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 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 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.

Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly‐horseradish peroxidase (PolyHRP)

“…Among these modifications, incorporation of Streptavidin, the addition of multiple HRPs, or conjugation of Streptavidin with PolyHRPs are suggested to be very effective for secondary signal generation . The modified conjugates were used in some immune detection methods such as ELISA , immunohistochemistry , nano‐immune detections , and immunosensors .…”

Biotinylated single-chain variable fragment-based enzyme-linked immunosorbent assay for glycocholic acid

“…The most common and versatile approach is the chemical modification using activated derivatives of biotin. 13, 31, 32 In this work, by using the commercially available sulfo-NHS-LC-Biotin, three different molar ratios of biotin to scFv were used to modify scFv according to manufacturer’s instructions. As shown in Fig.…”

Engineering hyperthermostable rcSso7d as reporter molecule forin vitrodiagnostic tests

“…[1][2][3][4] Many studies have proved antibody alternatives as promising entities for therapeutic applications; 5 however, due to differing design criteria, additional studies must be conducted to establish their utility in in vitro diagnostics. Previous studies for diagnostic applications often used the alternate scaffold as the capture protein but used a reporter antibody [6][7][8][9] or a phage-displayed variant of the scaffold with a reporter antibody 9,10 to quantify target proteins in solution, or a biotinylated antigen to quantify capture efficiency and tolerance for thermal challenges. [11][12][13] Soluble reporter proteins were investigated with sdAb by chemically conjugating biotins, 6 genetically adding tags, 7,14,15 or fusing the scaffold to a reporter enzyme.…”

“…Previous studies for diagnostic applications often used the alternate scaffold as the capture protein but used a reporter antibody [6][7][8][9] or a phage-displayed variant of the scaffold with a reporter antibody 9,10 to quantify target proteins in solution, or a biotinylated antigen to quantify capture efficiency and tolerance for thermal challenges. [11][12][13] Soluble reporter proteins were investigated with sdAb by chemically conjugating biotins, 6 genetically adding tags, 7,14,15 or fusing the scaffold to a reporter enzyme. [14][15][16] Although sdAb clones with high in vitro stability and excellent yields upon bacterial expression can be identified, these desirable properties are not universal, thus requiring additional screening during production of new clones.…”