Abstract
Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10−4 to 102 ng/mL).
Highlights
Human tissue kallikrens are secreted from human proteases with diverse expression and physiological roles [1]
We previously developed a P-Si antibody microarray platform for analyzing prostate specific antigen (PSA) in serum [24] and α-synuclein in cerebrospinal fluid (CSF) [35] with high sensitivity and reproducibility
We investigate the cross-reaction between human kallikrein 2 (hK2) capture antibodies and PSA antigen since PSA and hK2 antigen have high homology [9,10]
Summary
Human tissue kallikrens (hKs) are secreted from human proteases with diverse expression and physiological roles [1]. As a biomarker of prostate cancer, the levels of prostate specific antigen (PSA or hK3) in the serum, originating from leakage of pathological tissue to the vascular system, are routinely measured, and have been shown to be proportional to the tumor burden [3]. There are strong arguments challenging the usefulness of PSA screening tests because of the large discrepancies between decreasing disease aggressiveness and increasing levels of treatment This has led to widespread criticism that prostate cancer is an “overdiagnosed” and “overtreated” cancer [6]. We previously developed a P-Si (porous silicon) antibody microarray platform for analyzing prostate specific antigen (PSA) in serum [24] and α-synuclein in cerebrospinal fluid (CSF) [35] with high sensitivity and reproducibility.
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