Anti-Human VEGF Therapeutic Antibody Fab Fragment (Ranibizumab) (CAT#: TAB-012-F(E))

Recombinant monoclonal antibody Fragment Fab to Human VEGF. Ranibizumab (trade name ranibizumab) is a monoclonal antibody fragment (Fab) derived from the same parent mouse antibody as bevacizumab (bevacizumab). It is much smaller than the parent molecule and has been affinity matured to provide stronger binding to VEGF-A. It is an anti-angiogenic that has been approved to treat the "wet" type of age-related macular degeneration (AMD, also ARMD), a common form of age-related vision loss.

  • Published Data
  • Datasheet
  • MSDS
  • COA

Figure 1 The effects of VEGF Trap, ranibizumab and bevacizumab on luciferase activation induced by VEGF-A121 and VEGF-A165 in HEK293/VEGFR2 cells.

A Dose response curves for VEGF-A121 and VEGF-A165 with EC50 values of 70 and 30 pM, respectively. PlGF-2 was not active in this assay. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HEK293/VEGFR2 cells along with 20 pM of VEGF-A121. c Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HEK293/VEGFR2 cells along with 20 pM of VEGF-A165. The cells were incubated for 6 h and OneGlo luciferase substrate was then added to each well. The plates were read on a luminometer and the data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents a replica of 3 wells at each concentration.

Papadopoulos, N., Martin, J., Ruan, Q., Rafique, A., Rosconi, M. P., Shi, E., ... & Wiegand, S. J. (2012). Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis, 15(2), 171-185.

Figure 2 The effects of VEGF Trap, ranibizumab and bevacizumab on HUVEC migration.

A HUVEC were placed in the upper compartment of the Boyden chamber and allowed to migrate towards basal media containing 0.1% fetal bovine serum with or without VEGF-A165 or VEGF-A165 mixed with four concentrations each of VEGF Trap (circles, solid line), ranibizumab (triangles, dotted line) or bevacizumab (squares, dashed line) ranging from 0.013 to 13 nM. The percentage of total migration (y-axis) was calculated as (F Drug − F Basal)/(F Total− F Basal) × 100; where F Total is fluorescence in the presence of VEGF-A165, F Basal is fluorescence in the absence of VEGF-A165, and F Drug is fluorescence in the presence of VEGF-A165 mixed with drug at a specific molar ratio (x-axis). b HUVEC migration was assessed in the absence and presence of human PLGF-2 (hPLGF-2) or mouse PLGF-2 (mPLGF-2) with and without a 100-fold molar excess of VEGF Trap (VGT), ranibizumab (RAN) or bevacizumab (BEV). Fold migration (y-axis) was calculated as the ratio F/F Basal; where F is the total fluorescence measured for the indicated condition (x-axis) and F Basal is the fluorescence in the absence of either hPLGF-2 or mPLGF-2. Statistical significance: *P < 0.05; **P < 0.01; ns, no significance. Values and error bars represent the average value and standard error of the mean from at least three independent experiments with each experiment containing four biological replicates per condition (total n = 12–16 per condition) for all conditions tested. AU arbitrary units.

Papadopoulos, N., Martin, J., Ruan, Q., Rafique, A., Rosconi, M. P., Shi, E., ... & Wiegand, S. J. (2012). Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis, 15(2), 171-185.

Figure 3 Kinetic binding parameters for VEGF Trap, ranibizumab and bevacizumab binding to human VEGF family ligands determined by SPR-Biacore.

Numbers in parentheses represent the standard error of the kinetic fit; NB No binding under assay conditions used; aVEGF inhibitor captured on a Protein A-coupled sensor chip; bVEGF inhibitor captured on an anti-human Fab polyclonal antibody-captured sensor chip.

Papadopoulos, N., Martin, J., Ruan, Q., Rafique, A., Rosconi, M. P., Shi, E., ... & Wiegand, S. J. (2012). Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab. Angiogenesis, 15(2), 171-185.

Figure 4 Positive binding capacity of ranibizumab to rat VEGF-A.

A. To verify the binding of ranibizumab to rat VEGF-A a protein dot blot was used. Rat VEGF-A was spotted on a nitrocellulose coated-glass slide. Human VEGF-A, murine VEGF-A, ranibizumab, bevacizumab as well as the fluorophore-coupled anti-IgG secondary antibody served as positive control. Before incubation with ranibizumab, the dot blot was scanned to exclude possible unspecific fluorescence signals. As expected, a fluorescence signal was only detected for fluorophore-coupled secondary antibody. B. After incubation with ranibizumab and incubation with the secondary anti-IgG antibody, fluorescence signals were detected for all spots in comparison to the pre-scan. A positive binding of ranibizumab to rat VEGF-A and not only to human VEGF-A is proven.

Joachim, S. C., Renner, M., Reinhard, J., Theiss, C., May, C., Lohmann, S., ... & Dick, H. B. (2017). Protective effects on the retina after ranibizumab treatment in an ischemia model. PloS one, 12(8), e0182407.

Figure 5 Strong influence of ischemia-reperfusion on VEGF level and retinal layers.

A. Aqueous humor VEGF levels in control animals, ischemic animals, and animals treated with ranibizumab. Aqueous humor samples were collected 21 days after ischemia. A significant increase in VEGF was observed in ischemic animals compared to controls (p = 0.032). The ranibizumab and the control groups had comparable VEGF levels (p = 0.99). B. Exemplary H&E stained retinal cross-sections from a control, an ischemia and a ranibizumab animal with cut outs focused on the GCL. C. A significantly reduced thickness of
the total retina was detected in ischemic retinas compared to controls (p<0.001). Ranibizumab treated eyes showed similar reduction in retinal thickness like the ischemic eyes (p = 0.6). D. The measurements for the thickness of the GCL were comparable to the ones for the total retina. In ischemic retinas, a significant GCL reduction was noted in comparison to the control group (p<0.001). No significant differences could be detected between ischemic and ranibizumab treated eyes (p = 0.189). *: p< 0.05. Abbreviations: GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer; PR = photoreceptor layer. Scale bar: 50 μm (cut outs 10 μm).

Joachim, S. C., Renner, M., Reinhard, J., Theiss, C., May, C., Lohmann, S., ... & Dick, H. B. (2017). Protective effects on the retina after ranibizumab treatment in an ischemia model. PloS one, 12(8), e0182407.

Figure 6 Lower expression of macroglia and VEGF-receptor 2 after ischemiareperfusion with ranibizumab treatment.

Exemplary retinal sections labelled with anti-GFAP (red) and anti-VEGF-R2 (green).

Joachim, S. C., Renner, M., Reinhard, J., Theiss, C., May, C., Lohmann, S., ... & Dick, H. B. (2017). Protective effects on the retina after ranibizumab treatment in an ischemia model. PloS one, 12(8), e0182407.

Figure 7 Lower expression of macroglia and VEGF-receptor 2 after ischemiareperfusion with ranibizumab treatment.

GFAP+ area was significantly increased in ischemic retinas (p<0.001), while it was less prominent in ranibizumab ones (p = 0.1).

Joachim, S. C., Renner, M., Reinhard, J., Theiss, C., May, C., Lohmann, S., ... & Dick, H. B. (2017). Protective effects on the retina after ranibizumab treatment in an ischemia model. PloS one, 12(8), e0182407.

Figure 8 ELISA result of the ranibizumab.

Ranibizumab concentrations in the aqueous humor of nonvitrectomized eyes and vitrectomized eyes after intravitreal injection.

Niwa, Y., Kakinoki, M., Sawada, T., Wang, X., & Ohji, M. (2015). Ranibizumab and aflibercept: intraocular pharmacokinetics and their effects on aqueous VEGF level in vitrectomized and nonvitrectomized macaque eyes. Investigative ophthalmology & visual science, 56(11), 6501-6505.


Specifications

  • Immunogen
  • Purified intact human VEGF
  • Host Species
  • Mouse
  • Derivation
  • Humanized (from mouse)
  • Type
  • Fab - G1 - kappa
  • Specificity
  • Tested positive against native human antigen
  • Species Reactivity
  • Human
  • Applications
  • IP, IF, FuncS, FC, Neut, ELISA, IHC
  • Trade name
  • ranibizumab
  • CAS
  • 347396-82-1
  • Generic Name
  • Ranibizumab
  • Biological Half-Life
  • Approx 9 days
  • ATC Code
  • S01LA04
  • DrugBank
  • DB01270
  • UNII
  • ZL1R02VT79
  • ChEMBL
  • CHEMBL1201825
  • MW
  • 48350 g/mol
  • Related Disease
  • Diabetic retinopathy

Applications

  • Application Notes
  • The VEGFA antibody has been reported in applications of Activ, Migration, SPR, Dot, H&E staining, IHC, RT-PCR, ELISA.
    Migration: An aliquot of resuspended cells (250 μl; ~50,000 cells/well) was placed in the upper well of the ECM plate, and MBM (750 μl) with or without ligand (130 pM human VEGF-A165, 7.1 nM human PLGF-2, or 3.5 nM mouse PLGF-2), was mixed with VEGF Trap, bevacizumab, or ranibizumab (inhibitor concentration range 0.013–13 nM) and placed in the lower well following a 1 h incubation of the mixture at room temperature. The ECM plate was incubated for 18–20 h in a 37°C/5% CO2 incubator to allow cells from the upper well to migrate through the FluoroBlok™ membrane towards the lower well. Following migration, cells attached to the underside of the FluoroBlok™ membrane were stained with 500 μL of a 2 μg/mL solution of the fluorescent dye Calcein AM for 1.5 h in a 37°C/5% CO2 incubator.
    IHC: Retinal cross-sections (n = 7–10 eyes/group) were prepared for immunohistochemistry. After drying and rehydration in PBS, sections were blocked in 10–20% appropriate serum with 1% BSA in 0.1% or 0.2% Triton X-100 in PBS. Three retinal sections per eye were used for each staining. RGCs, cholinergic amacrine cells, (active) microglia as well as macroglia, the synaptic ribbon terminals, photoreceptors as well as the VEGF-receptor 2 were investigated using specific antibodies. The primary antibodies were incubated at room temperature overnight. Incubation with corresponding secondary antibodies was performed for 60 min. As a nuclear stain DAPI was added. Negative controls were implemented by using secondary antibodies only.
    ELISA: A total of 96 well plates were coated with recombinant human VEGF165 at a concentration of 1.0 μg/mL overnight at 4°C (100 μL/well). After washing three times with PBS containing 0.05% Tween-20, the wells were blocked with 3% BSA/PBS overnight at 4°C (200 μL/well), washed five times with PBS containing 0.05% Tween-20, and stored dry at 4°C for later use.

Target

  • Alternative Names
  • Ranibizumab;ranibizumab;347396-82-1;DB01270VEGFA;vascular endothelial growth factor A;vascular endothelial growth factor , VEGF;VEGF A;VPF;vascular permeability factor;VEGF;MVCD1;MGC70609;

For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

See other products for "VEGFA"

* Abbreviations
3D IHC3D Immunohistochemistry
ActivActivation
AgonistAgonist
ApopApoptosis
BABioassay
BIBioimaging
BlockBlocking
Cell ScreeningCell Screening
SeparationCell Separation
ChIPChromatin Immunoprecipitation
CMCDComplement Mediated Cell Depletion
CostimCostimulation
CytCytotoxicity
DepletionDepletion
DBDot Blot
EMElectron Microscopy
ELISAEnzyme-linked Immunosorbent Assay
ELISPOTEnzyme-linked Immunosorbent Spot
FCFlow Cytometry
FuncSFunctional Assay
GSGel Super Shift Assay
HAHemagglutination
IAImmunoassay
IBImmunoblotting
ICCImmunocytochemistry
IDImmunodiffusion
IFImmunofluorescence
IHCImmunohistochemistry
IHC-FrImmunohistochemistry-Frozen
IHC-PImmunohistochemistry-Paraffin
REImmunohistology - Resin Sections
IPImmunoprecipitation
IRMAImmunoradiometric Assay
SHIn situ hybridization
InhibInhibition
ICFCIntracellular Staining for Flow Cytometry
KO/KD-WBKnockout/Knockdown target confirmation by Western Blot
Live cell imagingLive cell imaging
CyTOF®Mass Cytometry
MeDIPMethylated DNA Immunoprecipitation
MultiplexMultiplex bead-based assay
NeutNeutralization
PPProtein Purification
PGProteogenomics
RIRadial Immunodiffusion
RIARadioimmunoassay
StimStimulation
SPRSurface Plasmon Resonance
TCTissue Culture
TBTurbidimetry
WBWestern Blot

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