Biomarkers play an important role in the detection at an early stage of
pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting
Hippocalcin-like 1 (HPCAL1),
phosphatidylethanolamine binding protein 1 (PEBP1),
lectin galactoside-binding soluble 7 (LGALS7), and
serpin peptidase inhibitor clade E member 2 (SERPINE2) as
biomarkers for
pancreatic cancer detection in a single assay and to investigate
antibodies' specificity and cross-reactivity. Capture
antibodies against HPCAL1, PEBP1, LGALS7 and SERPINE2 were printed on
nitrocellulose coated glass slides. HPCAL1, PEBP1, LGALS7 and SERPINE2
proteins with different concentrations were incubated with the capture
antibodies at different temperatures for different time periods. Biotinylated detection
antibodies recognizing a different
epitope on the captured
proteins and a secondary detection molecule (
Streptavidin-PE) were used to detect fluorescent signals. The arrays showed the strongest signals when the concentration of the capture
antibodies was at 500 µg/mL in PBST0.05 (PBS with 0.05%
Tween-20), and the slides were incubated overnight at 4°C. The lowest
protein concentration for detection was 2 ng/mL. Each antibody demonstrated high specificity to the corresponding
antigen in detecting a mixture of 4
proteins without significant cross-reactivity. The fluorescence and
biomarker concentration displayed a linear correlation. The antibody microarray system could be a useful tool for potential
biomarker detection for
pancreatic cancer.