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. 2016 Mar 23;5(2):7. doi: 10.3390/antib5020007

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© 2016 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

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Competitive binding assay to cluster mAb binding using flow cytometry. An excess of mAbs was added to Sn⁺-cells. After 60 min of incubation, biotinylated-mAbs were added and unbound mAbs were removed after 60 min. Fluorescently-labeled streptavidin was added to stain only the bound biotinylated-mAbs. Dead cells were excluded using PI. A high fluorescent signal indicated the mAb had a different epitope than the biotinylated-mAb. MAb 30E6 was biotinylated and added to (A) hSn⁺-CHO and (B) pSn⁺-CHO. Biotinylated mAbs 10H1 and SySy94 were added to (C) hSn⁺-CHO and (D) mSn⁺-CHO, respectively. The relative MFI was determined comparing the fluorescent signal of the cells to that of cells with no competition taking autofluorescence of the cells into account. The relative MFI and standard error of the mean was determined for three independent repeats.