Imaging of the Early Events of Classical Complement Activation Using Antibodies and Atomic Force Microscopy

In the present report we use atomic force microscopy (AFM) combined with antibody techniques to study the lateral distribution of specific serum proteins adsorbed onto flat silicon surfaces precoated with immunoglobulin G (IgG). Null-ellipsometry was used as a complimentary technique to quantify the adsorbed protein layers. After 15 s of incubation in human blood serum a partial monolayer of randomly distributed serum proteins was observed. The following exposure to antibodies to complement factor 1q (anti-C1q) resulted in a development of enlarged protein aggregates and a significant increase in adsorbed mass. Conversely, exposure to antibodies to complement factor 3c (anti-C3c) resulted in only a few randomly distributed protein aggregates and a much smaller increase in adsorbed mass. After 60 s of serum incubation the entire surface was covered with a proteinaceous film with irregular topography. This layer bound large amounts of anti-C3c but showed significantly smaller affinity for anti-C1q. Prolonging the serum incubation to 30 min resulted in an increased thickness and roughness of the protein layer and caused a massive deposition of anti-C3c but no anti-C1q. The results suggests that the transient affinity of anti-C1q, seen on various classically complement activating surfaces, is due to a shielding of the initially adsorbed proteins by subsequently deposited layers of C3. The results also show that qualitative information of the lateral organisation of specific proteins in a heterogeneous mixture can be assessed using AFM in combination with immunological techniques.