TY - JOUR
T1 - Biomarker analysis of fucosylated kininogen through depletion of lectin reactive heterophilic antibodies in hepatocellular carcinoma
AU - Wang, Mengjun
AU - Shen, Jiabin
AU - Herrera, Harmin
AU - Singal, Amit
AU - Swindell, Charles
AU - Renquan, Lu
AU - Mehta, Anand
N1 - Funding Information:
This work was supported by the National Cancer Institute grant numbers R01CA120206 and U01CA168856.
Funding Information:
This work was supported by the National Cancer Institute grant numbers R01 CA120206 and U01 CA168856 .
Publisher Copyright:
© 2018
PY - 2018/11
Y1 - 2018/11
N2 - Hepatocellular carcinoma (HCC) accounts for >700,000 deaths worldwide, largely related to poor rates of diagnosis. Our previous work identified glycoproteins with increased levels of fucosylation in HCC. Plate-based assays to measure this change were compromised by increased levels of heterophilic antibodies with glycan lacking terminal galactose residues, which allowed for increased binding to the lectins used in these assays. To address this issue, we developed a multi-step protein A/G incubation and filtration method to remove the contaminating signal. However, this method was time consuming and expensive so alternative methods were desired. Herein, we describe a simple method relying on PEG precipitation that allows for the removal of IgG and IgM but retention of glycoproteins of interest. This method was tested on three sample sets, two internal and one external. PEG depletion of heterophilic IgG and IgM reduced in the coefficient of variation as observed with the protein A/G filtration method from 26.82% to 7.50% and allowed for the measurement of fucosylated protein. This method allowed for the measurement of fucosylated kininogen, which could serve as a biomarker of HCC. In conclusion, a new and simple method for the depletion of heterophilic IgG and IgM was developed and allowed for the analysis of fucosylated kininogen in patients with liver disease.
AB - Hepatocellular carcinoma (HCC) accounts for >700,000 deaths worldwide, largely related to poor rates of diagnosis. Our previous work identified glycoproteins with increased levels of fucosylation in HCC. Plate-based assays to measure this change were compromised by increased levels of heterophilic antibodies with glycan lacking terminal galactose residues, which allowed for increased binding to the lectins used in these assays. To address this issue, we developed a multi-step protein A/G incubation and filtration method to remove the contaminating signal. However, this method was time consuming and expensive so alternative methods were desired. Herein, we describe a simple method relying on PEG precipitation that allows for the removal of IgG and IgM but retention of glycoproteins of interest. This method was tested on three sample sets, two internal and one external. PEG depletion of heterophilic IgG and IgM reduced in the coefficient of variation as observed with the protein A/G filtration method from 26.82% to 7.50% and allowed for the measurement of fucosylated protein. This method allowed for the measurement of fucosylated kininogen, which could serve as a biomarker of HCC. In conclusion, a new and simple method for the depletion of heterophilic IgG and IgM was developed and allowed for the analysis of fucosylated kininogen in patients with liver disease.
KW - Biomarker
KW - Glycosylation
KW - Hepatocellular carcinoma
KW - Lectin, liver cance
UR - http://www.scopus.com/inward/record.url?scp=85054361680&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85054361680&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2018.08.010
DO - 10.1016/j.jim.2018.08.010
M3 - Article
C2 - 30144410
AN - SCOPUS:85054361680
SN - 0022-1759
VL - 462
SP - 59
EP - 64
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
ER -