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Updated: Mar 21, 2026
Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples
Published on: May 4, 2012
Identification of IgM as a contaminant in lectin-FLISA assays for HCC detection
Mengjun Wang1, Mary Ann Comunale1, Harmin Herrera2
1Drexel University College of Medicine, Department of Microbiology and Immunology, Room 18307 New College Building, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
Insights
Early detection of liver cancer (hepatocellular carcinoma) is crucial. Researchers developed a new assay to detect specific protein changes, overcoming previous limitations for improved diagnosis of this deadly disease.
Area of Science:
- Biochemistry
- Oncology
- Immunology
Background:
- Hepatocellular carcinoma (HCC) causes over 700,000 deaths globally, often due to late diagnosis.
- Current biomarkers like alpha-fetoprotein (AFP) have limited sensitivity for HCC detection.
- Previous research identified altered protein glycosylation, specifically increased fucosylation, in HCC.
Purpose of the Study:
- To develop a reliable assay for detecting fucosylated glycoproteins as potential HCC biomarkers.
- To address and eliminate non-specific signals interfering with lectin-based assays in patient serum.
- To validate the clinical utility of the improved assay for early HCC detection.
Main Methods:
- Developed a plate-based assay to detect fucosylated glycoforms.
- Identified interfering substances in serum as increased levels of lectin-reactive IgM.
- Implemented a multi-step protein A/G incubation and filtration to remove IgG and IgM, reducing non-specific signals.
- Applied the refined assay to independent patient sample sets.
Main Results:
- Successfully removed interfering IgM and IgG, enabling specific detection of fucosylated glycoforms.
- The improved assay demonstrated applicability to patient samples previously untestable by lectin-FLISA.
- The assay was validated on a larger, independent patient cohort.
Conclusions:
- The developed assay effectively detects specific fucosylated protein glycoforms in HCC patients.
- Removal of interfering immunoglobulins is critical for accurate lectin-based glycan analysis.
- This enhanced assay holds promise for the early diagnosis and management of hepatocellular carcinoma.
Abstract:
Liver disease, in the form of hepatocellular carcinoma (HCC) accounts for > 700,000 deaths worldwide. A major reason for this is late diagnosis of HCC. The currently used biomarker, serum alpha-fetoprotein (AFP) is elevated in 40-60% of those with HCC and other markers that can either compliment or replace AFP are desired. Our previous work has identified a number of proteins that contain altered glycans in HCC. Specifically, these altered glycans were increased levels of core and outer arm fucosylation. To determine the clinical usefulness of those identified glycoproteins, a plate based assay was developed that allowed for the detection of fucosylated glycoforms. While this method was applicable to a number of independent patient sets, it was unable to specifically detect fucosylated glycoforms in many patient samples. That is, some material was present in serum that led to non-specific signal in the lectin- fluorescence -linked immunosorbent assay (lectin-FLISA). To address this issue, a systematic process was undertaken to identify the material. This material was found to be increased levels of lectin reactive IgM. Removal of both IgG and IgM using a multi-step protein A/G incubation and filtration step removed the contaminating signal and allowed for the analysis of specific protein glycoforms. This assay was subsequently used on two sample sets, one that was shown previously to be unable to be tested via a lectin FLISA and in a larger independent sample set. The clinical usefulness of this assay in the early detection of HCC is discussed.
