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