A sensitive biotinylated indirect competitive enzyme-linked immunosorbent assay (Bic-ELISA) was developed to detect acetamiprid pesticides in pollen, based on the heterogeneous coating antigen and biotinylated anti-acetamiprid monoclonal antibody. Under optimized experimental conditions, the detection limit for the Bic-ELISA was 0.17 ng/mL and the linear range was 0.25–25 ng/mL. The cross-reactivities could be regarded as negligible for the biotinylated antibodies with their analogues except for thiacloprid (1.66%). Analyte recoveries for extracts of spiked pollen (camellia pollen, lotus pollen, rape pollen) ranged from 81.1% to 108.0%, with intra-day relative standard deviations (RSDs) of 4.8% to 10.9%, and the average reproducibility was 85.4% to 110.9% with inter-assay and inter-assay RSDs of 6.1% to 11.7%. The results of Bic-ELISA methods for the Taobao’s website samples were largely consistent with HPLC-MS/MS. Therefore, the established Bic-ELISA methods would be conducive to the monitoring of acetamiprid in pollen.Keywords:pollen;acetamiprid;pesticide;heterogeneous coating antigen;biotinylated mAb;Bic-ELISA
Kanamycin is an aminoglycoside antibiotic used increasingly in human and veterinary medicine. However, kanamycin residues in food can cause serious side effects. Here we reported the preparation of polyclonal antibody and the development of an indirect competitive biotin-streptavidin-amplified-based enzyme-linked immunosorbent assay(BA-ELISA) that can sensitively and specifically detect kanamycin residues in milk and honey. The immunogen and coating antigen were synthesized by covalently linking kanamycin to carrier proteins using the carbodiimide method. The anti-kanamycin polyclonal antibodies were obtained from immunized rabbits. The key assay parameters were investigated and optimized. The results show that under optimum conditions, the limit of detection for kanamycin is 0.07 ng/mL and the IC 50 is 6.48 ng/mL. Cross-reactivity values of the antibody with four kanamycin analogues are all <1%. Trace amounts of kanamycin in milk and honey samples can be detected by this novel BA-ELISA method successfully with satisfactory recoveries of 91.0%—103.3%. The developed protocol was also validated against liquid chromatography-mass spectrometry, returning a significant correlation. These results indicate that BA-ELISA is a viable option for monitoring kanamycin residues in milk and honey.
Two kinds of new immunogens and coating antigens had been designed and synthesised by methods of lively ester (DCC) and carbodiimide (EDC), respectively. Preliminary immunosorbent assays indicated that the two target conjugates presented good inhibition ratio against the haptens of cypermethrin in vivo. The results showed that the best efficient titres of anti-cypermethrin rabbit serum in methods of DCC and EDC were 1:32000 and 1:64000, the IC50 of them were 18.907µg/L and 16.877µg/L against the haptens of cypermethrin, respectively. The data indicated that the anti-cypermethrin antibodies in method of EDC had higher sensitivity and higher selectivity than the anti-cypermethrin antibodies in method of DCC. Thus, we can synthesise antigens and anti-bodies with method of EDC. The antibodies of high quality can be effectively used in the field of the rapid detection of pyrethroid pesticide residues in the research of the reagent strips.
Jiawen Li,Huali Jin,Min Wei,Wenjie Ren,Jinshui Wang,Yurong Zhang,Ligen Wu,Baoshan He
In this work, a sensitive dual mode competitive electrochemical immunosensor based on differential pulse voltammetry (DPV) and square wave voltammetry (SWV) response modes for quantitative determination of dibutyl phthalate (DBP) was successfully established. According to diethylenetriamine (DETA) assisted hydrothermal strategy, a novel N-doped graphene/CoSe 2 nanobelt composite was prepared. Afterwards, PEI functionalized NG-CoSe 2 and gold nanowires (AuNWs) were further compounded as a preliminary signal amplification platform for immunosensor. Thionine (Thi)-loaded [email protected] and secondary antibody (Ab 2 ) connected as a signal label. The immobilized coating antigen DBP-BSA and the DBP compete for limited antibody binding sites. The concentration of DBP can be determined by DPV detect the Thi signal loaded on the [email protected] 2 label. It can also be determined by SWV mode to detect the current generated by the reaction system of H 2 O 2 and o-phenylenediamine (o-PD) catalyzed by [email protected] with peroxidase like properties. Under optimum conditions, the linear ranges of the DPV and SWV modes were 1 pg mL −1 ∼1 μg mL −1 . And LOD were 0.486 pg mL -1 (DPV) and 0.711 pg mL -1 (SWV), respectively. Besides, it exhibited outstanding selectivity, reproducibility, stability and can be used for sensitive detection of DBP in real samples.
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