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 wi th antibody techniques to study the lateral distribution of specific s erum proteins adsorbed onto flat silicon surfaces precoated with immun oglobulin G (IgG). Null-ellipsometry was used as a complimentary techn ique to quantify the adsorbed protein layers. After 15 s of incubation in human blood serum a partial monolayer of randomly distributed seru m proteins was observed. The following exposure to antibodies to compl ement factor 1q (anti-C1q) resulted in a development of enlarged prote in 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 s urface 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 m in resulted in an increased thickness and roughness of the protein lay er and caused a massive deposition of anti-C3c but no anti-C1q. The re sults suggests that the transient affinity of anti-C1q, seen on variou s classically complement activating surfaces, is due to a shielding of the initially adsorbed proteins by subsequently deposited layers of C 3. The results also show that qualitative information of the lateral o rganisation of specific proteins in a heterogeneous mixture can be ass essed using AFM in combination with immunological techniques. (C) 1997 Academic Press.