Atomic force microscopy (AFM) height images (3-dimensional view with 50° pitch) showing the effect of HCQ on aPL mAb and β2GPI complexes on 3 separate complexes. (A,E,I) The binding of β2GPI to phospholipid bilayers. Addition of β2GPI (10 μg/mL) to buffer covering the phospholipid bilayers resulted in the formation of distinct structures raised above the bilayers. (B,F,J) The binding of aPL IgG mAb, IS4, to β2GPI prebound to phospholipids. Addition of aPL mAb (20 μg/mL) to the phospholipid-bound β2GPI shown in panels A, E, and I resulted in large aggregates of increased height (indicated by white color) composed of aPL mAb–β2GPI complexes over the bilayers. (C,G,K) The effect of HCQ at 1 mg/mL on the phospholipid-bound aPL mAb and β2GPI complexes. At 12 minutes after the addition of HCQ to the supernatant fluid covering the bilayer, the immune complexes, shown in panels B, F and J, were significantly eroded after addition of the drug. (D,H,L) The immune complexes, shown in panels B, F, and J, were further disintegrated at thirty minutes after the addition of HCQ (1 mg/mL). All images show complexes electronically zoomed from an original 30 μm × 30 μm scan. Images were minimally processed to remove scan lines. The color shades represent height variations in the images, with the darker colors (rust) indicating lower heights and the lighter colors (white), the higher profiles. (M) Quantitative analysis of the effects of HCQ on 40 randomly encountered aPL mAb–β2GPI complexes. The height of the immune complexes at 12 minutes after the addition of HCQ (1 mg/mL) was significantly decreased (17.7 ± 5.8 nm; vs 9.3 ± 2.4 nm for the height before addition of HCQ).