CN107748259A - A kind of ELISA detection method of FcRn acceptors - Google Patents
A kind of ELISA detection method of FcRn acceptors Download PDFInfo
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- CN107748259A CN107748259A CN201710616108.7A CN201710616108A CN107748259A CN 107748259 A CN107748259 A CN 107748259A CN 201710616108 A CN201710616108 A CN 201710616108A CN 107748259 A CN107748259 A CN 107748259A
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- fcrn
- detection method
- coated
- vegf mab
- elisa
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
A kind of ELISA detection method of claimed FcRn acceptors, its be included on ELISA Plate be coated with FcRn coating step, the step of closing, sample preparation, secondary antibody are incubated, substrate colour developing, wherein, the sample preparation step is：After anti-VEGF mAb mixes with antigen, a hour formation immune complex is stood, is separately additionally added heparin.Separately in step is coated with, the mould Avidin of the company of being additionally added is coated with buffer solution to ELISA Plate.Using detection method can in vitro it is quick, directly detect anti-VEGF mAb and FcRn receptor-binding activities, and cost is relatively low, convenient experimental operation, reproducible, as a result judges objective, high sensitivity.Conformance Assessment and clinical drug effect and pharmacokinetic for anti-VEGF mAb also provide reference information.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of ELISA detection method of FcRn acceptors.
Background technology
VEGF (vascular endothelial growth factor, VEGF), is blood vessel endothelium
The HBGF of cell-specific, can in vivo induction of vascular it is newborn.VEGF also may be used by different tumor cell secretions
Developmental normal cell and tissue in express, in vivo adjust blood vessel permeability, be vascularization during it is a variety of
Cell provides a network of fibers；The degraded of matrix and propagation, migration, motion and the blood vessel of endothelial cell can be promoted in vitro
The formation of chamber spline structure, and the endothelial progenitor cells of derived from bone marrow can be mobilized.VEGF be the effect of induced tumor vascularization it is most strong,
Specific highest angiogenesis factor.So in theory, employ different approach to intervene or suppress vascular endothelial growth
The factor can suppress the growth of tumour.
Anti-VEGF mAb be using recombinant DNA technology by Chinese hamster ovary (Chinese hamster ovary,
CHO) the recombinant humanized monoclonal IgG1 antibody of cell expression system production, includes 93% Human monoclonal antibody sequence and 7%
People VEGF mouse source monoclonal antibody complementary determining region sequence can be combined, heavy chain is made up of 453 amino acid, and light chain is by 214 amino acid
Composition, molecular weight is about 149kDa.Therapeutic monoclonal antibodies are widely used in tumour, autoimmune disease, infectivity
Disease etc. is treated.For monoclonal antibody in addition to having the characteristics that specific height, adverse reaction are small and mechanism of action is clear and definite, long half time is also it
Unique advantage.Current therapeutic monoclonal antibody is mostly immunoglobulin G (immunoglobulin G, IgG), and it in vivo half
The phase decline mainly by the influence of Fc sections and neonatal Fc receptor (neonatal Fc receptor, FcRn) affinity, affinity drop
Low or raising can cause the shortening or extension of monoclonal antibody Half-life in vivo, can be predicted by IgG-FcRn affinity inside IgG
Half-life period.Therefore, the affinity for determining monoclonal antibody and FcRn can be as the indirect indexes of evaluation monoclonal antibody pharmacokinetics.In recent years
Come, the similar medicine of monoclonal antibody biology and the transformation of Fc sections and modification class monoclonal antibody continue to bring out, and it is affine with FcRn to evaluate such monoclonal antibody medicine
The important indicator for being changed as guide product research and development medicine comparative study similar with biology of power.IgG and FcRn combination is presented
The characteristics of pH is relied on, under the low ph condition in lysosome (pH 5.5-6.5), IgG is combined with FcRn makes IgG from degrading,
After IgG-FcRn compounds return to cell surface, under the conditions of neutral body fluid, adhesion is reduced, and IgG is released back into body fluid.
In recent years, biomembrane interference technique (biolayerinterferomeory, BLI), biacore technologies etc. are used for
The measure of FcRn receptor-binding activities, although simple and efficient, expensive large scale equipment is needed, is unfavorable for routine experimentation
The tracking and monitoring of room, the technical research of small-sized biological company and product quality.And enzyme-linked immunosorbent assay (ELISA) side
Method, because its is simple to operate, quick, sensitiveness is high, high specificity, equipment requirement are simple, therefore in laboratory extensive use.
The content of the invention
The technical problems to be solved by the invention are innovative utilization sandwich ELISA method detection anti-vegf lists of the invention
The anti-binding ability with FcRn acceptors, make antibody samples simpler easy with the detection of the binding activity of FcRn acceptors in vitro.
In order to solve the above-mentioned technical problem, technical scheme provided by the invention is that one kind ELISA method detects anti-vegf
The binding activity of monoclonal antibody and FcRn acceptors, i.e., a kind of ELISA detection method of FcRn acceptors, it, which is included on ELISA Plate, is coated with
The step of coating step of FcRn acceptors, closing, sample preparation, secondary antibody incubation, substrate develop the color, and the sample preparation step
For：After anti-VEGF mAb mixes with antigen, a hour formation immune complex is stood.
In currently preferred technical scheme, the mol ratio of anti-VEGF mAb and antigen is 0.1~10:Between 1；It is preferred that
Ground, the mol ratio of the anti-VEGF mAb and antigen is 0.5~5:Between 1, it is highly preferred that mole of anti-VEGF mAb and antigen
Than 1~3:Between 1.
In currently preferred technical scheme, in the sample preparation step, heparin is additionally added, and the heparin added is anti-
Final concentration >=0.05U/mL of system is answered, the final concentration of 0.05U/mL~5.00U/mL of heparin preferably added；It is highly preferred that
Final concentration of 0.05U/mL~the 0.50U/mL of heparin of addition.With this, during the course of the reaction, the super of Ag-Ab-heparin is formed
Level compound, adds acceptor affinity.
In currently preferred technical scheme, in step is coated with, the mould Avidin of the company of being additionally added is coated with buffer solution to enzyme mark
Plate, it is preferable that even mould Avidin concentration is 0.1~10 μ g/ml.In the present invention, pass through the mould Avidin of the company of addition, by increasing capacitance it is possible to increase by
Body is coated with treating capacity, i.e. increase coating acceptor density.
In currently preferred technical scheme, described immune complex is subjected to gradient dilution, addition has been coated with FcRn
ELISA Plate on be incubated.
In currently preferred technical scheme, the anti-VEGF mAb is Avastin (Avastin).
In currently preferred technical scheme, the molecular formula and molecular weight of the anti-VEGF mAb are：
C6638H10160N1720O2108S44149kDa, its light-chain amino acid sequence are as follows：
DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS
RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQGTKVEIKRTV AAPSVFIFPP SDEQLKSGTA
SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFN RGEC；
The amino acid sequence of heavy chain is：
EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY
AADFKRRFTF SLDTSKSTAY LQMNSLRAEDTAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSSASTKGPS
VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVPSSSLG
TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV
DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK
TISKAKGQPR EPQVYTLPPS REEMTKNQVSLTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF
FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK。
The anti-VEGF antibody FcRn receptor-binding activity detection methods of the present invention are to be based on ELISA method, utilize parallel song
Line (four parameter curves) model determination relative activity.Using detection method can in vitro it is quick, directly detect to resist
VEGF monoclonal antibody and FcRn receptor-binding activities, and cost is relatively low, convenient experimental operation, it is reproducible, as a result judge objective, sensitive
Degree is high.Conformance Assessment and clinical drug effect and pharmacokinetic for anti-VEGF mAb also provide reference information.
Brief description of the drawings
Fig. 1 is FcRn acceptors and anti-VEGF mAb binding activity detection method detection principle diagram.
Fig. 2 is the FcRn and anti-VEGF mAb binding curve figure of embodiment 1.It can be seen that using the side for adding heparin
Method, parameter curve can reach upper mounting plate very well, and the detection method of prior art is unable to reach.
Fig. 3 is the FcRn and anti-VEGF mAb binding curve figure of embodiment 2.It can be seen that using even mould Avidin bag
By the method for buffer solution, parameter curve can reach upper mounting plate very well, and known detection method is to be unable to reach this technology effect
Fruit.
Fig. 4 A are the light-chain amino acid sequence figure of anti-VEGF mAb；Fig. 4 B are the heavy chain amino acid sequence of anti-VEGF mAb
Figure.
Embodiment
Such scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are to be used to illustrate
The present invention and be not limited to limit the scope of the present invention.The implementation condition used in embodiment can be done according to the condition of specific producer
Further adjustment, unreceipted implementation condition is usually the condition in normal experiment.
Introduce and summarize
The present invention by way of example rather than provides the mode of limitation to illustrate.It should be noted that in present disclosure
Described " one " or " one kind " embodiment is not necessarily referring to same embodiment, and refers at least a kind of.
Various aspects of the invention are described below.However, as will be readily apparent to one of skill in the art, can
Implement the present invention according to the only some or all of aspects of the present invention.For purposes of illustration, provide herein specific numbering, material and
Configuration, enables one to thoroughly understand the present invention.However, be evident that for those of skill in the art,
The present invention can be implemented without concrete details.In other examples, not make the present invention is obscure many institutes have been omitted or simplified
Known feature.
Various operations are described successively as multiple discrete steps, and with most helpful in the side for understanding the present invention
Formula illustrates；However, in-order description should not be construed as to imply that these operations are necessarily dependent on order.
Reactant according to type species is illustrated to various embodiments.To show for those of skill in the art and
It is clear to, any number of different types of reactant can be used to implement for the present invention, and be more than those for the purpose of illustration
And the reactant provided herein.In addition, also it is evident that, the invention is not limited in any specific mixing is shown
Example.
Experiment material and equipment
Basic model eddy mixer, ELIASA (producer：Molecular Devices, model：Spectra Max M2e)、
ELISA Plate (is purchased from：Corning), microwell plate constant temperature oscillator, board-washing machine (are purchased from：Thermo)
Experiment reagent：All it is ordinary commercial products
Phosphate buffer (PBS solution), sample diluting liquid/washing lotion, confining liquid, nitrite ion TMB, terminate liquid (1M sulphur
Acid), antigen rhVEGF165, FcRn, heparin, even mould Avidin
Embodiment 1：By taking single sample as an example, illustrative step is as follows：
Standard items dilute with sample：
(1) Avastin and reference material are diluted to 2mg/ml concentration respectively with sample diluting liquid.
(2) 1.5mg/ml sample is taken, concentration is added thereto and is 0.3mg/ml VEGF, and adds sample diluting liquid,
Heparin of the final concentration no less than 0.05U/mL is stored at room temperature 1~2 hour after mixing.
(3) with sample diluting liquid by above-mentioned 8~11 concentration gradients of mixed liquor gradient dilution.
Experimental procedure：
(1) each hole of ELISA Plate is added with the μ g/ml of 100 μ l 3 FcRn coating buffer solutions；After being sealed with sealed membrane, 2- is placed in
It is incubated 16 hours under the conditions of 8 DEG C.
(2) after taking-up ELISA Plate discards coating buffer, 300 μ l washing lotions (wash buffer) board-washings 2 are added per hole with board-washing machine
Time, plate is patted dry on filter paper.
(3) 100 μ l confining liquids are added in each hole, after being sealed with sealed membrane, 200rpm is in micropore at ambient temperature
Shaking is incubated 1~2h in plate oscillator.
(4) microwell plate is removed from micropore plate oscillator, topples over content, patted dry.Add 300 μ l washing lotions per hole with board-washing machine
(wash buffer) is cleaned, and is patted dry, is repeated 2 times.
(5) for the microwell plate being coated with, each 100 μ l of need testing solution of gradient dilution are added in working hole respectively,
After being sealed with sealed membrane, 200rpm shakings at ambient temperature are incubated 2h.
(6) microwell plate is removed from micropore plate oscillator, topples over content, patted dry, add 300 μ l washing lotions per hole with board-washing machine
(wash buffer) is cleaned, and is patted dry, is repeated 4 times.
(7) the ELIAS secondary antibody dilutions of 100 μ l 1000~5000 times of dilutions are added in every hole, after being sealed with sealed membrane,
200rpm shakings at ambient temperature are incubated 60 minutes.
(8) microwell plate is removed from micropore plate oscillator, topples over content, patted dry, add 300 μ l washing lotions per hole with board-washing machine
(wash buffer) is cleaned, and is patted dry, is repeated 4 times.
(9) 100 μ l TMB nitrite ions, sealing are added in each working hole respectively, room temperature lucifuge is incubated 20~30 minutes, work
Make hole and blueness occur.
(10) add 100 μ l terminate liquid respectively per hole, rap microwell plate mixing, enzyme reaction terminates, and originally shows the hole of blueness
Will yellowing.
(11) in terminating reaction half an hour, light absorption value is surveyed in 450nm wavelength, produces the dosage of standard liquid and sample solution
Effect curve.
Embodiment 2：
Standard items dilute with sample：
(1) Avastin and reference material are diluted to 3 μ g/ml concentration respectively with sample diluting liquid pH5.8-6.0.
(2) 2 μ g/ml sample is taken, adds the VEGF that concentration is 0.3mg/ml thereto165, TAB008 samples are made：
VEGF mol ratio is 2:Mixed liquor between 1, it is stored at room temperature after mixing 1 hour.
(3) by above-mentioned 8~11 concentration gradients of mixed liquor gradient dilution.
Experimental procedure：
(1) each hole of ELISA Plate is added with the μ g/ml of the 100 μ l 0.5 mould Avidin coating buffer solution of company；Sealed with sealed membrane
Afterwards, 16-20 hours are incubated under the conditions of being placed in 2-8 DEG C.
(2) after taking-up ELISA Plate discards coating buffer, 300 μ l washing lotions (wash buffer) board-washings two are added per hole with board-washing machine
Time, plate is patted dry on filter paper.
(3) 100 μ l confining liquids are added in each hole, after being sealed with sealed membrane, 200rpm is in micropore at ambient temperature
Shaking is incubated 1~2h in plate oscillator.
(4) microwell plate is removed from micropore plate oscillator, topples over content, patted dry, add 300 μ l washing lotions per hole with board-washing machine
(wash buffer) is cleaned, and is patted dry, is repeated 2 times.
(5) the μ g/ml of 100 μ l0.5~2 biotinylation FcRn is added in each hole, after being sealed with sealed membrane, in room temperature
Under the conditions of 200rpm shaken in micropore plate oscillator be incubated 1~2h.
(6) microwell plate is removed from micropore plate oscillator, topples over content, patted dry, add 300 μ per hole with board-washing machine
LpH5.8-6.0 washing lotions (wash buffer) are cleaned, and are patted dry, are repeated 2 times.
(7) for the microwell plate being coated with, 3 times of gradient dilution need testing solution pH5.8- are added in working hole respectively
6.0 each 100 μ l.After being sealed with sealed membrane, 200rpm shakings at ambient temperature are incubated 1~2h.
(8) microwell plate is removed from micropore plate oscillator, topples over content, patted dry, add 300 μ per hole with board-washing machine
LpH5.8-6.0 washing lotions (wash buffer) are cleaned, and are patted dry, are repeated 4 times.
(9) the ELIAS secondary antibody dilution pH5.8-6.0 of 100 μ l 1000~5000 times of dilutions are added in every hole, with sealing
After film sealing, 200rpm shakings at ambient temperature are incubated 1~2h.
(10) microwell plate is removed from micropore plate oscillator, topples over content, patted dry, add 300 μ per hole with board-washing machine
LpH5.8-6.0 washing lotions (wash buffer) are cleaned, and are patted dry, are repeated 4 times.
(11) 100 μ l TMB nitrite ions, sealing are added in each working hole respectively, room temperature lucifuge is incubated 20~30 minutes, work
Make hole and blueness occur.
(12) add 100 μ l terminate liquid respectively per hole, rap microwell plate mixing, enzyme reaction terminates, and originally shows the hole of blueness
Will yellowing.
(13) in terminating reaction half an hour, light absorption value is surveyed in 450nm wavelength, produces the dosage of standard liquid and sample solution
Effect curve.
Specific embodiment described above is only the preferred embodiment of the present invention, it is noted that for the art
For those of ordinary skill, under the premise without departing from the principles of the invention, some improvement or replacement can also be made, these improvement
Or replace and should also be as being considered as protection scope of the present invention.
Claims (6)
1. a kind of ELISA detection method of FcRn acceptors, it is characterised in that it is included in the coating step that FcRn is coated with ELISA Plate
Suddenly, the step of closing, sample preparation, secondary antibody incubation, substrate colour developing, wherein, the sample preparation step is：Anti-VEGF mAb with
After antigen mixes, a hour formation immune complex is stood.
2. detection method according to claim 1, it is characterised in that in the sample preparation step, heparin is additionally added, and
Add final concentration of 0.05U/mL~5.00U/mL of heparin.
3. detection method according to claim 1 or 2, it is characterised in that in step is coated with, the mould Avidin of the company of being additionally added
Buffer solution is coated with to ELISA Plate.
4. detection method according to claim 1, it is characterised in that the mol ratio of the anti-VEGF mAb and antigen exists
0.1~10:Between 1.
5. detection method according to claim 1, it is characterised in that described immune complex is subjected to gradient dilution,
Addition, which has been coated with FcRn ELISA Plate, to be incubated.
6. detection method according to claim 1, it is characterised in that the anti-VEGF mAb is Avastin.
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Citations (6)
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| CN101268372A (en) * | 2005-07-21 | 2008-09-17 | 根马布股份公司 | Potency assays for antibody drug substance binding to FC receptor |
| US20130315907A1 (en) * | 2012-05-22 | 2013-11-28 | Regeneron Pharmaceuticals, Inc. | Vegf-a121 assay |
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| WO2017048699A2 (en) * | 2015-09-14 | 2017-03-23 | Galaxy Biotech Llc | Highly potent monoclonal antibodies to angiogenic factors |
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2017
- 2017-07-26 CN CN201710616108.7A patent/CN107748259A/en active Pending
Patent Citations (6)
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|---|---|---|---|---|
| CN1763097A (en) * | 1999-01-15 | 2006-04-26 | 杰南技术公司 | Polypeptide variants with altered effector function |
| US20060134709A1 (en) * | 2004-11-10 | 2006-06-22 | Jeffery Stavenhagen | Engineering Fc antibody regions to confer effector function |
| CN101268372A (en) * | 2005-07-21 | 2008-09-17 | 根马布股份公司 | Potency assays for antibody drug substance binding to FC receptor |
| CN104447990A (en) * | 2008-10-14 | 2015-03-25 | 霍夫曼-拉罗奇有限公司 | Immunoglobulin variants and uses thereof |
| US20130315907A1 (en) * | 2012-05-22 | 2013-11-28 | Regeneron Pharmaceuticals, Inc. | Vegf-a121 assay |
| WO2017048699A2 (en) * | 2015-09-14 | 2017-03-23 | Galaxy Biotech Llc | Highly potent monoclonal antibodies to angiogenic factors |
Non-Patent Citations (3)
| Title |
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| ADAM WALKER ET AL: "Novel Interaction Mechanism of a Domain Antibody-based Inhibitor of Human Vascular Endothelial Growth Factor with Greater Potency than Ranibizumab and Bevacizumab and Improved Capacity over Aflibercept", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| DOUGLAS A. MACDONALD ET AL: "Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes", 《ANGIOGENESIS》 * |
| ROBERT L.SHIELDS, ET AL.: "High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR.", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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Application publication date: 20180302 |