Normalization using a tagged-internal standard assay for analysis of antibody arrays and the evaluation of serological biomarkers for liver disease.
For minimizing systemic experimental variation in the analysis of antibody array data, we developed a novel median-centered/IgM-tagged-internal standard (TIS) assay normalization using median-centering and TIS assay-based determination of serum IgM concentrations. We evaluated five normalization methods by analyzing correlation coefficients and coefficients of variation for six serum proteins using human serum samples from normal controls (n=25) and patients with liver cirrhosis (n=25) or hepatocellular carcinoma (HCC; n=29). Median-centered normalization improved correlation coefficients, while IgM-based normalizations improved coefficients of variation. The TIS assay was more efficient, economical, and reproducible for determining IgM concentrations than enzyme-linked immunosorbent assay. Additionally, we normalized antibody array data for six serum proteins using the median-centered/IgM-TIS assay, and evaluated serum biomarkers through distribution analysis of normalized fluorescence intensities and receiver operating characteristic analyses for the diagnosis of liver cirrhosis and HCC. Apolipoprotein A-1 and a combination of alpha-fetoprotein and C-reactive protein were determined to be potential serological biomarkers for liver cirrhosis and HCC, respectively. Thus, median-centered/IgM-TIS assay normalization is a useful approach for analyzing antibody array data and evaluating serological biomarkers for the diagnosis of liver disease or cancers.
Department of Molecular and Cellular Biochemistry, Kangwon National University School of Medicine, Chuncheon, Kangwon-Do, Republic of Korea.
Deok-Hoon
DH
Kong
Jae-Wan
JW
Jung
Keun
K
Na
Seul-Ki
SK
Jeong
Young-Ki
YK
Paik
Se-Hui
SH
Jung
In-Bum
IB
Suh
Young-Myeong
YM
Kim
Kwon-Soo
KS
Ha
D000906:Antibodies / Q000276:immunology; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood* / Q000276:immunology; D008103:Liver Cirrhosis / Q000097:blood*; D008113:Liver Neoplasms / Q000097:blood*; D040081:Protein Array Analysis / Q000379:methods*
D000906:Antibodies; D007075:Immunoglobulin M