer.Histochemical staining is carried out to chip with 8-F3, its expression in other tumors is seen.8-F3 is diluted to optimal
Concentration, contaminates tumor tissue array, has different degrees of expression in having different tumors in 8-F3 group results, and Partial tumors are positive by force
Property expression, Partial tumors expression is general, while there is situation about not expressing, but at identical conditions, in pulmonary carcinoma, renal carcinoma, breast
There is sample to express in strong positive in adenocarcinoma, the esophageal carcinoma, as a result as shown in fig. 7, result shows that CMG2 monoclonal antibodies can be used
In the diagnosis and staging, the diagnosis of typing of tumor.
3) CMG2 monoclonal antibodies histochemical staining situation in the normal tissue
With the normal structure chip (multiple organ, OD-NH-Com01-001) of the Xin Chao companies of purchase, comprising it is many it is personal just
Often organize, including brain, cerebellum, brain stem, tongue, thyroid, esophaguses, stomach, intestinal, liver, pancreas, lung, trachea, vermiform appendix, skin, flesh
23 kinds of tissues such as meat, striped muscle, Zuo Xin, the right heart, aorta, testis, bladder, prostate, carry out a group change dye with 8-F3 to chip
Color, sees its expression in organizing more than.8-F3 is diluted to optimal concentration, contaminates normal structure chip, as a result such as Fig. 8 institutes
Show, its in the normal tissue histochemical staining be negative substantially, show specificity of the CMG2 monoclonal antibodies in lesion detection.8-
In F3 group results in addition to portion of tissue easily non-specific coloring, almost do not dye in the tissue of remaining normal person, carry
Show that expression of the 8-F3 to CMG2 under pathologic condition has to combine, with preferable tumour-specific, be for cmg2 positive tumors
Good target therapeutic agent or pharmaceutical carrier.
Embodiment described above only expresses embodiments of the present invention, and its description is more concrete and detailed, but can not
Therefore it is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art,
Without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the protection model of the present invention
Enclose.
Claims (10)
1. 2 monoclonal antibody of a kind of anthrax toxin acceptor, it is characterised in that be CCTCC by preserving number：The hybridoma of C201628
It is prepared by cell strain.
2. 2 monoclonal antibody of anthrax toxin acceptor according to claim 1, it is characterised in that the anthrax toxin acceptor 2
Monoclonal antibody is the monoclonal antibody of CMG2 specificitys.
3. the preparation method of 2 monoclonal antibody of a kind of anthrax toxin acceptor, it is characterised in that comprise the steps of：
1) extracellular region protein is recombinated as immunogen immune Balb/c mice with the CMG2 of purification；
2) by hybridoma technology by many times of the inguinal lymph node cells of the Balb/c mices and myeloma cell SP2/0 Jing
Cell fusion, carries out screening positive clone using indirect elisa method, and cloning is filtered out can stably excreting CMG2 monoclonal antibodies
Hybridoma cell strain.
4. the preparation method of 2 monoclonal antibody of anthrax toxin acceptor according to claim 3, it is characterised in that the step
The Balb/c mices in rapid 1 are 8 week old, respectively with Freund's complete adjuvant and incomplete Freund's adjuvant, point first immunisation and
The antigen of the vola of the 9th day Balb/c mice immune twice, the Freund's complete adjuvant and the incomplete Freund's adjuvant
Dosage is 100 micrograms.
5. the preparation method of 2 monoclonal antibody of anthrax toxin acceptor according to claim 3, it is characterised in that the step
It is rapid 2) to include：Inguinal lymph nodes separation cell is mixed with myeloma cell, with Macrogol 1450 as fusion agent, formation is melted
Cell is closed, fused cell is suspended from the HAT culture fluid containing calf serum, juxtaposition CO2In 37 DEG C cultivate；With it is described indirectly
ELISA method is screened, and during screening, is screened with CMG2 restructuring extracellular region protein coatings, and the positive colony hole to detecting is adopted
Cell-cloned is carried out with limiting dilution assay, CO is placed in2In in 37 DEG C cultivate, until all cell growth holes culture fluid it is equal
Till being positive.
6. a kind of screening technique of anthrax toxin acceptor 2 monoclonal antibody optimal with 2 protein binding of natural anthrax toxin acceptor,
Characterized in that, comprising the steps of：
1) 10 stomach organization paraffin sections are taken, the paraffin section is routinely dewaxed to water：The paraffin section is dipped in into two
10min in toluene, three times；Take out the paraffin section and be placed in 10min in 100% dehydrated alcohol, it is secondary；90%- is inserted successively
The each 10min of 70% ethanol at different levels；Taking-up is placed in 10min in distilled water；
2) incubate the dry paraffin section for 37 DEG C, paraffin is drawn a circle；
3) antigen retrieval：3M carbamide room temperature acts on 30min；Distilled water shakes washes 5min, three times；0.0lMpH7.4PBS shakes and washes 5min,
Three times；
4) Deca 3%H2O2Room temperature acts on 10min；Distilled water shakes washes 5min, three times；PBS shakes and washes 10min, secondary；
5) 1% bovine serum albumin of Deca, 37 DEG C of effect 30min, inclines, does not wash；
6) Deca adopts the different CMG2 monoclonal antibodies of identical dilution factor and solubility, while with Mus Ig supernatants as negative control, 4
DEG C overnight；PBS shakes and washes 10min, three times, gets rid of liquid (tissue is sure not drying), lies against in wet box；
7) Deca be marked with HRP sheep anti mouse two resist, room temperature effect 30min；PBS shakes and washes 10min, three times, gets rid of liquid (tissue
It is sure not drying), lie against in wet box；
8) Deca developer DAB working solutions, control colour developing under room temperature effect 5-30min, or light microscopic；After colour developing completely, distilled water
Bath color development stopping；
9) ethanol (70%-100%) dehydrations at different levels, every grade of 10min；Take out the paraffin section and insert 10min in dimethylbenzene, three
It is secondary；
10) gummy mounting.
7. anthrax toxin acceptor 2 monoclonal optimal with 2 protein binding of natural anthrax toxin acceptor according to claim 6
The screening technique of antibody, it is characterised in that step 9) in dehydration before if necessary haematoxylin redye.
8. 2 monoclonal antibody of anthrax toxin acceptor is in tumour diagnostic reagent and staging, the diagnostic reagent of typing is prepared
Using.
9. 2 monoclonal antibody of anthrax toxin acceptor treats answering for the medicine or pharmaceutical carrier of CMG2 positive tumors in preparation
With.
10. application according to claim 9, it is characterised in that：The tumor includes gastric cancer, colon and rectum carcinoma, esophaguses
Cancer, hepatocarcinoma, pulmonary carcinoma, renal carcinoma and breast carcinoma.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1717417A (en) * | 2001-10-26 | 2006-01-04 | 行星生物技术有限公司 | Novel immunoadhesins for treating and prventing toxicity and pathogen-mediated diseases |
| WO2008140483A2 (en) * | 2006-11-09 | 2008-11-20 | Human Genome Sciences, Inc. | Methods and antibodies for detecting protective antigen |
| US7601351B1 (en) * | 2002-06-26 | 2009-10-13 | Human Genome Sciences, Inc. | Antibodies against protective antigen |
-
2016
- 2016-10-21 CN CN201610919910.9A patent/CN106554418B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1717417A (en) * | 2001-10-26 | 2006-01-04 | 行星生物技术有限公司 | Novel immunoadhesins for treating and prventing toxicity and pathogen-mediated diseases |
| US7601351B1 (en) * | 2002-06-26 | 2009-10-13 | Human Genome Sciences, Inc. | Antibodies against protective antigen |
| WO2008140483A2 (en) * | 2006-11-09 | 2008-11-20 | Human Genome Sciences, Inc. | Methods and antibodies for detecting protective antigen |
Non-Patent Citations (2)
| Title |
|---|
| 屈野: "炭疽毒素受体的组织分布和活性位点的结构与功能研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 屈野等: "炭疽毒素受体单克隆抗体的制备、鉴定及结合位点分析", 《武警医学院学报》 * |
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