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 6 anti-chemokine-specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labelled in-situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on-line by laser-induced fluorescence 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 6 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 inter-assay coefficients of variance (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.