title: Imaging of the Early Events of Classical Complement Activation Using Antibodies and Atomic Force Microscopy
abstract: 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.
authors.affiliation: Laboratory of Applied Physics, Linkoping University, Linkoping, S-581 83, Sweden
authors.initials: B
authors.lastname: Walivaara
authors.initials: A
authors.lastname: Askendal
authors.initials: I
authors.lastname: Lundstrom
authors.initials: P
authors.lastname: Tengvall