A Rapid Sample Pretreatment Protocol: Improved Sensitivity in the Detection of a Low-abundant Serum Biomarker for Prostate Cancer

Abstract

The detection of small and low abundant biomarkers in clinical samples remains a challenge, mainly due to the presence of high-abundant proteins, such as albumin (HSA) and immunoglobulin G (IgG), accounting for at least 65% of plasma proteins. 1 The depletion of these proteins could facilitate biomarker discovery and detection. Depletion should aim to provide the selective removal of high-abundant proteins, and concentrate components of low abundance. Commercially available methods for IgG and HSA depletion have enabled the visualization of new protein signals, the identification of gene products and improved detection of low-abundant proteins. [2][3][4] Most of the methods utilize either antibody-or dye-based resins, 3,5 molecular size filtration, organic solvents, and ammonium sulfate precipitation, as well as reverse phase and ion exchange chromatography. 5,6 Also available are resins cross-linked with proteins A and G, and their recombinants for IgG removal. 3,7 Antibody-based resins display higher specificity and efficiency, 4,8 but are more expensive than the dye-based resins, limiting their usage. Commercially available pre-packed columns lack the flexibility to accommodate issues pertaining to sample size, for example. In addition, some rely on use of pumps and other equipment to operate smoothly, 2,3 thus limiting their use in many health centers. Most studies on IgG/HSA depletion have focused on the detection of lowabundant proteins using electrophoresis and mass spectrometry.2,3,9 As far as we are aware, the effects of depletion on point-of-care-testing (POCT) immunochromatography membrane-based assays is yet to be reported. Further, an evaluation of the potential benefits of HSA depletion over IgG, and vice versa has received very little attention to date.In this report, we present (a) a versatile method for HSA and IgG depletion from quality control (QC) and human serum samples, (b) assess the effect of depletion on human chorionic gonadotropin (HCG) and prostate specific antigen (PSA) using immunochromatography test strips (ICTS), and (c) evaluate the effects of HSA depletion compared to that of IgG on HCG and PSA detection. We employed a dye-based removal of HSA using Cibacron Blue 3G-A Sepharose (SB). SB has a strong affinity towards HSA. 10 For the removal of IgG, we exploited the high affinity between the Fc region of antibodies and protein G using sepharose beads cross-linked with protein G (S-PG). 7 Experimental MaterialsSB and S-PG were purchased from GE Healthcare BioSciences AB (Uppsala, Sweden). Handee TM spin columns (500 μL) and IgG specific kit (Easy-Titre ® IgG assay kit) were purchased from Pierce (IL, USA). HSA blue fluorescent assay kit was from Active Motif Japan (Tokyo, Japan). Prostate specific antigen (PSA) was obtained from Chemicon International (Temecula, CA, USA). Unless otherwise stated, all other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All solutions were prepared and diluted using ultra-pure water (18.3 MΩ cm -1 ) from the Millipore Milli-Q ...

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