Microfluidic analysis of antibody specificity in a compact disk format.
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.
Eriksson
Cecilia
C
Royal Institute of Technology, AlbaNova University Center, Department of Biotechnology, SE-106 91 Stockholm, Sweden.
Agaton
Charlotta
C
Kånge
Rikard
R
Sundberg
Mårten
M
Nilsson
Peter
P
Ek
Bo
B
Uhlén
Mathias
M
Gustafsson
Magnus
M
Hober
Sophia
S
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D016250:Compact Disks; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods*; D008904:Miniaturization; D040081:Protein Array Analysis; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000906:Antibodies