Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Mar 8, 2017; 9(7): 368-384
Published online Mar 8, 2017. doi: 10.4254/wjh.v9.i7.368
Characterization of a new monoclonal anti-glypican-3 antibody specific to the hepatocellular carcinoma cell line, HepG2
Preeyanat Vongchan, Robert J Linhardt
Preeyanat Vongchan, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand
Robert J Linhardt, Rensselaer Polytechnic Institute, Departments of Chemistry, Biology, Chemical and Biomedical Engineering, Troy, NY 12180, United States
Author contributions: Vongchan P (primary investigator) performed research, analyzed the data and prepared manuscript; Linhardt RJ contributed reagents and analytical tools, and revised manuscript.
Supported by National Research Council of Thailand (NRCT), No. 2559A10402115.
Institutional review board statement: Not available.
Institutional animal care and use committee statement: Not available.
Conflict-of-interest statement: The authors declare that there are no conflicts of interest.
Data sharing statement: No additional are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Correspondence to: Preeyanat Vongchan, PhD, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, 239 Huay Kaew Road, Muang District, Chiang Mai 50200, Thailand.
Telephone: +66-53-945080 Fax: +66-53-946042
Received: November 21, 2016
Peer-review started: November 23, 2016
First decision: December 15, 2016
Revised: December 19, 2016
Accepted: January 11, 2017
Article in press: January 14, 2017
Published online: March 8, 2017

To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan (HSPG), clone 1E4-1D9.


The antigen recognized by mAb 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3 ( The competitive reaction of mAb 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mAb 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mAb 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry.


Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by mAb 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350 (1478 amino acid) and gi60219551 (1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mAb 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of mAb 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mAb 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by mAb 1E4-1D9 was studied. The results demonstrated that mAb 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines.


All findings show that mAb 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies.

Keywords: Monoclonal anti-glypican-3, Hepatocellular carcinoma, HepG2, Heparan sulfate proteoglycan, Co-immunoprecipitation

Core tip: Heparan sulfate proteoglycan (HSPG) was isolated from human liver. Preliminary results showed that it was detected by rabbit anti-glypican. Monoclonal antibody, 1E4-1D9 was raised against human liver HSPG and its specific antigen was characterized. Amino acid sequence analysis revealed that the antigen recognized by mAb 1E4-1D9 specific molecule contained no transmembrane region. It has 15 cysteines and 11 putative glycosylation sites and 6 predicted N-glycosylation sites. The sequence matched to all PDZ domain proteins with an 85.6% match to glypican-3. Studies of co-expression and co-precipitation demonstrated that mAb 1E4-1D9 could compete with anti-glypican-3. It could also react with a various tumor cell lines including solid and hematopoietic cells. The findings suggested that the antigen recognized by 1E4-1D9 was glypican-3. Moreover, findings revealed that FYCO1 co-precipitated with glypican-3 using mAb 1E4-1D9, suggesting that FYCO1 is a partner molecule of glypican-3.