Identification of IgM as a contaminant in lectin-FLISA assays for HCC detection

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.

Changes in the Glycosylation of Kininogen and the Development of a Kininogen-Based Algorithm for the Early Detection of HCC

“…Fucosylated proteins were detected using a lectin-fluorescently linked immune-absorbent assay (FLISA) method that involves the antibody capture of specific proteins, i.e., kininogen (Abcam, Cambridge, MA, Cat# ab79650) or A1AT (Sigma-Aldrich, St. Louis, MO, Cat #: A0409) followed by detection of specific N-linked glycan with a sugar binding protein called a lectin, in this case the AAL (Vector Laboratories, Burlingame, CA). Details regarding this assay can be found in our previous publications(23, 24). GP73 was measured by western blot as done previously(25).…”

Biomarker analysis of fucosylated kininogen through depletion of lectin reactive heterophilic antibodies in hepatocellular carcinoma

“…The basic design of the commonly used lectin FLISA is described elsewhere[6]. Liver fibrosis (and cirrhosis) is associated with increased levels of heterophilic immunoglobulins and significantly, an alteration in the glycosylation of these molecules[15–17]. This change in glycosylation increases their reactivity to fucose binding lectins.…”

“…Two methods to deal with these lectin reactive heterophilic antibodies were used. For the protein A/G method[15–17], a Pall Omega Nanosep 100K spin filter (Pall Corporation, #OD100C35) is washed twice with 300 µ l of X PBS to prepare the filter. Subsequently, 150 µ l of Pierce Protein A/G Plus™ (Pierce Chemicals, # 20423/4) is added to the filter, spun at 14,000g for 5 minutes and the Protein A/G beads re-washed twice in 300 µ l 1X PBS.…”

“…Two assays for the measurement of fucosylated kininogen were performed. Our plate based assays that utilized detection of bound biotinylated lectin using IRDyelabelled streptavidin and visualized using the LI-COR Odyssey imaging system [8,14,7,18,19,15,12] and a second method using more conventional horse radish peroxidase-labelled streptavidin where development occurs with 3,3’,5,5’-tetramethylbenzidine (TMB) chromogenic substrate. In this case, fucosylated protein standards (66%, 55%, 44%, 33%, 22% 11% 7% and 0%) are used to generate the calibration curve and assign %fucosylation values for each sample.…”

“…We have previously identified heterophilic IgG and IgM as contaminants in a plate based lectin assay[15]. Our initial method to remove these factors involved depletion using Pierce Protein A/G Plus™ agarose and a 100K spin filter[15]. This method was complicated, costly and not suitable for high throughput clinical use.…”