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

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 biorecognition
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 ¼ 0.9925. Thus, integration of QDs with immuno-affinity
reactions revealed the versatility of nanoparticles as a potential fluorescence label for bioanalytical
applications.