A homogeneous time-resolved fluoroimmunoassay (TR-FIA) using antibody mediated luminescence quenching

Abstract

The determination of low-molecular weight substances (haptens) is demonstrated with a homogeneous time-resolved immunoassay using antibody-induced luminescence quenching. Our novel assay technology uses the newly developed monoclonal antibody (G24-BA9) to quench the luminescence of europium trisbipyridine (EuTBP). We performed a competitive biotin immunoassay including an EuTBP-biotin conjugate, the anti-EuTBP antibody G24-BA9 and streptavidin as assay components. Steric hindrance allows only the binding of either G24-BA9 (to the EuTBP moiety) or streptavidin (to the biotin moiety) to the EuTBP-biotin conjugate. Addition of the analyte biotin resulted in the binding of streptavidin to biotin and a concomitant preferred binding of G24-BA9 to EuTBP-biotin. Since G24-BA9 quenches the luminescence of EuTBP within the conjugate, the luminescence signal could be used to indicate and quantify the presence of free biotin in the system. All experiments were carried out in solution in the presence of 5% serum demonstrating the possibility of using our novel assay for a very fast determination of low molecular weight substances in biological fluids

Monoclonal antibody–europium conjugate-based lateral flow time-resolved fluoroimmunoassay for quantitative determination of T-2 toxin in cereals and feed

“…Time-resolved uorescence analysis based on lanthanide used as a marker has more advantages than traditional uorescence labeling. [23][24][25][26][27] Time-resolved means that uorescence signals are recorded aer attenuation in the uorescence of the background material. The Stokes shi of lanthanide is larger (as over 150 nm) and the uorescence lifetime of the marker is higher than that of the background material (5-6 orders of magnitude).…”

Dye Tool Box for a Fluorescence Enhancement Immunoassay

“…In quenching assays time-resolved fluorescence in combination with lanthanides is used to tackle this challenge. 61 The emission decay time of lanthanides is found in the μs to ms time range and is thus significantly longer than the emission decay times of any matrix constituent, which are on the nanosecond time scale. Therefore, lanthanides are frequently used as partners in sandwich fluorescence immunoassays.…”

“…A possible obstacle for applications of such homogeneous assays in biosensing or in clinical diagnostics is the influence of fluorescence from a “protein background” present in the sample matrix, so that measuring in serum or milk might be difficult. In quenching assays time-resolved fluorescence in combination with lanthanides is used to tackle this challenge . The emission decay time of lanthanides is found in the μs to ms time range and is thus significantly longer than the emission decay times of any matrix constituent, which are on the nanosecond time scale.…”