7 research outputs found
Comparison of the icELISA and HPLC method for quantitation of artemether in commercial drugs.
a<p>Each sample was analyzed in triplicate.</p>b<p>Data represent mean ± SD. The unit for ELISA and HPLC data was mg mL<sup>−1</sup>. The theoretical content of all drugs was 2 mg mL<sup>−1</sup>.</p
Cross reactivities of the icELISA with commonly used antimalarial drugs.
a<p>Cross-reactivity (%) = (IC<sub>50</sub> of artemether/IC<sub>50</sub> of other compound)×100.</p>b<p>Data represent means of triplicate ± SD.</p>c<p>No inhibitions were observed up to 20,000 ng mL<sup>−1</sup> of the analytes.</p
Bland-Altman bias plots for ELISA and HPLC.
<p>Quantitating artemether drugs concentration expressed as mg mL<sup>−1</sup>. The solid line represents the bias between the assays, and the dashed lines represent the bias ±1.96-s limits.</p
Standard inhibition curve of artemether in icELISA format.
<p>B<sub>0</sub> and B are absorbance in the absence and presence of competitors, respectively. Concentration causing 50% inhibition by artemether was 3.70 ng mL<sup>−1</sup>. Each value represents the mean of three replicates.</p
Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?
Soluble epoxide hydrolase
(sEH) is a potential pharmacological
target for treating hypertension, vascular inflammation, cancer, pain,
and multiple cardiovascular related diseases. A variable domain of
the heavy chain antibody (termed single domain antibody (sdAb), nanobody,
or VHH) possesses the advantages of small size, high stability, ease
of genetic manipulation, and ability for continuous manufacture, making
such nanobody a superior choice as an immunoreagent. In this work,
we developed an ultrasensitive nanobody based immunoassay for human
sEH detection using polymeric horseradish peroxidase (PolyHRP) for
signal enhancement. Llama nanobodies against human sEH were used as
the detection antibody in sandwich enzyme linked immunosorbent assays
(ELISA) with polyclonal anti-sEH as the capture antibody. A conventional
sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin
(HA) tag as the tracer showed a marginal sensitivity (0.0015 optical
density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL.
However, the introduction of the PolyHRP as the tracer demonstrated
a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold
decrease in LOD (0.05 ng/mL). Systematic comparison of three different
tracers in four ELISA formats demonstrated the overwhelming advantage
of PolyHRP as a label for nanobody based immunoassay. This enhanced
sEH immunoassay was further evaluated in terms of selectivity against
other epoxide hydrolases and detection of the target protein in human
tissue homogenate samples. Comparison with an enzyme activity based
assay and a Western blot for sEH detection reveals good correlation
with the immunoassay. This work demonstrates increased competiveness
of nanobodies for practical sEH protein detection utilizing PolyHRP.
It is worthwhile to rediscover the promising potential of PolyHRP
in nanobody and other affinity based methods after its low-profile
existence for decades
Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays
Single domain heavychain binders
(nanobodies) obtained from camelid
antibody libraries hold a great promise for immunoassay development.
However, there is no simple method to select the most valuable nanobodies
from the crowd of positive clones obtained after the initial screening.
In this paper, we describe a novel nanobody-based platform that allows
comparison of the reactivity of hundreds of clones with the labeled
antigen, and identifies the best nanobody pairs for two-site immunoassay
development. The output clones are biotinylated in vivo in 96-well
culture blocks and then used to saturate the biotin binding capacity
of avidin coated wells. This standardizes the amount of captured antibody
allowing their sorting by ranking their reactivity with the labeled
antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen,
we were able to classify 96 clones in four families and confirm this
classification by sequencing. This provided a criterion to select
a restricted panel of five capturing antibodies and to test each of
them against the rest of the 96 clones. The method constitutes a powerful
tool for epitope binning, and in our case allowed development of a
sandwich ELISA for sEH with a detection limit of 63 pg/mL and four
log dynamic range, which performed with excellent recovery in different
tissue extracts. This strategy provides a systematic way to test nanobody
pairwise combinations and would have a broad utility for the development
of highly sensitive sandwich immunoassays