Reducing nonspecific adhesion on cross-linked hydrogel platforms for real-time immunoassay in serum.
Biointerfaces that limit nonspecific adhesion of serum proteins have been developed by relying solely on cross-linked hydrogels. In addition to being characterized for adhesion of serum proteins, immunoassay sensitivity was also investigated through a sandwich assay for rhIL-1ra. Among the compositions developed, the optimal surface is comprised of pre-cross-linked carboxymethylcellulose (CMC) and polyethyleneimine (PEI) overlaid on a cross-linked layer of poly(ethylene glycol) (PEG) and PEI and employs an anti-IgG Fc specific ligand for oriented antibody immobilization; viscoelastic modeling provides a thickness estimate of 5 nm for the hydrogel alone, rising to 33 nm after the deposition of antibodies. Alternate compositions employing a Protein A ligand and PEG at the exposed surface of the biointerface were disfavored due to an 8-fold increase in serum adhesion and retarded immobilization kinetics, respectively. Through the rapid deposition provided by hydrogels, construction of the entire biointerface, including receptor immobilization, can be completed in 1 h. Based on QCM-D measurements, estimated nonspecific serum adsorption using these compositions is as low as 1.1 ng/mm2. The immunoassay as developed requires 10 min, providing a detection limit of 500 ng/mL rhIL-1ra in 25% human serum using only 5 microg of the secondary antibody.
Carrigan
Shawn D
SD
McGill University, Biomedical Engineering Department, 3775 University St., Duff Medical Building, Room 316, Montreal, QC, Canada H3A 2B4.
Tabrizian
Maryam
M
D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001798:Blood Proteins / Q000276:immunology / Q000378:metabolism*; D020100:Hydrogels; D007118:Immunoassay; D007700:Kinetics; D011485:Protein Binding
D000906:Antibodies; D000941:Antigens; D001798:Blood Proteins; D020100:Hydrogels