Prevention of nonspecific binding of immunoglobulin to Staphylococcus aureus protein A in ELISA assays.
The Fc region of IgG of most mammals binds protein A on S. aureus resulting in high backgrounds when measuring specific antibodies to S. aureus in the ELISA. Removal of protein A from S. aureus or modification of the Ig Fc to prevent binding to protein A could affect specific antibody binding. We compared effects of blockage of Fc binding to protein A with purified protein A to trypsin removal of protein A from S. aureus, on specific antibody binding. When NMS was incubated without and with protein A (0 microgram, 50 micrograms, 200 micrograms and 400 micrograms) and high protein A Cowan I was the bound S. aureus antigen in the ELISA, absorbance OD405 was 0.769, 0.240, 0.224 and 0.210 +/- SE 0.026. When mouse Mab (IgG1, kappa) to bovine IgA was incubated without and with protein A (400 micrograms) prior to reaction with bovine IgA in the ELISA, absorbance was 0.645 and 0.639, indicating protein A had no effect on specific antibody binding. To determine the effect of trypsin on specific binding, Becker S. aureus was trypsin treated before linking it to microtiter wells. When Mab (IgM) to Becker (Nelles et al., Infect. Immun. (1985) 49, 14) was incubated with protein A (400 micrograms) before use in the ELISA, trypsin treatment of Becker resulted in reduced specific antibody activity (untreated Becker = 1.306, trypsin treated Becker = 0.331). These results suggest that purified protein A can be used to block nonspecific binding via Fc of Ig to S. aureus, thus avoiding trypsin denaturation of surface antigens.
USDA-ARS, Beltsville, MD 20705.
A J
AJ
Guidry
K E
KE
Squiggins
W F
WF
Vann
D C
DC
Westhoff
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007070:Immunoglobulin A / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding / Q000187:drug effects; D013205:Staphylococcal Protein A / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D014357:Trypsin