Aflibercept (Eyelea ®, Zaltrap ®) ELISA Assay Kit

$895.00

The Eagle Biosciences Aflibercept ELISA Assay Kit is an enzyme immunoassay for the measurement of free Aflibercept in serum and plasma samples. The Aflibercept ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Aflibercept (Eyelea®, Zaltrap® ) ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 5 ng/mL
Dynamic Range: 6 – 200 ng/mL
Incubation Time: 2 hours
Sample Type: Serum, Plasma
Sample Size: 10 µL

Additional Information

Assay Background

The drug Aflibercept (trade names Eylea®, Zaltrap®) is a recombinant fusion protein consisting of VEGF-binding portions from the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept has broad affinity for all ligands that bind to these receptors, including isoforms of VEGF-A, VEGF-B, and placental growth factors. Aflibercept demonstrated antitumour effects and antiangiogenic activity as a single agent and enhanced activity in combination with chemotherapy.

Identification of biomarkers for (non-)response and risk factors for adverse drug reactions that might be related to serum concentrations and maintaining the effective concentration of Aflibercept in order to potentially avoid some side effects with a reliable method might be beneficial.

Assay Principle

This Eagle Biosciences Aflibercept ELISA Assay Kit is based on sandwich type ELISA. Diluted standards and samples (serum or plasma) are incubated in the microtiter plate coated with recombinant human vascular endothelial growth factor-A (rhVEGF-A). After incubation, the wells are washed. A horseradish peroxidase (HRP)conjugated anti-human IgG monoclonal antibody is added and binds to the Fc part of Aflibercept pre-captured by the rhVEGF-A on the surface of the wells. Following incubation, the wells are washed and the bound enzymatic activity is detected by addition of chromogen-substrate. The color developed is proportional to the amount of Aflibercept in the sample or standard. Results of samples can be determined by using the standard curve.

Assay Procedure

  1. Pipette 100 µL of Assay Buffer into each of the wells to be used.
  2. Pipette 75 µL of each 1:10 Diluted Standard, and 1:100 Diluted Samples into the respective wells of the microtiter plate.
  3. Cover the plate with adhesive seal. Shake plate carefully. Incubate 60 min at room temperature (RT, 20-25°C).
    Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well.
  4. Remove excess solution by tapping the inverted plate on a paper towel.
  5. Pipette 100 μL of Enzyme Conjugate (HRP-anti human IgG mAb) into each well.
  6. Cover plate with adhesive seal. Shake plate carefully. Incubate 30 min at RT.
    Remove adhesive seal. Aspirate or decant the incubation solution. Wash the plate 3 X 300 μL of Diluted Wash Buffer per well.
  7. Remove excess solution by tapping the inverted plate on a paper towel.
  8. Pipette 100 µL of Ready-to-Use TMB Substrate Solution into each well.
  9. Incubate 15 min at RT. Avoid exposure to direct sunlight.
  10. Stop the substrate reaction by adding 100 µL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Color changes from blue to yellow.
  11. Measure optical density (OD) with a photometer at 450 nm (Reference at OD620 nm is optional) within 15 min after pipetting the Stop Solution.

Typical Standard Curve

Manual

Product Manual


Publications

Citations

  • Park SJ, Choi Y, Na YM, Hong HK, Park JY, Park KH, Chung JY, Woo SJ. Intraocular Pharmacokinetics of Intravitreal Aflibercept (Eylea) in a Rabbit Model. Invest Ophthalmol Vis Sci. 2016;57(6):2612-7.
  • Stewart MW1, Rosenfeld PJ, Penha FM, Wang F, Yehoshua Z, Bueno-Lopez E, Lopez PF. Pharmacokinetic rationale for dosing every 2 weeks versus 4 weeks with intravitreal ranibizumab, bevacizumab, and aflibercept (vascular endothelial growth factor Trap-eye). Retina. 2012;32(3):434-57.
  • Arnott C, Punnia-Moorthy G, Tan J, Sadeghipour S, Bursill C, Patel S. The Vascular Endothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly Increase Expression of Atherosclerosis-Associated Inflammatory Mediators on Vascular Endothelial Cells. PLoS One. 2016;11(3):e0150688. doi: 10.1371.
  • Takahashi H, Nomura Y, Nishida J, Fujino Y, Yanagi Y, Kawashima H. Vascular Endothelial Growth Factor (VEGF) Concentration Is Underestimated by Enzyme-Linked Immunosorbent Assay in the Presence of Anti-VEGF Drugs. Invest Ophthalmol Vis Sci. 2016;57(2):462-6.
  • Puddu A, Sanguineti R, Traverso CE, Viviani GL, Nicolò M. Response to anti-VEGF-A treatment of endothelial cells in vitro. Exp Eye Res. 2016;146:128-36.
  • Niwa Y, Kakinoki M, Sawada T, Wang X, Ohji M. Ranibizumab and Aflibercept: Intraocular Pharmacokinetics and Their Effects on Aqueous VEGF Level in Vitrectomized and Nonvitrectomized Macaque Eyes. Invest Ophthalmol Vis Sci. 2015;56(11):6501-5.
  • Celik N, Scheuerle A, Auffarth GU, Kopitz J, Dithmar S. Intraocular Pharmacokinetics of Aflibercept and Vascular Endothelial Growth Factor-A. Invest Ophthalmol Vis Sci. 2015;56(9):5574-8.
  • Giurdanella G, Anfuso CD, Olivieri M, Lupo G, Caporarello N, Eandi CM, Drago F, Bucolo C, Salomone S. Aflibercept, bevacizumab and ranibizumab prevent glucose-induced damage in human retinal pericytes in vitro, through a PLA2/COX-2/VEGF-A pathway. Biochem Pharmacol. 2015;96(3):278-87.
  • Zehetner C, Bechrakis NE, Stattin M, Kirchmair R, Ulmer H, Kralinger MT, Kieselbach GF. Systemic counterregulatory response of placental growth factor levels to intravitreal aflibercept therapy. Invest Ophthalmol Vis Sci. 2015;56(5):3279-86.
  • Klettner A, Grotelüschen S, Treumer F, Roider J, Hillenkamp J. Compatibility of recombinant tissue plasminogen activator (rtPA) and aflibercept or ranibizumab coapplied for neovascular age-related macular degeneration with submacular haemorrhage. Br J Ophthalmol. 2015;99(6):864-9.
  • Mansour AM, Al-Ghadban SI, Yunis MH, El-Sabban ME. Ziv-aflibercept in macular disease. Br J Ophthalmol. 2015;99(8):1055-9.
  • Park SJ, Oh J, Kim YK, Park JH, Park JY, Hong HK, Park KH, Lee JE, Kim HM, Chung JY, Woo SJ. Intraocular pharmacokinetics of intravitreal vascular endothelial growth factor-Trap in a rabbit model. Eye (Lond). 2015;29(4):561-8.
  • Zehetner C, Kralinger MT, Modi YS, Waltl I, Ulmer H, Kirchmair R, Bechrakis NE, Kieselbach GF. Systemic levels of vascular endothelial growth factor before and after intravitreal injection of aflibercept or ranibizumab in patients with age-related macular degeneration: a randomised, prospective trial. Acta Ophthalmol. 2015;93(2):e154-9. doi: 10.1111/aos.12604.
  • Klettner A, Recber M, Roider J. Comparison of the efficacy of aflibercept, ranibizumab, and bevacizumab in an RPE/choroid organ culture. Graefes Arch Clin Exp Ophthalmol. 2014;252(10):1593-8.
  • Yoshida I, Shiba T, Taniguchi H, Takahashi M, Murano T, Hiruta N, Hori Y, Bujo H, Maeno T. Evaluation of plasma vascular endothelial growth factor levels after intravitreal injection of ranibizumab and aflibercept for exudative age-related macular degeneration. Graefes Arch Clin Exp Ophthalmol. 2014;252(9):1483-9.
  • Avery RL, Castellarin AA, Steinle NC, Dhoot DS, Pieramici DJ, See R, Couvillion S, Nasir MA, Rabena MD, Le K, Maia M, Visich JE. Systemic pharmacokinetics following intravitreal injections of ranibizumab, bevacizumab or aflibercept in patients with neovascular AMD. Br J Ophthalmol. 2014;98(12):1636-41.
  • Wang X, Sawada T, Sawada O, Saishin Y, Liu P, Ohji M. Serum and plasma vascular endothelial growth factor concentrations before and after intravitreal injection of aflibercept or ranibizumab for age-related macular degeneration. Am J Ophthalmol. 2014;158(4):738-744.
  • Shonka N, Piao Y, Gilbert M, Yung A, Chang S, DeAngelis LM, Lassman AB, Liu J, Cloughesy T, Robins HI, Lloyd R, Chen A, Prados M, Wen PY, Heymach J, de Groot J. Cytokines associated with toxicity in the treatment of recurrent glioblastoma with aflibercept. Target Oncol. 2013;8(2):117-25.
  • de Groot JF, Piao Y, Tran H, Gilbert M, Wu HK, Liu J, Bekele BN, Cloughesy T, Mehta M, Robins HI, Lassman A, DeAngelis L, Camphausen K, Chen A, Yung WK, Prados M, Wen PY, Heymach JV. Myeloid biomarkers associated with glioblastoma response to anti-VEGF therapy with aflibercept. Clin Cancer Res. 2011;17(14):4872-81.
  • Lassoued W, Murphy D, Tsai J, Oueslati R, Thurston G, Lee WM. Effect of VEGF and VEGF Trap on vascular endothelial cell signaling in tumors. Cancer Biol Ther. 2010;10(12):1326-33.