Application Notes

Using MST to analyse the binding of nanobodies and Nanobody-Fc fusion proteins to human CD38

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2 NT.115™ hydrophilic glass capillaries. In order to find the best thermophoretic setting, we analyzed the binding of the Nb to Alexa 647 -labeled CD38 at low (20 %), middle (40 %), and high (80 %) MST power. The best signal to noise ratio was obtained by using 80 % MST power. Therefore all other binding experiments were performed using these MST-settings. The results of the binding experiment with the monovalent Nb and Alexa 647 -labeled CD38 show a concentration-dependent change in the thermophoresis of the Alexa 647 -labeled CD38 of the in case of the Nb, whereas no change in thermophoresis of the Alexa 647 was observed in case of the anti-GFP control monoclonal antibody (Fig. 2). The calculated K d of the Nb-CD38 binding was 5.2 ± 0.3 nM. This correlates well with the binding affinity determined by semiquantative ELISA (not shown). The lack of change in thermophoresis in case of the anti-GFP antibody confirms that this antibody does not bind to Alexa 647 -labeled CD38. The results of the binding of the bivalent Nb-Fc fusion protein to Alexa 647 -labeled CD38 show a biphasic change in thermophoresis (Fig. 3). The calculated K d for the high affinity binding event was 4.9 +/- 0.3 nM, which corresponds well with the K d determined for the monovalent Nb. The calculated K d of the second weaker binding event was 810 +/- 82 nM. It is conceivable that binding of CD38 to one of the two binding sites on the Fc- fusion protein sterically interferes with binding to the second site. Fig. 2 Binding of monovalent Nanobody to Alexa 647 -labeled CD38. The concentration of the fluorescently labelled CD38 was kept constant at 5 nM, while the concentration of the Nanobody or mAb Rat-α-GFP was varied from 0.3 nM – 1000 nM. After a 10 min equilibration, MST-analysis was performed (n = 2). The calculated K d for the binding of the Nanobody to CD38 was 5.2 ± 0.3 nM. As expected, no binding of the Rat-α-GFP to CD38 was detected. Fig. 3 Binding of bivalent Nb-Fc fusion protein to Alexa 647 - labelled CD38. The concentration of the fluorescently labelled CD38 was kept constant, while the concentration of the Nb-Fc fusion protein was varied from 0.3 nM - 1000 nM. After a 10 min equilibration, MST-analysis was performed (n = 2). A K d of 4.9 ± 0.3 nM was calculated for the high affine binding event (black) and a K d of 810 nM ± 82 nM for the second, weaker binding event (blue) (n = 2). Conclusion This study demonstrates that MicroScale Thermophoresis is capable of measuring antibody-antigen interactions in solution. Other advantages of this approach include simple handling, low material consumption, and fast analysis. Material and Methods Assay conditions The recombinant soluble ecto-domain of human CD38 (8) was labelled with Alexa 647 according to the manufacturer's (Molecular Probes, Invitrogen, Germany) instructions. The concentration of the labelled CD38 was kept constant at a concentration of 5 nM. CD38-specific Nanobody 1067 and the 1067-mouse IgG1-Fc fusion protein were produced in E. coli and HEK cells, respectively as described previously (9, 10). A monoclonal antibody directed against GFP Rat-α- GFP (Chromotek, Germany) was used as control. The unlabeled binding partners were titrated in 1:1 dilutions starting at 1000 nM. Samples were diluted in MST optimized buffer (50 mM Tris-HCl buffer pH 7.6 containing 150 mM NaCl, 10 mM MgCl 2 and 0.05 % Tween-20). For the measurement the samples were filled into hydrophilic treated capillaries (K004, NanoTemper technologies, Germany) and measured after a 10 min equilibration at room temperature. The measurements were performed at 40 % LED, and 20 %, 40 %, and 80 % MST power. Laser-On time was 30 sec, Laser-Off time 5 sec.

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