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This report describes a method of controlling the sensitivity and reproducibility of a microchip-based immunoassay by using isotachophoresis to preconcentrate the antigen and antibody prior to binding. Gel electrophoresis separation is coupled to the preconcentration step to separate the immunocomplex products formed. The system employs a quartz-based LabChip that automates the metering, preconcentration, reaction, separation, and detection. The system also uses a handoff mechanism that switches the immunocomplex from the stacking mode to the separation mode. We show that the handoff timing affects the data quality and repeatability of the electropherograms, and we demonstrate an automatic handoff mechanism to precisely control the signal intensity and separation of peaks of interest. In so doing, the automatic handoff mechanism also improves the reproducibility of the assay. When applied to the homogeneous liquid-phase detection of [alpha]-fetoprotein, a common tumor marker, the system shows a greater than 200-fold stacking of specific analytes of interest.

The kinetics of T-cell activation employing exogenous antigenic peptides in the presence of antigen-presenting cells (APCs) was studied. Extracellular acidification rate increased immediately when A.E7 T cells and 4R3.9 T cells were exposed to their cognate peptides in the presence of CH27 cells. Triggering events were completely inhibited when the cloned T cells were pretreated with herbimycin. Use of stipulated kinetic constraints with IA(super k) shows that at the minimum peptide levels, the kinetics of T-cell triggering is limited by kinetics of peptide binding to major histocompatibility complex class II molecules.

The epsilon isoenzyme of protein kinase C (PKC) was found to take part in the signal transduction pathway of granulocyte-macrophage colony-stimulating factor (GM-CSF) in TF-1 human bone marrow cells. Microphysiometer analysis revealed that exposure of TF-1 cells to GM-CSF resulted in a burst of metabolic activity. In addition, treatment with isoenzyme-specific antisense oligonucleotides inhibited the expression of PKC alpha and epsilon by TF-1 cells. However, only the inhibition of PKC epsilon resulted in decrease in the burst of metabolic activity induced by GM-CSF.