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

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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.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the incidence in the United States (USA) is increasing [1], [2]. The progression of liver disease into liver cancer has been monitored with serum levels of alpha-fetoprotein (AFP). However, AFP’s limited sensitivity and specificity has resulted in the questioning of AFP as a primary screen for HCC [3] and more sensitive biomarkers for HCC are desired.

Using fucose-specific lectins we have previously identified more than 50 glycoproteins that contained increased fucosylation with HCC [4] and have used these in plate-based assays to diagnosis HCC [5], [6], [7]. While this method was applicable to a number of independent patient sets, it was unable to specifically detect fucosylated glycoforms in certain patient samples. That is, some material was present in serum that led to non-specific signals in the lectin-FLISA [8], [9]. In the current study, we have identified the contaminating lectin reactive factors present in the serum. This lectin reactive factor was shown to be IgM and when this was removed from the serum prior to lectin-FLISA, specific glycoprotein associated lectin reactive signal could be detected. This method was used in two independent sample sets to validate the method and also to validate the performance of the fucosylated glycoforms as biomarkers of HCC. The potential use of this method as a diagnostic tool for the detection of liver cancer is discussed.

Section snippets

Patient samples

Serum samples were obtained from the University of Michigan and the University of California San Diego under a study protocol approved by the respective Institutional Review Board and written informed consent was obtained from each subject. Patients details regarding samples from the University of Michigan are found in our previous publication [10]. Detailed information regarding patients from the University of California at San Diego are found in Supplementary Table 2.

Lectin FLISA

The traditional lectin

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

We have previously identified proteins that become hyper-fucosylated in HCC and developed a plate-based assay to assess the level of fucosylation in samples [5], [12]. This method worked well in many sample set but in others, was unable to detect a lectin reactive signal that could be attributed specifically to the protein that was captured [10]. An example of such a result is shown in Fig. 1A. Here a lectin-FLISA was performed for fucosylated alpha-1-anti-trypsin (A1AT). As Fig. 1A show, when

Discussions

Glycosylation changes have been observed in many cancers, including but not limited to liver [13], [14], [15], [16], pancreas [17], [18], [19], [20], lung [21], [22], breast [23], ovarian [24], colon [25] and prostate cancer [26], [27]. In many, proteins that contain these glycan changes have been identified. If these changes are to be used clinically, they must be examined in a rapid and in-expensive manner. To that end, several plate-based systems, have been developed. These methods all

Acknowledgements

This work was supported by grants R01 CA120206 (ASM) and U01 CA168856 (ASM).

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