Sensitive array-based assay for determination of serological protein kinase A autoantibody levels based on its antigen protein activation.
OBJECTIVE
We investigated the effect of cPKAα conformational states during protein immobilization on an array platform for cPKA autoantibody assays for sensitive and high-throughput profiling of protein kinase A (PKA) autoantibody levels in human sera.


METHODS
We prepared activated human cPKAα protein arrays by addition of cofactors including ATP, MgCl2, and Triton X-100 to incubation buffer. Anti-human cPKAα antibody or PKA autoantibody levels in human sera were analyzed using activated human cPKAα protein arrays.


RESULTS
Activation of cPKAα with ATP, Mg(2+), and Triton X-100 enhanced the sensitivity of the assay by increasing the signal/noise ratio and lowering the limit of detection. cPKAα activation also enhanced the sensitivity of cPKA autoantibody detection in human sera. We successfully applied this assay to determine cPKA autoantibody levels in human sera from normal individuals (n=30) and hepatic cancer patients (n=30).


CONCLUSIONS
Our results demonstrate that cPKAα activation enhanced the sensitivity of array-based PKA autoantibody assays, and that this assay is suitable for high-throughput analyses of cPKA autoantibodies in human sera.
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Su-Hyeon
SH
Kim
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Se-Hui
SH
Jung
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Deok-Hoon
DH
Kong
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Hye-Yoon
HY
Jeon
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea; Department of Anesthesiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Min-Soo
MS
Kim
Department of Medical Environmental Biology and Tropical Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Eun-Taek
ET
Han
Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Won Sun
WS
Park
Department of Internal Medicine, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Seok-Ho
SH
Hong
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea.
Young-Myeong
YM
Kim
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do 200-701, South Korea. Electronic address: ksha@kangwon.ac.kr.
Kwon-Soo
KS
Ha
D000328:Adult; D000368:Aged; D001323:Autoantibodies / Q000097:blood*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000276:immunology*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged
D001323:Autoantibodies; D017868:Cyclic AMP-Dependent Protein Kinases
Hepatic cancer; Human serum; Protein arrays; cPKA autoantibody; cPKAα