Preparation and characterization of anti-sulphamethoxazole (SMX) monoclonal antibody

Abstract

Anti-sulphamethoxazole (SMX) monoclonal antibody (MAb) has a significant role in monitoring of SMX residue levels in edible animal products. In this study, SMX was diazotizated and coupled to human serum albumin to form an antigen for animal immunization. Anti-SMX hybridomas were prepared by cell fusion using an enzyme-linked immunosorbent assay (ELISA) method to screen for positive hybridomas. A monoclonal antibody (MAb) against SMX was secreted by the hybridomas and its specificity was characterized by ELISA, SDS-PAGE and Western-blot. The results showed that the SMX-MAb was of the IgG1 type and its molecular weight was 162 000 Daltons. The affinity constant of MAb for the conjugated antigen was 2.3)/10 8 l/mol. The detection limit of 50% inhibition rate in the range 10 pg/mL to 10 ng/mL was 0.82 ng/mL and the recoveries of SMX were above 90%. There was no crossreaction between MAb and SMX analogues.

Development of immunoassays to determinate sulfamethoxazole residues in wastewaters

“…Using the optimized conditions (PBS 20 mmol L À1 , pH 5.5, 0.025% Tween 20 and 30 minutes of competition time), competitive calibration curves against SMX were carried out in order to demonstrate the analytical features of the ELISA. The assay showed great sensitivity (IC 50 ¼ 0.75 ng mL À1 ), with a LD of 0.001 ng mL À1 , comparable to that achieved by chromatographic methods, 12,33,34 and a dynamic range (DR) between 0.02-17.80 ng mL À1 (see Table 1), better than those obtained by previous ELISAs [15][16][17] and BIAs. [18][19][20] Cross-reactivity studies.…”

“…Several methods have been developed to identify SMX, generally high-performance liquid chromatography (HPLC) interfaced with various detectors, such as mass spectrometry, 9,10 tandem mass spectrometry, 7,12 ultraviolet (UV) and fluorescence; 13 SMX has also been determined by capillary electrophoresis, 14 ELISA-plate [15][16][17] and biosensor immunoassays (BIAs). [18][19][20] The detection limits for SMX range from 0.01 ng mL À1 achieved by chromatography with previous analyte extraction and preconcentration, 12 to 0.067 ng mL À1 by ELISA 17 or 0.15 ng mL À1 with BIA directly determined in the sample.…”