The effect of surface charge on non-specific binding of rabbit immunoglobulin G in solid-phase immunoassays.
The binding of IgG to a series of surface-immobilized proteins and synthetic polypeptides with differing isoelectric points was investigated by ELISA. The binding of rabbit IgG to a protein-coated test well surface was found to be highly charge dependent and relatively linear over a wide range of isoelectric points of the surface coating species. Binding was high to positively charged surfaces and low to negatively charged ones. When an intermediately charged protein, the semen antigen p30 (also known as prostate-specific antigen), was co-coated on the test surface with polypeptides of varying charge, the specificity of surface binding between anti-p30 IgG and non-immune IgG was altered. Co-coating p30 with the anionic glycoprotein AGP significantly enhanced binding specificity as reflected in an increased signal-to-noise ratio, whereas co-coating p30 with less negatively charged proteins led to a progressive decrease in the signal-to-noise ratio. The sialic acid moiety of AGP was primarily responsible for its ability to inhibit non-specific IgG binding. The results indicate that non-specific binding of IgG to polypeptide-coated ELISA test surfaces is highly charge dependent. In solid-phase immunoassays which measure antigen-specific antibody, non-specific binding of immunoglobulins may be diminished by co-coating surface antigens with a hydrophilic anionic surface matrix which resembles in some respects the cell surface glycocalyx.
Graves
H C
HC
Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley 94720.
D000818:Animals; D000951:Antigens, Neoplasm / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D007074:Immunoglobulin G; D007163:Immunosorbent Techniques; D007477:Ions; D007526:Isoelectric Point; D017430:Prostate-Specific Antigen; D011485:Protein Binding; D011817:Rabbits; D013329:Structure-Activity Relationship; D013499:Surface Properties
D000951:Antigens, Neoplasm; D007074:Immunoglobulin G; D007477:Ions; D017430:Prostate-Specific Antigen