A Europium Nanosphere-Based Time-Resolved Fluorescent Immunochromatographic Assay for the Rapid Screening of 4,4'-Dinitrocarbanilide: Aiming at Improving Strip Method Performance.

Considering that the strip method is simple and convenient for users, a Europium nanosphere-based time-resolved fluorescent immunochromatographic assay (TRFICA) for the rapid screening of 4,4'-dinitrocarbanilide (DNC) was developed to improve the performance of strip assays. After optimization, TRFICA showed IC50, the limit of detection, and cut-off values of 0.4, 0.07, and 5.0 ng mL-1, respectively. No significant cross-reactivity (CR < 0.1%) with 15 DNC analogs was observed in the developed method. TRFICA was validated for DNC detection in spiked chicken homogenates, and recoveries ranged from 77.3% to 92.7%, with coefficients of variation of <14.9%. Moreover, the time needed for the detection procedure, including the sample pre-treatment, was less than 30 min for TRFICA, which had never been achieved before in other immunoassays. The newly developed strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique for DNC analysis in chicken muscle.

Synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for amantadine with high sensitivity in chicken.

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N'-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC50 value (1.0 ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9 µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. PRACTICAL APPLICATION: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10 min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

Discovery of a novel recombinant avian orthoreovirus in China.

In mid-2020, using next-generation sequencing (NGS) technology, we identified a recombinant cluster 2 avian orthoreovirus (ARV) variant named PHC-2020-0545, isolated from tendons of 33-day-old broilers with leg swelling in China. Complete genomic sequencing and analyses demonstrated that the isolate was genetically significantly distinct from known ARV strains in M1 and M3 genes and its σC coding gene had an extremely high variability, compared with the identified ARV strains grouped into other genotyping cluster. Further analysis showed that many base substitutions were silent and non-silent substitutions are most likely to occur in the first positions of codons. Multiple segmental recombination, intra-segmental recombination and accumulation of point mutations might contribute to the emergence of this isolate. The PHC-2020-0545 strain had a strong replication ability in 1-day-old broilers, and mainly affected the movement, digestion and metabolism of broilers. In addition, the infection route of the isolate is related to its pathogenicity to broilers. Therefore, combined with its unique genetic characteristics and potential origin, we determined that the PHC-2020-0545 field strain is a novel recombinant ARV strain, which has certain reference value for the preparation and evaluation of new vaccines.

An Innovative Nanobody-Based High-Biocompatibility Gold Interdigitated Microelectrode Electrochemical Bioimpedance Sensor for the Ultrasensitive Detection of Difenacoum in Human Serum.

Difenacoum (DIF) is one of the most widely used anticoagulant rodenticides. However, accidental or intentional ingestion of DIF seriously threatens humans and other non-target species. Therefore, a rapid and sensitive detection method to quantify DIF is urgently needed. In this study, one anti-DIF nanobody (Nb) was assembled on the surface of a gold interdigitated microelectrode (IDME) using an Au-S bond to fabricate a bioimpedance sensor. To improve the immobilization amount of Nbs on the electrode, a polycrystalline gold IDME was prepared to provide a larger surface and better biocompatibility. Thus, a novel and ultrasensitive bioimpedance sensor based on electrochemical impedance spectroscopy (EIS) was designed for the determination of DIF, and it displayed good reproducibility and stability in human serum. The proposed bioimpedance sensor displayed a wide working range, between 0.1-1000 pg/mL, with a limit of detection (LOD) of 0.1 pg/mL of DIF. This method exhibited excellent performance, good sensitivity, and reproducibility and achieved the highest sensitivity of all currently existing methods used to quantify DIF. The highly sensitive DIF detection of this proposed bioimpedance sensor indicates its potential as an efficacious approach for DIF monitoring in human serum with high accuracy and precision.

Hapten synthesis, monoclonal antibody production and immunoassay development for direct detection of 4-hydroxybenzehydrazide in chicken, the metabolite of nifuroxazide.

Derivatization is usually employed in immunoassay for detection of metabolites of nitrofurans and avoiding derivatization could be preferable to achieve an efficient screening. In the study, we designed four haptens of 4-hydroxybenhydrazide (HBH), the nifuroxazide metabolite. The effect of hapten structures on antibody affinity were evaluated and one monoclonal antibody was produced by using the Hapten C with a linear alkalane spacer arm. After optimization, an enzyme linked-immunosorbent assay (ELISA) was established with an 50% inhibition concentration of 0.25 ng mL-1 for HBH, which could ensure the direct detection of HBH without derivatization. The limit of detection of the ELISA for HBH was 0.12 µg kg-1 with the recoveries of 90.1-96.2% and coefficient of variation (CV) values lower than 9.1%. In conclusion, we produced several high affinity antibodies to HBH with new designed hapten and developed an icELISA for the direct detection of HBH without derivatization in chicken.

Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays.

Enzyme labeling of an antigen or an antibody helps to visualize and amplify the signal and is an important reagent used in immunoassays for the detection of a target of interest. In this research, soybean peroxidase (SBP), a less commonly used enzyme reporter, was compared in immunoassays with the two most commonly used reagents, horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The enzyme-antibody conjugates were evaluated by their performance in an indirect competitive enzyme-linked immunosorbent assay (icELISA) and in an indirect competitive chemiluminescent enzyme immunoassay (icCLEIA) for ractopamine (RAC). The results revealed that the more affordable SBP offers a long-lasting chemiluminescent signal, which outperformed ALP and HRP. SBP-antibody conjugate (SBP-Ab) based immunoassays produced lower limits of detection (LODs) and better accuracy in the detection of RAC in animal urine samples. Additionally, SBP-Ab has advantages in being more resistant to heat, acid and organic solvents. These results suggest that SBP could be a potentially excellent alternative to HRP and ALP for the development of immunoassay in food safety field.