An immunoreactor-based competitive fluoroimmunoassay for monitoring staphylococcal enterotoxin B using bioconjugated quantum dots

Abstract

Extensive research on avian systems has proved hens as an alternate source for polyclonal antibody generation necessary for immunosensing applications. Herein, we present the immobilization of avian antibody raised against staphylococcal enterotoxin B (SEB) and its applicability for a competitive fluoroimmunoassay technique. White leghorn hens immunized with SEB generated high affinity antibodies with a highest yield of 3.2 mg ml⁻¹ having affinity constant of 0.976 × 10¹⁰ M l⁻¹. A competitive fluoroimmunoassay format was developed comprising CdTe₅₅₇ as a fluorescence detector for monitoring SEB, a bacterial super-antigen. CdTe₅₅₇ was bioconjugated to SEB according to the carbodiimide protocol and confirmed by absorption spectral analysis. An immunoreactor column was designed by immobilizing anti-SEB antibodies and was successfully employed as an efficient bio-recognition tool. An immuno-affinity reaction involving competitive binding between free SEB and CdTe₅₅₇-bioconjugated SEB for immobilized antibody was relied upon to attain assay specificity and sensitivity. It was possible to quantify SEB from 1000 to 10 ng based on the integrated fluorescence of the SEB–CdTe₅₅₇ bioconjugate eluted from the immunoreactor column with a limit of detection of 8.15 ng and a regression coefficient R² = 0.9925. Thus, integration of QDs with immuno-affinity reactions revealed the versatility of nanoparticles as a potential fluorescence label for bioanalytical applications.