Analysis of Glycan Variation on Glycoproteins from Serum by the Reverse Lectin-Based ELISA Assay

TLDR

Altered glycosylation in glycoproteins is linked to carcinogenesis, with specific glycan structures serving as tumor progression markers. The study aims to develop a reverse lectin‑based ELISA to identify diagnostic glycoprotein markers from crude serum. The assay uses lectin‑coated microplates to capture glycoproteins, followed by biotin/HRP‑antibody detection, with optimized lectin density and specificity confirmed by sugar competition and desialylation controls. The assay detected increased fucosylated haptoglobin in ovarian cancer sera; combined with CA125, it achieved AUC 0.88 for stage‑III and 0.855 for early‑stage cancer, outperforming CA125 alone.

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.