Multi-residue detection of benzodiazepines by ELISA based on class selective antibodies

Abstract

A class selective enzyme-linked immunosorbent assay (ELISA) was developed for the detection of benzodiazepines, such as nitrazepam, clonazepam, 7-aminonitrazepam, 7-amidoclonzepam, chlordiazepoxide, delorazepam and nordazepam. Class selective artificial antigens were prepared by haptens of 7-aminonitrazepam and 7-aminoclonazepam conjugating to the carrier protein bovine serum albumin (BSA). Class selective polyclonal antibodies (PcAbs) were generated by immunising male New Zealand white rabbits with the class selective immunogens. Characterisation studies of the PcAbs showed that PcAbs raised by 7-aminonitrazepamÁBSA had high affinity and specificity to the six relative benzodiazepines, while PcAbs raised by 7-amidoclonazepamÁBSA had high affinity and specificity to five. So, PcAbs raised by 7-aminonitrazepamÁBSA were selected to establish an indirect competitive ELISA for multi-residue determination. The IC 50 s of the optimised immunoassay were 2.50 mg/l, 3.13 mg/l, 1.92 mg/l, 2.78 mg/l, 3.13 mg/l and 4.55 mg/l for nitrazepam, clonazepam, 7-aminonitrazepam, 7-aminoclonazepam, delorazepam and nordazepam, respectively. Assay performance was validated using spiked pig urine by adding nirazepam. Recoveries were between 75 and 103%, and the coefficient variation was below 14%. This assay can be applied to detect multiresidues of benzodiazepines in urine.

Development of an ELISA for nitrazepam based on a monoclonal antibody

“…The hapten was synthesized by reduction reaction from NZP and iron powder (Yue et al, 2009). A mixture of 1 g of Fe and 5 g of NH 4 Cl in 20 mL of methanol was prepared and 1 N HCl was added until a pH of about 3 was achieved.…”

“…Email: kuangh@jiangnan.edu.cn Food and Agricultural Immunology, 2015http://dx.doi.org/10.1080/09540105.2014 In announcement No. 176 of the Chinese agriculture ministry (Yue, Wu, Li, & Xu, 2009), NZP, Clonazepam, and Diazepam were listed as illegal veterinary drugs, banning them from use in feed of livestock and poultry.…”

“…The hapten was synthesized by the iron powder reduction method (Yue et al, 2009). This hapten was then conjugated to protein to obtain the immunogen and coating antigen.…”

Development and application of one-step ELISA for the detection of neomycin in milk

“…For a large-scale screening, however, separations are difficult to achieve because of the structural similarity of the aminoglycoside antibiotics, and the analysis requires derivatisation of the sample to improve detectability. ELISA has become the most universal assay method for antibiotics because of its simplicity, rapidity, sensitivity and low cost (Cui, Chu, Hu, & Xu, 2010;Hao et al, 2009;Kuang et al, 2009;Kuang, Xu, Cui, Ma, & Xu, 2010;Li et al, 2009;Tian, Liu, Peng, Chen, & Xu, 2009;Xu, Peng, Wang, Hao, & Jin, 2006;Yue, Wu, Li, & Xu, 2009). For neomycin immunoassay, some shortcomings still remain, including high background, lower sensitivity and extensive time required.…”

Multiplex bioanalytical methods for food and environmental monitoring

“…The use of generic structures for the development of group-specific antibodies enables detection of groups of compounds, widening the screening range [52,53]. In general, they are sensitive and easy to use, have high specificity, require minimal sample preparations, and are therefore suitable for the screening of a large number of samples in a short time (about 2-3 h).…”