Bright or dark immune complexes of anti-TAMRA antibodies for adapted fluorescence-based bioanalysis.
Anal Bioanal Chem
; 407(12): 3313-23, 2015 May.
Article
em En
| MEDLINE
| ID: mdl-25711988
ABSTRACT
Fluorescence labels, for example fluorescein or rhodamin derivatives, are widely used in bioanalysis applications including lateral-flow assays, PCR, and fluorescence microscopy. Depending on the layout of the particular application, fluorescence quenching or enhancement may be desired as the detection principle. Especially for multiplexed applications or high-brightness requirements, a tunable fluorescence probe can be beneficial. The alterations in the photophysics of rhodamine derivatives upon binding to two different anti-TAMRA antibodies were investigated by absorption and fluorescence-spectroscopy techniques, especially determining the fluorescence decay time and steady-state and time-resolved fluorescence anisotropy. Two monoclonal anti-TAMRA antibodies were generated by the hybridoma technique. Although surface-plasmon-resonance measurements clearly proved the high affinity of both antibodies towards 5-TAMRA, the observed effects on the fluorescence of rhodamine derivatives were very different. Depending on the anti-TAMRA antibody either a strong fluorescence quenching (G71-DC7) or a distinct fluorescence enhancement (G71-BE11) upon formation of the immune complex was observed. Additional rhodamine derivatives were used to gain further information on the binding interaction. The data reveal that such haptens as 5-TAMRA could generate different paratopes with equal binding affinities but different binding interactions, which provide the opportunity to adapt bioanalysis methods including immunoassays for optimized detection principles for the same hapten depending on the specific requirements.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Rodaminas
/
Espectrometria de Fluorescência
/
Anticorpos Monoclonais
Limite:
Animals
Idioma:
En
Revista:
Anal Bioanal Chem
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Alemanha