posted on 2006-07-07, 00:00authored byCecilia Eriksson, Charlotta Agaton, Rikard Kånge, Mårten Sundberg, Peter Nilsson, Bo Ek, Mathias Uhlén, Magnus Gustafsson, Sophia Hober
A new and flexible technology for high throughput analysis of antibody specificity and affinity is
presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which
the centrifugal force moves fluids through microstructures containing immobilized metal affinity
chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to
the column via a genetically attached His6-tag. The antibodies to be analyzed are applied onto the
columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies,
and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling
analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through
the three-dimensional visualization of the binding patterns in a column it is possible to separate high
affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible
and reproducible.
Keywords: microfluidics • miniaturization • compact disk • biochip • gyrolab • antibody specificity