Receptor affinity CE for measuring bioactive inflammatory cytokines in human skin biopsies.
A chip-based receptor affinity CE system has been employed to measure the concentrations of bioactive pro-inflammatory cytokines in biopsy materials obtained from human atopic skin lesions. The device employs a replaceable affinity disk to which recombinant cytokine receptors have been chemically immobilized. Homogenates obtained from micro-dissected human skin samples were injected into the system where the bioactive cytokines were captured in the receptor affinity port and labeled in situ with a laser dye. The captured cytokines were released and separated by CE, the resolved peaks being detected and measured by laser-induced fluorescence. When compared with conventional cell-based bioassays, the affinity receptor chip showed reasonable correlation with r(2) values of 0.998 for interferon gamma, 0.994 for IL-6 and 0.991 for tumor necrosis factor alpha. The complete process including cytokine capture, labeling, and analysis took approximately 12.5 min with intra- and inter-assay CVs below 5.3% and recoveries of 84.9-98.4% at the 100 pg/mL concentration in buffer solutions and 84.5-95% in normal human tissue extract. The system could indicate clear differences between the various clinical stages of atopic dermatitis in human patients and could run 4-6 samples per hour. This system, like previous chip-based systems designed in our laboratory, 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
Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA. phillipt@mail.nih.gov
Kalish
Heather
H
Wellner
Edward
E
D000328:Adult; D001706:Biopsy; D016207:Cytokines / Q000032:analysis*; D003876:Dermatitis, Atopic / Q000473:pathology; D046209:Electrophoresis, Microchip / Q000379:methods*; D005629:Frozen Sections; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis; D015850:Interleukin-6 / Q000032:analysis; D018121:Receptors, Cytokine / Q000737:chemistry*; D012867:Skin / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D016207:Cytokines; D015850:Interleukin-6; D018121:Receptors, Cytokine; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma