Chip-based immunoaffinity CE: application to the measurement of brain-derived neurotrophic factor in skin biopsies.
A chip-based immunoaffinity CE system has been employed to measure the concentrations of brain-derived neurotrophic factor in human skin biopsies, taken during atopic inflammatory events. The device employs a replaceable immunoaffinity disk to which capture antibodies have been chemically immobilized. Homogenates obtained from micro-dissected human skin samples were injected into the system, where the analyte of interest was captured in the immunoextraction port, thus allowing non-reactive materials to be removed prior to analysis. The captured analyte was labeled in situ with a red-emitting laser dye before being released from the capture antibody, separated by electrophoresis, and the resolved peaks detected by online LIF. Comparison of this chip-based system with conventional immunoassay demonstrated good correlation when analyzing both standards and patient samples. The system was semi-automated resulting in a CE analysis within 1.5 min and a total of circa 5 min. Intra- and inter-assay CV's of 3.85 and 4.19 were achieved with circa 98.8% recovery of brain-derived neurotrophic factor at a concentration of 100 pg/mL. The assay demonstrated clear differences between clinical stages of atopic dermatitis in human patients and could run 10-15 samples per hour. This system holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.
Phillips
Terry M
TM
Ultramicro Immunodiagnostics Section, Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD 20892, USA. phillipt@mail.nih.gov
Wellner
Edward F
EF
D000328:Adult; D001706:Biopsy; D019208:Brain-Derived Neurotrophic Factor / Q000032:analysis*; D003876:Dermatitis, Atopic / Q000378:metabolism / Q000473:pathology; D019075:Electrophoresis, Capillary / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007249:Inflammation / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology
D019208:Brain-Derived Neurotrophic Factor