Figure 2

Dual biochemical and microscopy-based selection of nanobodies. (a) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25 and W23 as the primary antibody. Mouse anti-Myc (1:3000) followed by anti-mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. Lineal contrast and grey scale were applied to the image, original dot blot scan is shown in the supplemental Fig. 3a. (b) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by mouse anti-Myc (1:3000) and anti-mouse-Alexa 647. The image shows two positive clones (W25 and W23), and an example of a negative Nanobody the screening assay was performed once, scale bar indicates 20 µm. (c) Sequence alignment of aminoacidic sequence of W25 and W23. CDR sequences are marked with a black line. (d) Purification from periplasm of bacteria, elution fraction of a single liter of bacterial culture n = 5. (e) Immunodetection as in (b), using purified protein n = 3, scale bar indicates 20 µm. (f) ELISA assay of full-length Spike of SARS-CoV-2 using conjugated W25-HRP nanobody n = 3. (g) ELISA assay of RBD of Spike using W25-HRP conjugate Nanobody n = 3. Statistic t-test, *** P ≤ 0.001; ** P ≤ 0.005; * P ≤ 0.01 to -W25 control. Illustrations (f,g) by Felipe G. Serrano BSc., MSc Scientific illustrator.