3 research outputs found
Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition
On the basis of the structural features
of (fluoro)Âquinolones (FQs),
pazufloxacin was first used as a generic immunizing hapten to raise
a broad-specificity antibody. The obtained polyclonal antibody exhibited
broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs.
Furthermore, the antibody was able to recognize these FQs below their
maximum residue limits (MRLs) in an indirect competitive chemiluminescence
enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging
from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with
spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range
of 84.6–106.9% and an acceptable coefficient of variation below
15%, suggesting its suitability and reliability for the use of a promising
tool to detect FQs. Meanwhile, comparative molecular field analysis
(CoMFA) and comparative molecular similarity indices analysis (CoMSIA)
models, with statistically significant correlation coefficients (<i>q</i><sup>2</sup><sub>CoMFA</sub> = 0.559, <i>r</i><sup>2</sup><sub>CoMFA</sub> = 0.999; <i>q</i><sup>2</sup><sub>CoMSIA</sub> = 0.559, <i>r</i><sup>2</sup><sub>CoMSIA</sub> = 0.994), were established to investigate the antibody recognition
mechanism. These two models revealed that in the antibody, the active
cavity binding FQs’ 7-position substituents worked together
with another cavity (binding FQs’ 1-position groups) to crucially
endow the high cross-reactivity. This investigation will be significant
for better exploring the recognition mechanism and for designing new
haptens
Molecular Modeling Application on Hapten Epitope Prediction: An Enantioselective Immunoassay for Ofloxacin Optical Isomers
To deepen our understanding of the
physiochemical principles that
govern hapten–antibody recognition, ofloxacin enantiomers were
chosen as a model for epitope prediction of small molecules. In this
study, two monoclonal antibodies (mAbs) mAb-WR1 and mAb-MS1 were raised
against <i>R</i>-ofloxacin and <i>S</i>-ofloxacin,
respectively. The enantioselective mAbs have a high sensitivity and
specificity, and the enantioselectivity is not affected by heterologous
coating format reactions. The epitopes of the ofloxacin isomers were
predicted using the hologram quantitative structure–activity
relationship (HQSAR) and comparative molecular field analysis (CoMFA)
approaches. The results consistently show that the epitope of the
chiral hapten should be primarily composed of the oxazine ring and
the piperazinyl ring and mAbs recognize the hapten from the side of
this moiety. The enantioselectivity of mAbs is most likely due to
the steric hindrance caused by the stereogenic center of the epitope.
Modeling of chiral hapten–protein mimics reveals that ofloxacin
isomers remain upright on the surface of the carrier protein. Suggestions
to improve the enantioselectivity of antibodies against ofloxacin
isomers were also proposed. This study provided a simple, efficient,
and general method for predicting the epitopes of small molecules
via molecular modeling. The epitope predictions for small molecules
may create a theoretical guide for hapten design
Investigation of an Immunoassay with Broad Specificity to Quinolone Drugs by Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Data Sets and Advanced Quantitative Structure–Activity Relationship Analysis
A polyclonal antibody against the
quinolone drug pazufloxacin (PAZ)
but with surprisingly broad specificity was raised to simultaneously
detect 24 quinolones (QNs). The developed competitive indirect enzyme-linked
immunosorbent assay (ciELISA) exhibited limits of detection (LODs)
for the 24 QNs ranging from 0.45 to 15.16 ng/mL, below the maximum
residue levels (MRLs). To better understand the obtained broad specificity,
a genetic algorithm with linear assignment of hypermolecular alignment
of data sets (GALAHAD) was used to generate the desired pharmacophore
model and superimpose the QNs, and then advanced comparative molecular
field analysis (CoMFA) and advanced comparative molecular similarity
indices analysis (CoMSIA) models were employed to study the three-dimensional
quantitative structure–activity relationship (3D QSAR) between
QNs and the antibody. It was found that the QNs could interact with
the antibody with different binding poses, and cross-reactivity was
mainly positively correlated with the bulky substructure containing
electronegative atom at the 7-position, while it was negatively associated
with the large bulky substructure at the 1-position of QNs