Modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesize the artificial antigen of norfloxacin (NOR), and New Zealand rabbits were used to produce anti-NOR polyclonal antibody (pAb). Based on the checkerboard titration, an indirect competitive enzyme-linked immunosorbent assay (icELISA) standard curve was established. This assay was sensitive and had a working range from 0.12 to 68.40 ng/ml, with the half maximal inhibitory concentration (IC 50 ) and limit of detection (LOD) values of 2.7 ng/ml and 0.06 ng/ml, respectively. The produced pAb exhibited high cross-reactivity to fluoroquinolones (FQs) tested, and the IC 50 values to enoxacin, ciprofloxacin, and pefloxacin were 3.1, 3.4, and 4.1 ng/ml, respectively. It also indicated that the concentrations of NaOH and methanol in assay buffer should not be higher than 10% and 30%. When spiked in milk at 5, 20, and 50 ng/ml, the recoveries for NOR, enoxacin, ciprofloxacin, and pefloxacin ranged 90.5%-98.0%, 84.0%-95.2%, 94.0%-106.0%, and 89.5%-100.0%, respectively. The results suggest that this class-specific pAb-based icELISA could be utilized for the primary screening of FQ residues in animal-original products.

This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC 50 ) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products.

The results of this study suggest that the immunoassay described is a promising alternative for screening CLOP residues in biological matrices and is suitable for routine diagnostics.

To prepare broad-spectrum and specific monoclonal antibodies (mAb) against deoxynivalenol (DON), DON-BSA was synthesised by the carbonyl diimidazole method as an artificial antigen to immunise Balb/C mice, and the coated DON-OVA was synthesised by the carbodiimide method to detect anti-DON antibodies. Monoclonal antibodies were screened by indirect ELISA and indirect competitive ELISA. A hybridoma cell line (4F3) capable of stably secreting DON antibody was obtained. The titre of antibody in the culture supernatant was 1:1.28 Â 10 3 , and the titre of the ascites antibody was 1:3.2 Â 10 5 . The monoclonal antibody homology was IgG1/k. The half inhibitory concentration (IC 50 ) of DON was 9.84 ng/mL. The cross-reaction rates with 3-Ac-DON and 15-Ac-DON were 60.44% and 52.04%, respectively, but no cross-reaction with other mycotoxins was observed. The results showed that the anti-DON monoclonal antibodies prepared in this experiment could recognise not only DON but also 3-Ac-DON and 15-Ac-DON, which could provide materials for the next step to establish a method to detect DON and its similar compounds. HIGHLIGHTSThis study demonstrated that an artificial antigen was synthesised by carbonyl diimidazole method, and a novel broad-spectrum, high affinity monoclonal antibody was developed. The anti-DON mAb developed in this study has better sensitivity, moreover the antibody was sensitive to DON and its derivatives. Therefore, it could be used for simultaneous monitoring of three mycotoxins. Results of this study will further lay the foundation for the establishment of immunological assays for the total amount of similar compounds.

has been illegally used in horse racing to boost physical performance, and in animal husbandry to accelerate weight gain. To monitor the abuse of NT, our goal was to develop a commercial enzyme linked immunosorbent assay (ELISA) kit. For this purpose, hybridomas were prepared by fusing NS0 mouse myeloma cells with splenocytes isolated from immunized BALB/c mouse. Noncompetitive and competitive indirect ELISA were used to screen positive cell clones. To optimize the indirect competitive ELISA (icELISA) method, various methanol concentrations in assay buffer were evaluated. Matrix effects in urine and spiking test were also investigated. Finally, five hybridoma cell lines named NT-1, NT-2, NT-3, NT-4 and NT-5 were screened out. The corresponding monoclonal antibodies (mAbs) were of the IgG 1 isotype with a k light chain, and the antibody affinity of all mAbs were between 2.6×10 9 and 4.7×10 9 L/mol. The titer and IC 50 values of purified ascites were in the range of 0.64×10 5 −2.56×10 5 and 0.55-1.0 ng/mL, respectively. Based on the NT-1 hybridoma, a heterologous icELISA method was developed for the quantitative detection of NT in cattle urine. The dynamic range was from 0.004 to 85.8 ng/mL, with a detection limit for the assay and IC 50 values of 0.002 and 0.55 ng/mL, respectively. Except for a high cross-reactivity (62%) to α-NT, negligible cross-reactivity to other compounds was observed. After optimization, 10% of methanol was used in the assay buffer, and a 20-fold dilution in cattle urine gave an inhibition curve almost the same as that in phosphate buffered saline. The correlation coefficient between the established icELISA and LC-MS/MS method was 0.9871. The results showed that the established heterologous icELISA method provides an excellent alternative for the detection of NT residues in food producing animals. 19-nortestosterone

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Two novel Gram-positive, non-spore-forming, facultatively anaerobic, non-motile, and short rods to coccoid strains were isolated from the feces of the greater white-fronted geese ( Anser albifrons ) at Poyang Lake. The 16S rRNA gene sequences of strains 4H37-19 T and 3HC-13 shared highest identity to that of Corynebacterium uropygiale Iso10 T (97.8%). Phylogenetic and phylogenomic analyses indicated that strains 4H37-19 T and 3HC-13 formed an independent clade within genus Corynebacterium and clustered with Corynebacterium uropygiale Iso10 T . The average nucleotide identity and digital DNA-DNA hybridization value between strains 4H37-19 and 3HC-13 and members within genus Corynebacterium were all below 95% and 70%, respectively. The genomic G + C content of strains 4H37-19 T and 3HC-13 was 52.5%. Diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylcholine, and phosphatidyl inositol mannosides (PIM) were the major polar lipids, with C 18:1 ω 9 c , C 16:0 , and C 18:0 as the major fatty acids, and MK-8 (H 4 ), MK-8(H 2 ), and MK-9(H 2 ) as the predominant respiratory quinones. The major whole cell sugar was arabinose, and the cell wall included mycolic acids. The cell wall peptidoglycan contained meso -diaminopimelic acid ( meso -DAP). The polyphasic taxonomic data shows that these two strains represent a novel species of the genus Corynebacterium , for which the name Corynebacterium poyangense sp. nov. is proposed. The type strain of Corynebacterium poyangense is 4H37-19 T (=GDMCC 1.1738 T = KACC 21671 T ). Supplemental material for this article may be found at 10.1007/s12275-022-2089-9.