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Ultrasensitive Immunoassay for Ex‐Vivo Human Soluble Epoxide Hydrolase Detection
Author(s) -
Li Dongyang,
Cui Yongliang,
Morisseau Christophe,
Bellien Jérémy,
Hammock Bruce
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.558.5
Subject(s) - immunoassay , chemistry , epoxide hydrolase 2 , horseradish peroxidase , ex vivo , polyclonal antibodies , detection limit , chromatography , antibody , in vivo , microbiology and biotechnology , biochemistry , enzyme , biology , in vitro , immunology
Soluble epoxide hydrolase (sEH) is a potential pharmacological target for the treatment of hypertension, inflammation, cancer, pain and multiple cardiovascular related diseases and a marker of inflammation. Detecting and quantifying sEH appears an important diagnostic tool for numerous pathologies. However, current methods rely on expensive technologies such as radioactive markers or mass spectrometry that are available by the bench side. Here, we report the development of an ultrasensitive nanobody based immunoassay for human sEH ex vivo. Nanobodies are the variable domain of the heavy chain antibody, which permit high stability, ease of genetic manipulation, bacterial expression, and ability for continuous manufacture, thus providing a constant high quality biochemical reagent. Llama nanobodies against non‐denatured human sEH were used as the detection antibody in a sandwich ELISA format with polyclonal anti‐sEH as the capture antibody. To enhance the reporting signal, the conventional HRP labeled anti‐HA tag tracer was replaced with polymeric horseradish peroxidase (PolyHRP) reporter system. This yields a 141‐fold increase in the sensitivity and 57‐fold decrease in limit of detection. Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. The 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 a radioactive enzyme activity based assay and a Western‐blot for sEH detection reveals good correlation with the immunoassay. Moreover, the enhanced ELISA was used successfully to detect the sEH level in peripheral blood mononuclear cells isolated from 90 patients with different disease status, which have in general very low abundance of sEH hard to detect by classic methods. The work demonstrates the promise of nanobody based ELISA as a diagnostic tool for detection of and other pharmacologically and toxicologically important proteins. Support or Funding Information National Institute of Health (Superfund P42ES04699 and R01 ES002710) and National Natural Science Foundation of China (81402722) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .