title: Analysis of glycan variation on glycoproteins from serum by the reverse lectin-based ELISA assay.
abstract: Altered glycosylation in glycoproteins is associated with carcinogenesis, and certain glycan structures and glycoproteins are well-known markers for tumor progression. To identify potential diagnostic candidate markers, we have developed a novel method for analysis of glycosylation changes of glycoproteins from crude serum samples using lectin-based glycoprotein capture followed by detection with biotin/HRP-conjugated antibodies. The amount of lectin coated on the microplate well was optimized to achieve low background and improved S/N compared with current lectin ELISA methods. In the presence of competing sugars of lectin AAL or with sialic acid removed from the glycoproteins, we confirmed that this method specifically detects glycosylation changes of proteins rather than protein abundance variation. Using our reverse lectin-based ELISA assay, increased fucosylated haptoglobin was observed in sera of patients with ovarian cancer, while the protein level of haptoglobin remained the same between cancers and noncases. The combination of fucosylated haptoglobin and CA125 (AUC = 0.88) showed improved performance for distinguishing stage-III ovarian cancer from noncases compared with CA125 alone (AUC = 0.86). In differentiating early-stage ovarian cancer from noncases, fucosylated haptoglobin showed comparable performance to CA125. The combination of CA125 and fucosylated haptoglobin resulted in an AUC of 0.855, which outperforms CA125 to distinguish early-stage cancer from noncases. Our study provides an alternative method to quantify glycosylation changes of proteins from serum samples, which will be essential for biomarker discovery and validation studies.
authors.affiliation: University of Michigan , Department of Surgery, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Jianhui
authors.initials: J
authors.lastname: Zhu
authors.forename: Haidi
authors.initials: H
authors.lastname: Yin
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Buckanovich
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008875:Middle Aged; D010051:Ovarian Neoplasms; D011134:Polysaccharides / Q000097:blood / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D014408:Biomarkers, Tumor; D006023:Glycoproteins; D037102:Lectins; D011134:Polysaccharides