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. 2021 Mar 1;12:1359. doi: 10.1038/s41467-021-21497-6

Fig. 2. Construction and characterization of the antibody immobilization platform (αFc-NP).

Fig. 2

a Schematic depicting the construction of αFc-conjugated nanoparticles (αFc-NP). αFc was oxidized and immobilized onto aminated polystyrene NP via aldol condensation. b Determination of the αFc binding efficacy by ELISA. c, Average hydrodynamic size of NP and αFc-NP as determined by DLS, confirming that NP had an average diameter of 123.4 ± 4.1 nm and αFc-NP had an average diameter of 152.8 ± 2.0 nm. d Representative scanning electron microscopy (SEM) image of NP and αFc-NP. Scale bar, 100 nm. e Reducing SDS-PAGE gel stained with Coomassie Brilliant Blue showing the heavy chain (HC) and light chain (LC) released from soluble αFc or αFc-NP. β-mercaptoethanol treatment breaks the interchain disulfide bonds and separates HC and LC of αFc. The molecular weights of HC and LC are approximately 50 kDa and 25 kDa, respectively. f Size distribution of αFc-NP and αFc-NPαPD1 determined by DLS, confirming the αFc-NPαPD1 had an average diameter of 176.1 ± 5.3 nm, 30 nm larger than αFc-NP. g Stochastic optical reconstruction microscopy (STORM) images of IgG-NPαPD1 and αFc-NPαPD1. NP and αPD1 were labeled with AF750 and AF647, respectively. An NP conjugating IgG control antibody (IgG-NP) was used as a control. Scale bar, 200 nm. h Nanoflow Cytometry showed that αFc-NP could simultaneously bind two types of mAbs. αFc-NP was incubated with FITC-labeled αPD1 and PerCP-Cy5.5-labeled αPDL1 separately or in combination. i Binding efficacies of αPD1 and αPDL1 versus incubation time. αFc-NP were incubated with αPD1 and αPD1 at an αFc: αPD1: αPDL1 ratio of 1:0.5:0.5 for different periods, and then the unbound αPD1 and αPD1 were then examined by ELISA. j The ratio of αPD1 and αPDL1 immobilized by αFc-NP. αFc-NP was incubated with αPD1 and αPDL1 at an αFc: (αPD1 & αPDL1) ratio of 1:1, while the ratio of αPD1 and αPDL1 ranged from 0.3 to 3.0. k, l The capabilities of αFc-NPαPD1 and αFc-NPαPDL1 to bind corresponding antigens. Data are the means ± s.d of three different experiments with similar results. Source data are provided as a Source Data file.