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To ensure proper dosage of a drug, analytical quantification of it in biofluid is necessary. Liquid chromatography mass spectrometry (LC-MS) is the conventional method of choice as it permits accurate identification and quantification. However, it requires expensive instrumentation and is not appropriate for bedside use. Using soluble epoxide hydrolase (sEH) inhibitors (EC5026 and TPPU) as examples, we report development of a nanobody-based enzyme-linked immunosorbent assay (ELISA) for such small molecules and its use to accurately quantify the drug chemicals in human samples. Under optimized conditions, two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL, respectively, and two order of magnitude linear ranges with high precision and accuracy. The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026. In addition, the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency. The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds. These methods could be easily implemented by the bedside, in the field in remote areas or in veterinary practice. This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy. Attributes of nanobody based pharmaceutical assays are discussed.

The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.

A microsomal epoxide hydrolase (mEH) metabolizes in vivo in both xenobiotic and endogenous epoxides associated with signaling function. Findings in patients suggest that mEH might be a biomarker for several diseases, including metastatic cancer and viral hepatitis. To easily quantify mEH, nanobodies specific to the human mEH were isolated from a phage library of llama VHHs. Four unique clones were obtained and used for developing ELISAs. Three formats of double antibody sandwich assays were investigated using different detection strategies. Using PolyHRP, the signal was strongly amplified, yielding a 22-fold lower LOD (12 pg mL-1) than the 'conventional'. To further validate the performance of the immunoassays, human tissue samples were analyzed by nanobody-based ELISAs and compared to the enzyme activities (R2 > 0.95). The results demonstrate that these nanobodies are powerful tools for the quantification of human mEH and could eventually result in a bedside assay.

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

Glycocholic acid (GCA) has been identified as a novel selective and sensitive biomarker for hepatocellular carcinoma (HCC). In this work, a recombinant antibody, scFv-G11, which was shown previously to have selective reactivity for GCA, was labeled with biotin using a chemical and an enzymatic method, respectively. The enzymatic method proved superior giving sensitive scFv-biotin preparations. Based on biotinylated scFv against GCA and a biotin-streptavidin system for signal amplification, an indirect competitive biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) has been established for the sensitive and rapid detection of GCA. Several physiochemical factors that influenced assay performance, such as organic cosolvent, ionic strength, and pH, were studied. Under the optimized conditions, the indirect competitive BA-ELISA based on the obtained biotinylated scFv antibodies indicated that the average concentration required for 50% inhibition of binding (IC50) and the limit of detection (LOD) for GCA were 0.42 μg mL-1 and 0.07 μg mL-1, respectively, and the linear response range extended from 0.14 to 1.24 μg mL-1. Cross-reactivity of biotinylated scFv antibodies with various bile acid analogues was below 1.89%, except for taurocholic acid. The recoveries of GCA from urine samples via this indirect competitive BA-ELISA ranged from 108.3% to 131.5%, and correlated well with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS), which indicated the accuracy and reliability of biotinylated scFv-based ELISA in the detection of GCA in urine samples. This study also demonstrates the broad utility of scFv for the development of highly sensitive immunoassays.

Glycocholic acid (GCA) is an important metabolite of bile acids, whose urine levels are expected to be a specific diagnostic biomarker for hepatocellular carcinoma (HCC). A high-throughput immunoassay for determination of GCA would be of significant advantage and useful for primary diagnosis, surveillance, and early detection of HCC. Single-chain variable fragment (scFv) antibodies have several desirable characteristics and are an attractive alternative to traditional antibodies for the immunoassay. Because chicken antibodies possess single heavy and light variable functional domains, they are an ideal framework for simplified generation of recombinant antibodies for GCA detection. However, chicken scFvs have rarely been used to detect GCA. In this study, a scFv library was generated from chickens immunized with a GCA hapten coupled to bovine serum albumin (BSA), and anti-GCA scFvs were isolated by a phage-displayed method. Compared to the homologous coating antigen, use of a heterologous coating antigen resulted in about an 85-fold improvement in sensitivity of the immunoassay. This assay, under optimized conditions, had a linear range of 0.02-0.18 μg/mL, with an IC50 of 0.06 μg/mL. The assay showed negligible cross-reactivity with various related bile acids, except for taurocholic acid. The detection of GCA from spiked human urine samples ranged from 86.7% to 123.3%. These results, combined with the advantages of scFv antibodies, indicated that a chicken scFv-based enzyme-linked immunosorbent assay is a suitable method for high-throughput screening of GCA in human urine.

Antibiotics are currently used for the treatment of Helicobacter pylori (H. pylori), which is confirmed to be the major cause of gastric disorders. However, the long-term consumption of antibiotics has already caused antibiotic resistance and side effects in vivo. Therefore, there is an emerging need for searching for safe and effective anti-H. pylori agents. Inspired by the excellent bioactivities of cinnamic acid, a series of cinnamic acid derivatives (compounds 1–30) were synthesized and determined for H. pylori inhibition. The initial screening revealed that compound 23, a 2,4-dinitro cinnamic acid derivative containing 4-methoxyphenol, showed excellent H. pylori inhibition with an MIC value of 4 μM. Further studies indicated that compound 23 showed anti-bacterial activity and had a bactericidal effect on H. pylori due to the destruction of the bacterial structure. Molecular docking analysis revealed that the 2,4-dinitro groups in cinnamic acid moiety formed hydrogen bonding with amino acid residues in an active pocket of H. pylori protein. Interestingly, the ester moiety fitted into the hydrophobic pocket, attaining additional stability to compound 23. Above all, the present study reveals that compound 23 could be considered a promising anti-H. pylori agent to treat H. pylori causing gastritis. [ABSTRACT FROM AUTHOR]