Development and optimization of an antibody array method for potential cancer biomarker detection

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.

HSP90B1-mediated plasma membrane localization of GLUT1 promotes radioresistance of glioblastomas

Ionizing radiation is a popular and effective treatment option for glioblastoma(GBM).However,resistance to radiation therapy inevitably occurs during treatment.It is urgent to investigate the mechanisms of radioresistance in GBM and to find ways to improve radiosensitivity.Here,we found that heat shock protein 90 beta family member 1(HSP90B1)was significantly upregulated in radioresistant GBM cell lines.More importantly,HSP90B1 promoted the localization of glucose transporter type 1,a key rate-limiting factor of glycolysis,on the plasma membrane,which in turn enhanced glycolytic activity and subsequently tumor growth and radioresistance of GBM cells.These findings imply that targeting HSP90B1 may effectively improve the efficacy of radiotherapy for GBM patients,a potential new approach to the treatment of glioblastoma.

Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

Murine monoclonal antibodies(mAbs)are widely used but have limitations if administered in humans.The use of chimeric or humanized mAbs can reduce immunogenicity.The first step in producing such mAbs is to clone murine variable genes from a hybridoma,but it is possible to amplify both functional and aberrant variable genes,as they coexist in the hybridoma.During the development of a murine–human chimeric antibody,we have cloned from a hybridoma the functional heavy chain variable region(VH)and light chain variable region(VL)genes of a mAb that blocks the binding of anthrax lethal factor to protective antigen.In this study,we report the detection of two aberrant transcripts from a hybridoma produced using myeloma cell line OUR-1,the development of a method to distinguish between the functional and abundant aberrant VL transcripts,and the origins of these aberrant genes.The aberrant VL gene is derived from OUR-1 cells,while the aberrant VH gene might derive from antibody repertoires in B cells or from gene rearrangement in the hybridoma cells.The aberrant VH and VL genes in this study may facilitate discrimination between the functional and aberrant variable genes from hybridoma cells.