Dynamic force spectroscopy of the DIGOXIGENIN (cas 1672-46-4)–antibody complex

Add time:10/01/2019    Source:sciencedirect.com

Small ligands and their receptors are widely used non-covalent couplers in various biotech applications. One prominent example, the digoxigenin–antibody complex, was often used to immobilize samples for single molecule force measurements by optical trap or AFM. Here, we employed dynamic AFM spectroscopy to demonstrate that a single digoxigenin–antibody bond is likely to fail even under moderate loading rates. This effect potentially could lower the yield of measurements or even obscure the unbinding data of the sample by the rupture events of the coupler. Immobilization by multiple antibody–antigen bonds, therefore, is highly recommended. The analysis of our data revealed a pronounced loading rate dependence of the rupture force, which we analyzed based on the well-established Bell–Evans-model with two subsequent unbinding barriers. We could show that the first barrier has a width of Δx1 = 1.15 nm and a spontaneous rate of koff1 = 0.015 s−1 and the second has a width of Δx2 = 0.35 nm and a spontaneous rate of koff2 = 4.56 s−1. In the crossover region between the two regimes, we found a marked discrepancy between the predicted bond rupture probability density and the measured rupture force histograms, which we discuss as non-Markovian contribution to the unbinding process.

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