scholarly journals Development of Aptamer-Based TID Assays Using Thermophoresis and Microarrays

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 124 ◽  
Author(s):  
Kurth ◽  
Witt ◽  
Bolten ◽  
Waniek ◽  
Kortmann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Detection ◽  
Model System ◽  
Vascular Endothelial ◽  
Protein Biomarkers ◽  
High Affinity ◽  
Model Protein ◽  
Recognition Elements ◽  
Systematic Development ◽  
Endothelial Growth Factor

Aptamers are single-stranded oligonucleotides which can be used as alternative recognition elements for protein detection, because aptamers bind their targets with a high affinity similar to antibodies. Due to the targetinduced conformational changes of aptamers, these oligonucleotides can be applied in various biosensing platforms. In this work, aptamers directed against the vascular endothelial growth factor (VEGF) were used as a model system. VEGF plays a key role in physiological angiogenesis and vasculogenesis. Furthermore, VEGF is involved in the development and growth of cancer and other diseases like agerelated macular degeneration, rheumatoid arthritis, diabetes mellitus, and neurodegenerative disorders. Detecting the protein biomarker VEGF is therefore of great importance for medical research and diagnostics. In this research, VEGFbinding aptamers were investigated for the systematic development of a targetinduced dissociation (TID) assay utilizing thermophoresis and microarrays. The established aptamer-microarray allowed for the detection of 0.1 nM of VEGF. Furthermore, the systematic development of the TID method using the VEGF model protein could help to develop further TID assays for the detection of various protein biomarkers.

Inhibition of receptor binding by high-affinity RNA ligands to vascular endothelial growth factor

Biochemistry ◽  
1994 ◽  
Vol 33 (34) ◽  
pp. 10450-10456 ◽  
Author(s):  
Derek Jellinek ◽  
Louis S. Green ◽  
Carol Bell ◽  
Nebojsa Janjic
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Receptor Binding ◽  
Vascular Endothelial ◽  
High Affinity ◽  
Rna Ligands ◽  
Endothelial Growth Factor

scholarly journals A Humanized Antibody against LRG1 that Inhibits Angiogenesis and Reduces Retinal Vascular Leakage

2020 ◽  
Author(s):  
David Kallenberg ◽  
Vineeta Tripathi ◽  
Faiza Javaid ◽  
Camilla Pilotti ◽  
Jestin George ◽  
...  
Keyword(s):  
Choroidal Neovascularisation ◽  
Vascular Leakage ◽  
Age Related Macular Degeneration ◽  
Vascular Endothelial ◽  
Fab Fragment ◽  
High Affinity ◽  
Pathological Angiogenesis ◽  
Age Related ◽  
Vessel Growth ◽  
Endothelial Growth Factor

ABSTRACTPathological angiogenesis contributes to morbidity in a number of diseases including cancer, diabetic retinopathy and the neovascular form of age-related macular degeneration, leading to significant efforts to develop effective anti-angiogenic therapeutics for these conditions. The field is dominated by inhibitors of vascular endothelial growth factor (VEGF), yet angiogenesis can also be driven and modified by other factors. We have previously demonstrated that leucine-rich alpha-2-glycoprotein 1 (LRG1) contributes to abnormal vessel growth by activating a TGFß switch. Here we report the development and characterisation of a function-blocking fully humanised IgG4 and its Fab fragment, that bind to LRG1 with high affinity and specificity and inhibit vascular leakage in the mouse model of laser-induced choroidal neovascularisation. In summary, we have developed a therapeutic antibody that targets a VEGF-independent signalling axis, which may be effective in a number of conditions either as monotherapy or in combination with other vascular targeted therapies.

scholarly journals Inflammatory and Angiogenic Protein Detection in the Human Vitreous: Cytometric Bead Assay

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
M. J. Koss ◽  
M. Pfister ◽  
F. H. Koch
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Frozen Storage ◽  
Protein Detection ◽  
Age Related Macular Degeneration ◽  
Vascular Endothelial ◽  
Vein Occlusion ◽  
Age Related ◽  
23 Gauge ◽  
Endothelial Growth Factor ◽  
Central Retinal Vein

Introduction.To evaluate clinical feasibility and reproducibility of cytometric bead assay (CBA) in nondiluted vitreous samples of patients with age-related macular degeneration (ARMD), diabetic macular edema (DME), and central retinal vein occlusion (CRVO).Methods.Twelve patients from a single clinics day qualified for intravitreal injections (ARMDn=6, DMEn=3, CRVOn=3) and underwent a combination treatment including a single-site 23 gauge core vitrectomy which yielded a volume of 0.6 mL undiluted vitreous per patient. Interleukin-6 (IL-6), vascular endothelial growth factor isoform A (VEGF-A), and monocyte chemo-attractant protein-1 (MCP-1) were assessed directly from 0.3 mL at the same day (fresh samples). To assess the reproducibility 0.3 ml were frozen for 60 days at −80°, on which the CBA was repeated (frozen samples).Results.In the fresh samples IL-6 was highest in CRVO (median IL-6 55.8 pg/mL) > DME (50.6) > ARMD (3.1). Highest VEGF was measured in CRVO (447.4) > DME (3.9) > ARMD (2.0). MCP-1 was highest in CRVO (595.7) > AMD (530.8) > DME (178). The CBA reproducibility after frozen storage was examined to be most accurate for MCP1 (P=0.91) > VEGF (P=0.68) > IL-6 (P=0.49).Conclusions.CBA is an innovative, fast determining, and reliable technology to analyze proteins in fluids, like the undiluted vitreous, which is important to better understand ocular pathophysiology and pharmacology. There is no influence of intermittent storage at −80° for the reproducibility of the CBA.

scholarly journals Protein detection using tunable pores: resistive pulses and current rectification

2016 ◽  
Vol 193 ◽  
pp. 487-505 ◽  
Author(s):  
Emma L. C. J. Blundell ◽  
Laura J. Mayne ◽  
Michael Lickorish ◽  
Steven D. R. Christie ◽  
Mark Platt
Keyword(s):  
Rapid Detection ◽  
Particle Surface ◽  
Protein Detection ◽  
Layer By Layer ◽  
Vascular Endothelial ◽  
Translocation Event ◽  
Lbl Assembly ◽  
Current Rectification ◽  
Endothelial Growth Factor ◽  
Tunable Pores

We present the first comparison between assays that use resistive pulses or rectification ratios on a tunable pore platform. We compare their ability to quantify the cancer biomarker Vascular Endothelial Growth Factor (VEGF). The first assay measures the electrophoretic mobility of aptamer modified nanoparticles as they traverse the pore. By controlling the aptamer loading on the particle surface, and measuring the speed of each translocation event we are able to observe a change in velocity as low as 18 pM. A second non-particle assay exploits the current rectification properties of conical pores. We report the first use of Layer-by-Layer (LbL) assembly of polyelectrolytes onto the surface of the polyurethane pore. The current rectification ratios demonstrate the presence of the polymers, producing pH and ionic strength-dependent currents. The LbL assembly allows the facile immobilisation of DNA aptamers onto the pore allowing a specific dose response to VEGF. Monitoring changes to the current rectification allows for a rapid detection of 5 pM VEGF. Each assay format offers advantages in their setup and ease of preparation but comparable sensitivities.

Sign in / Sign up

Export Citation Format

Share Document