PMC:4589137 / 13773-15748 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4589137","sourcedb":"PMC","sourceid":"4589137","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4589137","text":"3.4. Exploring the Relationship between Ligand Affinity and Bmax\nThe 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].\nFigure 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.\nFigure 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.\n\n4","divisions":[{"label":"Title","span":{"begin":0,"end":64}},{"label":"Figure caption","span":{"begin":1121,"end":1621}},{"label":"Figure caption","span":{"begin":1620,"end":1974}}],"tracks":[{"project":"2_test","denotations":[{"id":"26435848-19108655-118316782","span":{"begin":401,"end":403},"obj":"19108655"},{"id":"26435848-19108655-118316782","span":{"begin":401,"end":403},"obj":"19108655"},{"id":"26435848-17631555-118316783","span":{"begin":404,"end":406},"obj":"17631555"},{"id":"26435848-17631555-118316783","span":{"begin":404,"end":406},"obj":"17631555"},{"id":"26435848-19108655-69476149","span":{"begin":1113,"end":1115},"obj":"19108655"},{"id":"26435848-19108655-69476149","span":{"begin":1113,"end":1115},"obj":"19108655"},{"id":"26435848-17631555-69476150","span":{"begin":1116,"end":1118},"obj":"17631555"},{"id":"26435848-17631555-69476150","span":{"begin":1116,"end":1118},"obj":"17631555"},{"id":"26435848-19108655-69476147","span":{"begin":401,"end":403},"obj":"19108655"},{"id":"26435848-19108655-69476147","span":{"begin":401,"end":403},"obj":"19108655"},{"id":"26435848-17631555-69476148","span":{"begin":404,"end":406},"obj":"17631555"},{"id":"26435848-17631555-69476148","span":{"begin":404,"end":406},"obj":"17631555"},{"id":"T21136","span":{"begin":1113,"end":1115},"obj":"19108655"},{"id":"T19260","span":{"begin":1113,"end":1115},"obj":"19108655"},{"id":"T15457","span":{"begin":1116,"end":1118},"obj":"17631555"},{"id":"T9004","span":{"begin":1116,"end":1118},"obj":"17631555"}],"attributes":[{"subj":"26435848-19108655-118316782","pred":"source","obj":"2_test"},{"subj":"26435848-19108655-118316782","pred":"source","obj":"2_test"},{"subj":"26435848-17631555-118316783","pred":"source","obj":"2_test"},{"subj":"26435848-17631555-118316783","pred":"source","obj":"2_test"},{"subj":"26435848-19108655-69476149","pred":"source","obj":"2_test"},{"subj":"26435848-19108655-69476149","pred":"source","obj":"2_test"},{"subj":"26435848-17631555-69476150","pred":"source","obj":"2_test"},{"subj":"26435848-17631555-69476150","pred":"source","obj":"2_test"},{"subj":"26435848-19108655-69476147","pred":"source","obj":"2_test"},{"subj":"26435848-19108655-69476147","pred":"source","obj":"2_test"},{"subj":"26435848-17631555-69476148","pred":"source","obj":"2_test"},{"subj":"26435848-17631555-69476148","pred":"source","obj":"2_test"},{"subj":"T21136","pred":"source","obj":"2_test"},{"subj":"T19260","pred":"source","obj":"2_test"},{"subj":"T15457","pred":"source","obj":"2_test"},{"subj":"T9004","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93ea","default":true}]}]}}