Avian Antibodies for Staphylococcal Enterotoxin B as an Efficient Tool for FRET-Based Fluoroimmunosensor

In recent years, antibodies have become an indispensable
tool in research, diagnostic, therapy and other laboratory
applications. In this direction, hens have become an
alternate source for polyclonal antibody production necessary
for immunosensing applications. Herein, we present generation
of avian antibody (IgY) against staphylococcal enterotoxin
B (SEB) used as an efficient bio-recognition molecule while
developing Forster resonance energy transfer (FRET)-based
fluorescence inhibition assay format for monitoring SEB, a
bacterial super-antigen responsible for food poisoning.
Immunization of white leghorn hens with SEB, generated
antibodies (IgY) with highest yield of 51.1 mg in
hyperimmune egg having affinity constant of 1.81×1010 M−1
that was bioconjugated with gold nanoparticle (GNP) by electrostatic
interaction. Quantum dot (QD, CdTe523) and GNP
bioconjugated to SEB and anti-SEB IgY, respectively, were
used as donor–acceptor pair during FRET to study resonance
interactions and fluorescence quenching in an
immunocomplex. The mutual affinity between IgY-GNP and
SEB-CdTe523 was made use to obtain efficient energy transfer
between respective chromophores. The presence of SEB in the
range 1,000 to 20 ng mL−1 varied the rate of fluorescence
quenching due to FRET in SEB-CdTe523 demonstrating efficiency
of fluorescence inhibition assay. It was possible to
achieve a detection limit of 8.53 ng mL−1 with a regression
coefficient R2=0.9813. Thus, bioconjugation of nanoparticles
demonstrated their efficiency in sensitive monitoring of SEB
through immuno-affinity reactions to address the potential
health risk and economic impacts of staphylococcal food
poisoning.