Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity

Abstract

Immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 position of the sulfonamides, were selected to evaluate the effectiveness for producing group-specific monoclonal antibodies (MAbs). Mice immunized with three different two-ring haptens were used for hybridoma production, which resulted in three unique MAbs recognizing 10, 13, and 15 sulfonamides showing 50 % inhibition (IC50) at concentrations below 100 ng mL(-1). MAb 4D11 derived from one novel immunizing hapten could recognize 12 sulfonamides with IC50 values ranging from 1.2 to 12.4 ng mL(-1), almost within 1 order of magnitude. These produced MAbs show lower IC50 values in addition to significantly improved group specificity compared with previously generated MAbs. This study clearly indicates that the careful selection of the immunizing hapten has an important effect on the specificity of the generated antibodies.

Enzyme-Linked Immunosorbent Assay for the Determination of Five Organophosphorus Pesticides in Camellia Oil

“…However, the sensitivity and selectivity of the heterologous ELISAs based on haptens 6 and 7 were lower than the homologous result, and the ELISA from hapten 7 could not even recognize fenitrothion. The ability of different competitor antibody combinations to modify immunoassay selectivity and detectability were also reported in ELISA development for sulfonamides (25), sarafloxacin (15), and triacetone triperoxide (21).…”

A highly sensitive and class-specific fluorescence polarisation assay for sulphonamides based on dihydropteroate synthase

“…S1 were purchased from SigmaAldrich (St. Louis, MO, USA). Haptens SA10, SSS and CS were previously synthesised in our laboratory (Wang et al, 2013a(Wang et al, , 2013b. The 6-hydroxymethylpterin pyrophosphate was obtained from Schirks Laboratories (Jona, Switzerland).…”

“…S1). Currently, more than 20 sulphonamides are used in clinical studies (Wang et al, 2013a). For example, at least nine and six sulphonamides have been approved for use in veterinary medicine in the Netherlands and in the Czech Republic, respectively (Cliquet et al, 2003;Franek et al, 2006).…”

“…To minimise the risk of sulphonamides, the maximum residue limits (MRLs) are set to 100 mg kg À 1 for total sulphonamides by the EU and China (Bangemann, 1999; Ministry of Agriculture of the People's Republic of China, 2002). To achieve the goal of class-sulphonamide detection, many antibodies class-specific to sulphonamides have been produced; however, each of these antibodies was capable of recognising only a single type of sulphonamide (Cliquet et al, 2003;Franek et al, 2006;Wang et al, 2013a). In our previous study, we prepared dihydropteroate synthase (DHPS) and developed a microplate assay for multi-sulphonamides for the first time (Liang et al, 2013).…”

“…In the assay based on DHPS, 9 sulphonamides were detected at concentrations less than 100 ng mL À 1 and 28 sulphonamides exhibited half-maximal inhibitory concentration (IC 50 ) values ranging from 426 to 50,000 ng mL À 1 . Obviously, the sensitivity of the assay is inferior to that of antibody-based assays and chromatographic methods (Salvia et al, 2012;Wang et al, 2013a;Zhou et al, 2014); thus, this method could not satisfy the requirements of regulatory agencies. Levy et al and other groups have demonstrated that the sulphonamide target for competitive inhibition of DHPS with PABA should be the DHPS-DHPPP binary complex (DHPPP is the acronym for dihydropteridine pyrophosphate, which acts as the first substrate for DHPS), rather than DHPS alone (Levy et al, 2008).…”