Multilectin Assay for Detecting Fibrosis-Specific Glyco-Alteration by Means of Lectin Microarray Atsushi Kuno, 1 Yuzuru Ikehara, 1 Yasuhito Tanaka, 2 Takashi Angata, 1 Sachiko Unno, 1 Maki Sogabe, 1 Hidenori Ozaki, 1 Kiyoaki Ito, 3 Jun Hirabayashi, 1 Masashi Mizokami, 2,3 and Hisashi Narimatsu 1* BACKGROUND: Despite the progress made in under- standing glyco-alterations of specific glycoproteins such as 1-acid glycoprotein (AGP) associated with liver fibro- sis, there has been no useful diagnostic assay with a lectin recognizing the fibrosis-specific alteration and an anti- body against the core protein. We therefore developed a compatible multiple lectin-antibody sandwich immuno- assay on the basis of the results obtained by the lectin microarray analysis for monitoring fibrosis. METHODS: AGP-enriched fractions derived from 0.5-L sera of 125 patients with staging-determined fibrosis (26.4% F0 –F1, 25.6% F2, 24% F3, and 23.2% F4) were subjected to systematic analysis by antibody- overlay lectin microarray. Data were analyzed to statis- tically relate to the degree of fibrosis progression. Ad- ditionally, we applied an optimal lectin signal set on the microarray to distinguish 45 patients with cirrhosis from 43 patients with chronic hepatitis. RESULTS: Signal patterns of the 12 selected lectins re- flected fibrosis-associated glyco-alteration of AGP. Among the 12 lectins, we found a specific lectin at each stage of fibrosis (i.e., significant fibrosis, severe fibrosis, and cirrhosis) (P  0.0001). The test for the detection of cirrhosis showed that combinational use of 3 lectins (AOL, MAL, and DSA) on the array enhanced the di- agnostic value for liver cirrhosis to 95% diagnostic sen- sitivity and 91% diagnostic specificity. CONCLUSIONS: The multiple lectin-antibody sandwich immunoassay targeting AGP enables monitoring of disease progression in chronic hepatitis patients at risk of developing hepatocellular carcinoma. © 2010 American Association for Clinical Chemistry It is estimated that 350 million and 170 million people are chronically infected with the hepatitis B virus (HBV) 4 and hepatitis C virus (HCV), respectively (1, 2 ). People with HBV or HCV have an increased risk of developing of chronic hepatitis, cirrhosis, and hepa- tocellular carcinoma. Globally, 57% of cirrhosis cases are attributable to either HBV (30%) or HCV (27%), and 78% of hepatocellular carcinoma (HCC) cases are attributable to HBV (53%) or HCV (25%) (3). In Ja- pan, 90% of HCC patients had chronic hepatitis as- sociated with HBV (13%) or HCV (81%) during the period 1996 –2000 (4). Although HCV infection is asymptomatic for about 10 years before chronic hepa- titis develops, it causes cirrhosis and irreversible fibro- sis during this interval. According to the association between staging fibrosis (F0 –F4) and carcinogene- sis, 7.9% of F4 stage fibrosis patients in Japan will develop HCC each year, whereas the corresponding rate for hepatitis patients with the primary F0/F1 stage is much lower (0.5%) (5). An accurate method for monitoring the progression of fibrosis is needed to identify F4 stage fibrosis patients as a high-risk group for the development of HCC, especially in the case of hepatitis C. HCV patients are currently subjected to continu- ous monitoring for chronic inflammation by using biochemical markers, such as aspartate aminotransfer- ase and alanine aminotransferase, to evaluate inflam- matory activity and platelet count to assess the progres- sion of fibrosis. However, platelet count is affected by physical status and some medical treatments. Conse- quently, despite the invasiveness and high risk, liver biopsies are regarded as the conventional method of evaluating the progression of fibrosis in patients with chronic hepatitis (6). As an alternative to liver biopsy, Imbert-Bismut et al. developed a method that involves 1 Research Center for Medical Glycoscience (RCMG), National Institute of Ad- vanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan; 2 Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi, Mizuho, Japan; the 3 Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan. * Hisashi Narimatsu, Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan. Fax +81-29-861-3191; e-mail h.narimatsu@aist.go.jp. Received June 1, 2010; accepted October 11, 2010. Previously published online at DOI: 10.1373/clinchem.2010.151340 4 Nonstandard abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; HCC, hepatocellular carcinoma; AGP, 1-acid glycoprotein; AIST, National Institute of Advanced Industrial Science and Technology; PBSTx, PBS containing 1% Triton X-100; ROC, receiver operating characteristic; AUC, area under the curve; LR, likelihood ratio. Clinical Chemistry 57:1 48–56 (2011) Proteomics and Protein Markers 48 Downloaded from https://academic.oup.com/clinchem/article/57/1/48/5621251 by guest on 11 June 2022