Optimization of phage-immobilized ELISA for autoantibody profiling in human sera.
Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.
Daejeon-KRIBB-FHCRC Cooperation Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon, South Korea.
Mi-Kyung
MK
Woo
Chang-Kyu
CK
Heo
Hae-Min
HM
Hwang
Jeong-Heon
JH
Ko
Hyang-Sook
HS
Yoo
Eun-Wie
EW
Cho
D001323:Autoantibodies / Q000097:blood*; D001435:Bacteriophages / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins; D019151:Peptide Library; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D055767:Immobilized Proteins; D019151:Peptide Library