ISSN:
0173-0835
Keywords:
Affinity capillary electrophoresis
;
Microfluidics
;
Immunoassay
;
Affinity constant
;
Microchip
;
Ascites fluid
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
The affinity constant of a monoclonal antibody to fluorescently labeled bovine serum albumin (BSA*) was measured in diluted mouse ascites fluid using a microfluidic chip to perform affinity capillary electrophoresis. Borofloat glass-based devices could be used repeatedly with samples for many months. On-chip separations were performed in less than 60 s, and 30-60 s was required for manual sample exchange. The change in peak height for BSA* with increasing BSA*/anti-BSA concentration ratio was used to determine concentration changes in bound and free BSA*. A Scatchard plot analysis gave an affinity constant (more exactly the intrinsic association constant) of 3.5 ± 0.6 × 107 M-1 for a 1:1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1:1 stoichiometric ratio. The other complex is proposed to have a stoichiometry with an excess of anti-BSA to BSA*, most likely (anti-BSA)2-BSA*, on the basis of a faster migration time than the 1:1 complex, a decrease in the amount of this complex with increasing [BSA*], and predictions of theoretical models for multi-valent antigens. Potential applications of microchip-based devices in affinity measurements are discussed.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150191641
Permalink
