Microfluidic measurement of antibody-antigen binding kinetics from low-abundance samples and single cells.
Anal Chem
; 82(20): 8671-9, 2010 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-20857931
ABSTRACT
We present a simple microfluidic fluorescence bead assay for accurately measuring antibody-antigen binding kinetics with a standard inverted fluorescent microscope. We measured association and dissociation rate constants from antibody-antigen interactions spanning nearly 4 orders of magnitude in equilibrium binding affinity (30 pM-100 nM). Two versions of this assay are presented, which allow for dissociation rate measurements either directly, by use of fluorescently labeled antigen, or indirectly, by use of unlabeled antigen. We also demonstrate simultaneous, multiplexed binding measurements of multiple antibody-antigen interactions using a combination of spectral separation and spatial localization. Complete antibody-antigen binding kinetics were measured for as little as 8 × 104 antibody molecules (~132 zeptomoles) immobilized on a single bead and less than 2 × 106 antibodies (~3 attomoles) loaded into the microfluidic device, a reduction in detection limit and sample consumption of 4 orders of magnitude when compared to surface plasmon resonance (SPR) spectroscopy and alternative measurement techniques. We show that the microfluidic bead assay, when combined with small volume compartmentalization, enables direct measurement of antigen binding kinetics of antibodies secreted from single hybridoma cells. We anticipate that this assay will be useful as a routine analytical tool for studying molecular interactions as well as for screening primary antibody-secreting plasma cells isolated from immunized animals.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Microfluídica
/
Reações Antígeno-Anticorpo
Tipo de estudo:
Evaluation_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article