sodium-dodecyl-sulfate and biomer

Previously, we found that when fibrinogen-coated polyurethanes resided in a buffer for a period of time (the 'residence time') platelet adhesion to these materials decreased. Other changes in adsorbed fibrinogen such as decreases in polyclonal antibody binding and SDS elutability supported the conclusion that fibrinogen undergoes postadsorptive conformational changes. Subsequently we measured the binding of monoclonal antibodies to the three putative platelet binding sites on fibrinogen, using a single mid-range concentration of antibody. We found that binding of a monoclonal antibody to the platelet binding site at the C-terminus of the gamma chain of fibrinogen changed little with residence time, while binding of monoclonal antibodies to the other two putative binding sites on fibrinogen either increased with residence time (RGDF at A alpha 95-98), or first increased and then decreased with residence time (RGDS at A alpha 572-575). In the current study, we measured antibody binding affinity, Ka, by measuring antibody binding at a series of antibody concentrations. This is a more sensitive method for detecting changes in adsorbed fibrinogen than measuring antibody binding from a single antibody concentration. The Ka was determined for two antibodies, M1 (4A5), which binds to a platelet binding domain of fibrinogen (gamma 402-411) and R1 (155 B 1616), which binds to residues 87-100 of the A alpha chain (containing an RGDF site). A summary of the results for the M1 antibody are as follows. The Ka was higher for M1 binding to fibrinogen adsorbed to Immulon I than to Biomer, Biospan or poly(ethylene terephthalate), suggesting that fibrinogen adsorbed to Immulon I is more platelet adhesive than fibrinogen adsorbed to the other polymers. On Biospan, the Ka decreased from 2.8 x 10(9) to 1.0 x 10(9) M-1 after a 24 h 37 degrees C residence time, which correlated with the decrease in platelet adhesiveness of adsorbed fibrinogen observed previously under these conditions. The change in Ka was greater when adsorbed fibrinogen was kept under denaturing conditions. For example, the Ka decreased from 2.8 x 10(9) to 0.8 x 10(9) M-1 after a 1 h 70 degrees C residence time whereas it remained approximately the same, 2.9 x 10(9) M-1, after a 24 h 0 degree C residence time.

Topics: Adsorption; Antibodies, Monoclonal; Antibody Specificity; Binding Sites; Biocompatible Materials; Blood Platelets; Blood Proteins; Cell Adhesion; Enzyme-Linked Immunosorbent Assay; Fibrinogen; Humans; Kinetics; Polyethylene Terephthalates; Polyurethanes; Reproducibility of Results; Sodium Dodecyl Sulfate

Residence-time-dependent changes in fibrinogen after its adsorption to Biomer were examined by measuring platelet adhesion and antibody binding to the adsorbed protein, and the amount of adsorbed fibrinogen which could be eluted by sodium dodecyl sulfate (SDS). Baboon fibrinogen was first adsorbed (from either pure solution or dilute plasma) to Biomer, which was then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the adherent protein layer was either probed for fibrinogen capable of mediating platelet adhesion using 111In radiolabeled, washed platelet suspensions under both static and shearing conditions, or for fibrinogen capable of binding antibody using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed protein layer was soaked in a 3% SDS solution, and the amount of 125I radiolabeled fibrinogen retained was measured. Decreases in platelet and antibody binding, and in the SDS elutability of the adsorbed fibrinogen after it was stored in buffer were detected, although different rates of decrease were observed for each method. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen was prevented. While each of the three assays measures a different property of adsorbed fibrinogen, this study suggests that the adherent protein undergoes time dependent conformational changes which render it less reactive toward platelets and antibodies, and more resistant to elution by SDS.

Topics: Adsorption; Animals; Antigen-Antibody Reactions; Buffers; Enzyme-Linked Immunosorbent Assay; Fibrinogen; In Vitro Techniques; Molecular Conformation; Papio; Platelet Adhesiveness; Polyurethanes; Sodium Dodecyl Sulfate