Abstract
Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates.
Highlights
Protein-attached glycans are bio-synthesized by a subset of glycosyltransferases mostly located in the endoplasmic reticulum and the Golgi apparatus, and play various functional roles at molecular and cellular levels including molecular interactions, stability, immune function, adhesion, etc
Because it has been reported that the ratio of Alpha feto protein (AFP)-L3 to total AFP could be highly specific for hepatocellular carcinoma (HCC), AFP-L3 has been a preferred HCC biomarker to AFP levels
It is the first report that aberrant glycan codes are utilized as an in vitro diagnostic multivariate index assay (IVDMIA) in the development of cancer biomarkers
Summary
Semi-quantitative Measurement of a Specific Glycoform Using a DNA-tagged Antibody and Lectin Affinity Chromatography for Glyco-biomarker Development*□S. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. DNA-tagged Antibody-based Glyco-biomarker Detection Method and extensive investigations culminated in FDA-approval of an AFP-L3 lab test to determine the risk of developing liver cancer in patients with chronic liver disease [11]. We report a novel quantitative method by which a specific glycoform can be quantitatively measured by using a DNA-tagged antibody and lectin chromatography With this approach, we validated fucoform biomarkers through a casecontrol study in a sensitive and multiplexing manner. It is the first report that aberrant glycan codes are utilized as an in vitro diagnostic multivariate index assay (IVDMIA) in the development of cancer biomarkers
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