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RANIBIZUMAB ELISA (MAB-BASED)
Enzyme immunoassay for the specific and quantitative determination of free Ranibizumab (Lucentis®) in aqueous humour.
The solid phase (MTP) is coated by a highly specific monoclonal antibody directed against Ranibizumab. Therefore any cross reactivity to the other therapeutical monoclonal antibodies is excluded except for Bevacizumab.
| Required Volume (µL) | 5 |
| Incubation Time (min) | 135 |
| Sample | aqueous humour |
| Plate Size | 96 Tests |
| Standard Range (ng/mL, 10x) | 0-300 |
| Detection Limit (ng/mL) | 0,33 |
| Spike Recovery (%) | >95 |
| Shelf Life (years) | 2 |
Intended Use: This kit has been developed for the measurement of drug levels in research and diagnostic uses. It is suitable for Therapeutical Drug Monitoring (TDM) purposes.
Lucentis® is a trademark of ©2018 Genentech USA, Inc.
ESSAY CHARACTERISTICS
SPECIFICITY
There is no cross reaction with any other proteins present in native aqueous humour samples. A screening test was performed with different aqueous humour samples. All produced OD450/620 nm values (ranged from 0.016 to 0.023) less than the mean OD (0.134) of standard D (1 ng/mL). In addition, binding of Ranibizumab to the solid phase is inhibited by recombinant human VEGF. Therefore, the ImmunoGuide Ranibizumab ELISA (mAb-Based) measures the biologically active free form of Ranibizumab, i.e. not pre-occupied by human VEGF antigen. No cross reaction was observed with the other therapeutic antibodies including Infliximab, Rituximab, Veolizumab, Trastuzumab, Nivolumab and Aflibercept at concentrations tested up to 50 µg/mL. All produced mean OD450/620 nm values (ranged from 0.052 to 0.068) less than standard D. Because the 2A1 mAb, used for coating solid phase, is also reactive against Bevacizumab, it causes full cross reaction. But a quantification of Bevacizumab in aqueous homour by this testkit is possible only by using the drug-specific standards (i.e. Bevacizumab).
SENSITIVITY
The lowest detectable level that can be clearly distinguished from the zero standard is 0,33 ng/mL (zero standard +2SD read from the curve) under the above-described conditions. If needed, the minimum measurable level of Ranibizumab in samples can be improved up to 10 times by diluting aqueous humour samples at 1:200 instead of 1:2000. Under the conditions described in section 10.2 the analytical sensitivity is 0,33 ng/mL, and corresponding to the detection limit (limit of quantification) of 0.66 µg/mL for undiluted clinical samples because the serum or plasma samples are instructed to be diluted at 1:2000 before starting the assay.
PRECISION
Intra-assay CV: <10%.
Inter-assay CV: <10%.
RECOVERY
Recovery rate was found to be >90% with aqueous humour when spiked with exogenous Ranibizumab.
AUTOMATION
The ImmunoGuide Ranibizumab ELISA (mAb-based) is suitable also for being used by an automated ELISA processor.
REFERENCES
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2. Krohne TU, Liu Z, Holz FG, Meyer CH, Intraocular Pharmacokinetics of Ranibizumab Following a Single Intravitreal Injection in Humans, Am J Ophthalmol 2012; 154: 682-686.
3. Ahn SJ, Ahn J, Park S, et al. Intraocular pharmacokinetics of ranibizumab in vitrectomized versus nonvitrectomized eyes. Invest Ophthalmol Vis Sci. 2014; 55: 567-573.
4. Wang X, Sawada T, Sawada O, et al., 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: 738-744.
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8. Xiying Wang, Tomoko Sawada, Masashi Kakinoki,et al, Aqueous vascular endothelial growth factor and ranibizumab concentrations after monthly and bimonthly intravitreal injections of ranibizumab for age-related macular degeneration, Graefes Arch Clin ExpOphthalmol, 2014; 252: 1033-1039.
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10. Arnott C, Punnia-Moorthy G, Tan J, Sadeghipour S, Bursill C, Patel S, The VascularEndothelial Growth Factor Inhibitors Ranibizumab and Aflibercept Markedly IncreaseExpression of Atherosclerosis-Associated Inflammatory Mediators on Vascular EndothelialCells, 2016; 11(3): e0150688. doi:10.1371.
11. Takahashi H, Nomura Y, Nishida J, Fujino Y, Yanagi Y, Kawashima H. Vascularendothelial growth factor (VEGF) concentration is underestimated by enzyme-linkedimmunosorbent assay in the presence of anti- VEGF drugs. Invest Ophthalmol Vis Sci.2016; 57: 462-466.
12. Siti Munirah Md Noh, Siti H. Sheikh Abdul Kadir, Jonathan G. Crowston, VisvarajaSubrayan, Sushil Vasudevan, Effects of ranibizumab on TGF-β1 and TGF-β2 production byhuman Tenon’s fibroblasts: An in vitro study, Molecular Vision 2015; 21: 1191-1200.
13. Dickmann LJ, Yip V, Li C, et al. Evaluation of fluorophotometry to assess the vitrealpharmacokinetics of protein therapeutics. Invest Ophthalmol Vis Sci. 2015; 56: 6991-6999.
14. Niwa Y, Kakinoki M, Sawada T, Wang X, Ohji M. Ranibizumab and aflibercept:intraocular pharmacokinetics and their effects on aqueous VEGF level in vitrectomized andnonvitrectomized macaque eyes. Invest Ophthalmol Vis Sci. 2015; 56: 6501-6505.
15. Ying Zhou, Yanrong Jiang, Yujing Bai, Jing Wen, Li Chen, Vascular endothelial growthfactor plasma levels before and after treatment of retinopathy of prematurity withranibizumab, Graefes Arch Clin Exp Ophthalmol, 2016; 254: 31-36.
16. Ciro Costagliola, Francesco Semeraro, Roberto dell’Omo, Mario R Romano, AndreaRusso, Fabiana Aceto, Rodolfo Mastropasqua, Antonio Porcellini, Effect of intravitrealranibizumab injections on aqueous humour concentrations of vascular endothelial growthfactor and pigment epithelium-derived factor in patients with myopic choroidalneovascularisation, Br J Ophthalmol 2015; 99: 1004-1008.
17. Terasaki H, Sakamoto T, Shirasawa M, et al., Penetration of bevacizumab andranibizumab and through retinal pigment epithelial layer in vitro, Retina, 2015; 35: 1007–1015.
18. Claus Zehetner, Martina T. Kralinger, Yasha S. Modi, Inga Waltl, Hanno Ulmer, RudolfKirchmair, Nikolaos E. Bechrakis and Gerhard F. Kieselbac, Systemic levels of vascularendothelial growth factor before and after intravitreal injection of aflibercept or ranibizumabin patients with age-related macular degeneration: a randomised, prospective trial, ActaOphthalmol. 2015; 93: e154–e159.
19. Izumi Yoshida, Tomoaki Shiba, Hikari Taniguchi, Mao Takahashi, Takeyoshi MuranoNobuyuki Hiruta, Yuichi Hori, Hdieaki Bujo, Takatoshi Maeno, Evaluation of plasmavascular endothelial growth factor levels after intravitreal injection of ranibizumab andaflibercept for exudative age-related macular degeneration, Graefes Arch Clin ExpOphthalmol, 2014; 252: 1483–1489.
20. Yi Zhang, Zhenling Yao, Nitin Kaila, Peter Kuebler, et al, Pharmacokinetics ofRanibizumab after Intravitreal Administration in Patients with Retinal Vein Occlusion orDiabetic Macular Edema, Ophthalmology 2014; 121: 2237-2246.
INSTRUCTIONS FOR USE
SAFETY DATA SHEET
BATCH/LOT INFORMATION