Novel haptens were designed and synthesized to prepare antibodies against free histamine, but none resulted in producing suitable antibodies for developing an enzyme-linked immunosorbent assay (ELISA). However, an antiserum was obtained having high specificity and affinity to p-nitrobenzoylated histamine (NPHA), which can be easily formed from reaction between histamine and p-nitrobenzoic acid N-hydroxysuccinimide ester (PNBA-OSu) under mild conditions. Based on rabbit polyclonal antibodies, a competitive indirect ELISA (ciELISA) for histamine determination in foods was developed. After ciELISA and derivatization optimization, the assay showed good sensitivity, with limits of detection of 1.8 mg/kg, 93.6 μg/L, and 93.6 μg/kg in fish, red wine, and yoghurt, respectively, with negligible cross-reactivity with related biogenic amines and amino acids. Average recovery of histamine in fortified food samples ranged from 80.9% to 110.1% with coefficients of variation below 16.3%. Good correlation between the ciELISA and liquid chromatography–tandem mass spectrometry was obtained for spiked food samples.

A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC50 of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography–tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.

The hapten of Flumequine (FLU) with four carbon atoms spacer arm (FLUABA) was synthesized and coupled to bovine serum albumin (BSA) for immunogen by the activated ester method. BALB/C mice were immunized with the artificial immunogen and the splenocytes of immunized mice were fused with SP2/0 cells to obtain the monoclonal antibody (McAb). A hybridoma cell line (named DB6-E7) secreting anti-FLU McAbs was obtained by limited dilution method and screened by heterologous enzyme-linked immunosorbent assay (ELISA). The results showed that the subtype of the obtained McAb was IgG1, and the affinity was about 8.19 × 108 M−1. The haptens of FLU, FLUABA, and FLUACA with different space arms were linked to ovalbumin (OVA) for heterologous or homologous coating antigens. The results of indirect ELISA and indirect competitive ELISA indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. Using FLU-OVA as coating antigen, the ELISA showed an IC50 value of 26.33 μg L−1, a limit of detection (LOD) of 4.0 μg L−1, and the workable range of 8–114 μg L−1 (IC20–IC80). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity (< 0.1%) was detected between the obtained McAbs and the several major quinolones compounds or other structural analogs. The developed ELISA can satisfy the detection criteria of FLU in animal food-products.

A good strategy was brought forward for designing efficient haptens and complete antigens for 3-amino-2-oxazolidinone (AOZ). Haptens designed newly were achieved facilely in good yield by using LiCl–N(Et)3 as new catalysis system, the structure of which was elucidated by spectroscopy analysis, such as NMR and MS. Novel antigens for AOZ were prepared successfully by convenient active ester method. The ratios of haptens 3 and 4 to carrier proteins were proven respectively as 41:1 (5a), 39:1 (6a), 11:1 (5b) and 9:1 (6b) by trinitrobenzene sulfonic acid (TNBS) method. The results of indirect competitive ELISA (ic-ELISA) of antiserums indicated that the haptens with a short unsaturated side chain can evoke specific immune response effectively.