3.4. Exploring the Relationship between Ligand Affinity and Bmax
The results from Figure 3 suggested that ligand affinity (KD), and therefore the ratio of affinity to Bmax, might have a profound impact on the sensitivity of the SMM screening assay. To explore this relationship we utilized a series of PSMA ligands previously reported by our group (Figure 4A), which spanned a wide range of affinity [22,31]: β-AG (KD = 2 µM), GPI (KD = 9 nM), β-AG trimer (KD = 60 nM), and GPI trimer (KD = 0.4 nM). The SMM was probed with PSMA-positive cells (LNCaP) and PSMA-negative cells (PC3), using the optimized parameters described for Figure 3. As shown in Figure 4, the SMM assay was able to perform well over 3 logs of affinity space, with the number of cells bound per spot being proportional to affinity. These results reinforce the importance of defining any cell-bound ligand spot as “positive” during initial screening of diverse chemical libraries because low affinity ligands might have only a few cells bound. And, if Bmax is low, even high affinity interactions may result in only a few cells bound per spot [22,31].
Figure 3  Robustness of the SMM Screening Assay: Three different model systems, varying in ligand chemical structure, cell type, receptor transmembrane topology, Bmax, and ligand affinity were tested as described in the text. Shown are mean ± SD for each data point from four randomly chosen spots on the slide. Receptor-positive and receptor-negative cells were labeled with 700 nm and 800 nm NIR fluorophores and pseudo-colored red and green, respectively, during microscopy. Scale bars = 200 μm.
Figure 4  The effect of affinity on cell-ligand spot binding: PSMA-positive LNCaP cells and PSMA-negative PC3 cells were labeled with 700 nm and 800 nm NIR fluorophores and pseudo-colored in red and green, respectively. (A) Chemical structures of targeting ligands employed, (B) 4× microscopy images, and (C) 20× microscopy images. Scale bars = 200 μm.

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