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Fluoroquinolones (FQs) are among the antibiotics whose widespread use in farm-raised animals results in potentially harmful residues in the end products. Additionally, most Thai farmers use antibiotics. Amoxicillin and enrofloxacin were commonly used by pig farms, and hens were given enrofloxacin to prevent immunization side effects. Moreover, antibiotic overuse has harmed food safety in the long term, and the use of low-dose antibiotics causes bacterial resistance. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to make a fast, easy, sensitive, and cost-effective method for monitoring FQs residues. After immunizing hens with mixed multi-hapten ciprofloxacin-bovine serum albumin (CPFX-BSA) with norfloxacin-bovine serum albumin (NFX-BSA), the IgY antibody purified from egg yolk was used for the detection of FQs residues in chicken and pork samples. The efficiency of the IgY antibody showed excellent sensitivity, with 50% inhibitory concentration (IC50) of enrofloxacin at 0.05 µg/mL, far below the MRLs defined by the EU for muscle samples, which was not to exceed 100 µg/kg. The recovery range for chicken muscle samples spiked with ENFX concentrations of 1.00–0.01 µg/mL was 86.65–112.71%, similar to pork samples, which were 84.24–117.22.2%. This method has a lot of potential for analyzing fluoroquinolones in complex samples quickly, easily, and at a low cost on-site. The IgY-based ic ELISA was developed to detect ciprofloxacin (CPFX), norfloxacin (NFX), and enrofloxacin (ENFX) residues; it confirms that IgY could be a promising choice for the detection of antibiotic residues in food samples. [ABSTRACT FROM AUTHOR]

Aristolochic acid (AA) toxicity has been shown in humans regarding carcinogenesis, nephrotoxicity, and mutagenicity. Monitoring the AA content in drug homologous and healthy foods is necessary for the health of humans. In this study, a monoclonal antibody (mAb) with high sensitivity for aristolochic acid I (AA-I) was prepared. Based on the obtained mAb, a chemiluminescent immunoassay (CLEIA) against AA-I was developed, which showed the 50% decrease in the RLUmax (IC50) value of 1.8 ng/mL and the limit of detection (LOD) of 0.4 ng/mL. Carbon dots with red emission at 620 nm, namely rCDs, were synthesized and employed in conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) to improve the assay sensitivity of a fluoroimmunoassay (FIA). Oxidized 3,3′′,5,5′′-tetramethylbenzidine dihydrochloride (oxTMB) can quench the emission of the rCDs through the inner-filter effect; therefore, the fluorescence intensity of rCDs can be regulated by the concentration of mAb. As a result, the assay sensitivity of FIA was improved by five-fold compared to CLEIA. A good relationship between the results of the proposed assays and the standard ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-QQQ-MS/MS) of real samples indicated good accuracy and practicability of CLEIA and FIA. [ABSTRACT FROM AUTHOR]

Tylosin and tilmicosin (T&T) residues in livestock products have received extensive attention from consumers. Time-resolved fluorescence immunochromatographic assay (TRFICA), as a fast, efficient and sensitive immunoassay method, has played an increasingly important role in the food safety field. Therefore, herein a quantitative and visual TRFICA was established for simultaneously detecting T&T in milk in a group-screening manner. Under the optimal conditions, the standard curve range of developed TRFICA based on the T&T was 1.87~7.47 ng/mL, and the half-maximal inhibition concentrations (IC50) were 4.06 ng/mL and 3.74 ng/mL, respectively. The limits of detection (LOD) of the TRFICA method were from 1.72 ng/mL to 1.39 ng/mL, and the visual cut-off values were 31.25 ng/mL and 62.50 ng/mL for T&T in milk, respectively. Moreover, the stability experiments showed that the strips could be stored at 4 °C for more than 6 months, the total detection time was less than 13 min, and the cross-reactivities (CRs) with related compounds were less than 0.1%, which concluded that the developed TRFICA method could be used in real milk sample detection. [ABSTRACT FROM AUTHOR]

Nanobody (Nb), a new type of biorecognition element generally from Camelidae, has the characteristics of small molecular weight, high stability, great solubility and high expression level in E. coli. In this study, with 19-nortestosterone (19-NT), an anabolic androgenic steroid as target drug, three specific Nbs against 19-NT were selected from camel immune library by phage display technology. The obtained Nbs showed excellent thermostability and organic solvent tolerance. The nanobody Nb2F7 with the best performance was used to develop a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for 19-NT detection. Under optimized conditions, the standard curve of ic-ELISA was fitted with a half-maximal inhibitory concentration (IC50) of 1.03 ng/mL and a detection limit (LOD) of 0.10 ng/mL for 19-NT. Meanwhile, the developed assay had low cross- reactivity with analogs and the recoveries of 19-NT ranged from 82.61% to 99.24% in spiked samples. The correlation coefficient between ic-ELISA and the ultra-performance liquid chromatography/mass spectrometry (UPLC-MS/MS) method was 0.9975, which indicated that the nanobody-based ic-ELISA could be a useful tool for a rapid analysis of 19-NT in animal urine samples. [ABSTRACT FROM AUTHOR]

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples. [ABSTRACT FROM AUTHOR]

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules. [ABSTRACT FROM AUTHOR]

Nanobodies are one-tenth the size of conventional antibodies and are naturally obtained from the atypical heavy-chain-only antibodies present in camelids. Their small size, high solubility, high stability, and strong resilience to organic solvents facilitate their use as novel analytical reagents in immunochemistry. In this study, specific nanobodies against pesticide carbofuran were isolated and characterized from an immunized library via phage display platform. We further established an indirect competitive enzyme-linked immunosorbent assay (ELISA) using nanobody Nb316 to detect carbofuran in vegetable and fruit samples. The results showed a half-maximal inhibitory concentration (IC50) of 7.27 ng/mL and a detection limit of 0.65 ng/mL. A simplified sample pretreatment procedure omitting the evaporation of organic solvent was used. The averaged recovery rate of spiked samples ranged between 82.3% and 103.9%, which correlated with that of standard UPLC–MS/MS method. In conclusion, a nanobody with high specificity for carbofuran was characterized, and a nanobody-based sensitive immunoassay for simple and rapid detection of carbofuran in real samples was validated. [ABSTRACT FROM AUTHOR]

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics. [ABSTRACT FROM AUTHOR]

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O , O -diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β -d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.