Edward Wellner

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 CVs 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.

A chip-based immunoaffinity capillary electrophoresis assay for assessing hormones in human biological fluids

A chip‐based capillary electrophoresis system has been designed for assessing the concentrations of four hormones in whole human blood, saliva, and urine. The desired analytes were isolated by immunoextraction using a panel of four analyte‐specific antibodies immobilized onto a glass fiber insert within the injection port of the chip. Following extraction, the captured analytes were labeled prior to electro‐elution into the chip separation channel, where they were resolved into four individual peaks in circa 2 min. Quantification of each peak was achieved by on‐line LIF detection and integration of the area under each peak. Comparison to commercial high‐sensitivity immunoassays demonstrated that the chip‐based assay provided fast, accurate, and precise measurements for the analytes under investigation. As the availability of commercially available antibodies rapidly expands, the application of this system will greatly increase. Chip‐based CE separations of multiple analytes from a single sample also provide a significant advantage in the analysis of small samples.

Detection of Cerebral Spinal Fluid-Associated Chemokines in Birth Traumatized Premature Babies by Chip-Based Immunoaffinity CE

A major concern in treating premature infants with birth‐associated head trauma is the rapid determination of reliable biomarkers of neuroinflammation. To this end a chip‐based immunoaffinity CE device has been applied to determine the concentrations of inflammation‐associated chemokines in samples of cerebral spinal fluid collected from such subjects. The chip utilizes replaceable immunoaffinity disks, to which reactive antibody fragments (FAb) of six antichemokine‐specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labeled in situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on‐line by LIF detection and the results compared to standard curves produced by running known chemokine standards through the immunoaffinity system. The complete processing of a sample took 10 min with separation of all six analytes being achieved in less than 2 min. The system compared well to commercial ELISA, analysis of the results by linear regression demonstrating r2 values in the range of 0.903–0.978, and intra and interassay CV of the migration times and the measured peak areas being less than 2.3 and 5%, respectively. Application of the system to analysis of cerebrospinal fluid from head traumatized babies clearly indicated the group with mild trauma versus those with severe injury. Additionally, CE analysis demonstrated that the severe trauma group could be divided into individuals with good and poor prognosis, which correlated with the clinical finding for each patient.

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 r2 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.

Measurement of Inflammatory Chemokines in Micro-dissected Tissue Biopsy Samples by Chip-Based Immunoaffinity Capillary Electrophoresis

To aid in the biochemical analysis of human skin biopsies, a chip-based immunoaffinity capillary electrophoresis (ICE) system has been developed for measuring inflammatory chemokines in micro-dissected areas of the biopsy. Following isolation of the areas of interest, the tissue was solubilized and the analytes of interest were isolated by the immunoaffinity disk within the chip. The captured analytes were labeled in situ with a 635 nm light-emitting laser dye and electro-eluted into the chip separation channel. Electrophoretic separation of all of the analytes was achieved in 2.5 min with quantification of each peak being performed by online LIF detection and integration of each peak area. The degree of accuracy and precision achieved by the chip-based system is comparable to conventional immunoassays and the system is robust enough to be applied to the analysis of clinical samples. Further, with the expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different biomedical and clinical analyses.