Submitted to: Analytical Chemist
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2017
Publication Date: 5/2/2017
Citation: Li, D., Cui, Y., Morisseau, C., Gee, S., Bever, C.R., Liu, X., Wu, J., Hammock, B., Ying, Y., Stanker, L.H. 2017. Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP. Analytical Chemist. 14:24-30. doi: 10.1021/acs.analchem.7b01247. Interpretive Summary: The ability to detect and quantify low amounts of specific proteins are needed for diagnostic evaluations. Antibodies are a common tool for detecting specific targets, or antigens. An enzyme reporter generates a visual signal of the detection event. This study evaluates the feasibility of combining two detection technologies: a novel heavy chain only antibody (called a nanobody) with a multiplexed reporter enzyme (polyHRP). By combining these two reagents, the developed tool demonstrated a vast improvement in the detection of the target protein, soluble epoxide hydrolase, compared to previous methods. The new method could detect much lower (57-fold lower) amounts of protein. Additionally, the new method was very selective for its target antigen, demonstrating little cross-reactivity with epoxide hydrolases from other animal species. The method was performed using whole human tissue homogenates and the results correlated well to conventional protein quantification methods. The combination of signal enhancement with this novel nanobody demonstrates the ability to greatly improve protein detection and quantification and should be applicable to many other protein targets
Technical 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 a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses 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 ELISAs with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a HRP labeled anti-HA tag as the tracer showed a marginal sensitivity (0.0015 OD•mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 140-fold increase in the sensitivity (0.2098 OD•mL/ng) and 57.0-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 the enzyme activity based assay and the 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 of decades.