Dataset Information

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

ABSTRACT: 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.

SUBMITTER: Li D

PROVIDER: S-EPMC5611449 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

Li Dongyang D Cui Yongliang Y Morisseau Christophe C Gee Shirley J SJ Bever Candace S CS Liu Xiangjiang X Wu Jian J Hammock Bruce D BD Ying Yibin Y

Analytical chemistry 20170510 11

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 u ...[more]

PMID: 28460522

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Project description:BACKGROUND:Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. RESULTS:In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R2?=?0.914) was found between the percent competitive inhibition values of cELISA and HI titers. CONCLUSIONS:A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.

Dataset Information

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

Publications

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

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