chromatography • spectroscopy 29
equilibrium dissociation constant determined by CG-MALS, KD
= 8nM, agrees well with the manufacturer’s data of KD
= 15nM,
as measured by ELISA. Furthermore, both thrombin and the antibody
exhibit no propensity for self- association, a finding that could not be evaluated by conventional ELISA or SPR.
Tus, CG-MALS as automated by
the Calypso system provides direct observation of multiple interactions in solution. By measuring the weight-average molar mass of the ensemble of molecules in solution, the presence of both the (1 Ab):
(1 Tr) and (1 Ab):(2 Tr) species can be tracked as a function of Ab and Tr concentration.
In addition, the monomeric state of both the antibody and antigen is confirmed. Tis simultaneous tracking of all affinities and stoichiometries present in solution makes CG-MALS unique among biophysical characterisation techniques and enables unambiguous quantification of macromolecular interactions.
Sophia Kenrick, Application Development Engineer, and Daniel Some, Principal Scientist, are with Wyatt Technology
Corporation, Santa Barbara, CA, USA.
www.wyatt.com
NEW S-Live 1/2 page 11/20/06 12:27 PM Page 1
Fig. 3. Mole fraction of each species present during hetero-association gradient as a function of total thrombin and antibody concentrations.
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