Protein A antibody-capture ELISA (PACE): an ELISA format to avoid denaturation of surface-adsorbed antigens.
Adsorption to a polymeric surface may severely alter the antigenic structure of proteins through unfolding. A conventional capture ELISA in which a protein antigen is adsorbed to the microtiter plate may be unsuitable for testing the specificity of antibodies directed against native proteins (C. Schwab and H.R. Bosshard (1992) J. Immunol. Methods 147, 125). This problem can be overcome by PACE, a new ELISA procedure in which monoclonal or polyclonal antibodies are first allowed to equilibrate with biotinylated antigen in solution. Thereafter, the antigen-antibody complex (and free antibody) is bound to the microtiter plate through protein A. Captured antigen-antibody complex is detected by streptavidin-alkaline phosphatase and p-nitrophenylphosphate. A competition assay is accomplished by co-incubation of biotinylated and non-biotinylated antigens before capture to the protein A-coated plate. PACE combines the advantages of a solution-phase immunoassay (Farr assay) with the ease of a solid-phase ELISA. PACE has been used to test the conformational specificity of polyclonal and monoclonal antibodies against native and denatured cytochrome c, and of a polyclonal antiserum against a coiled coil leucine zipper peptide. Since a biotin group can be attached specifically to the N-terminal residue of synthetic peptides, PACE is also useful for assaying reactivity against peptide antigens which are difficult to adsorb to microtiter plates.
Ngai
P K
PK
Biochemisches Institut, Universität Zürich, Switzerland.
Ackermann
F
F
Wendt
H
H
Savoca
R
R
Bosshard
H R
HR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D000941:Antigens / Q000032:analysis* / Q000737:chemistry; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D001667:Binding, Competitive; D001710:Biotin; D003574:Cytochrome c Group / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D045304:Cytochromes c; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011863:Radioimmunoassay; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000941:Antigens; D001059:Apoproteins; C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; D001710:Biotin; D045304:Cytochromes c