Generation of Broad Specificity Antibodies for Sulfonamide Antibiotics and Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Analysis of Milk Samples

Abstract

Immunoreagents appropriately produced to detect a wide range of sulfonamide antibiotic congeners have been used to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA). The selectivity has been achieved by combining antibodies raised against 5-[6-(4-aminobenzenesulfonylamino)pyridin-3-yl]-2-methylpentanoic acid (SA1), covalently coupled to horseshoe crab hemocyanin (HCH), and 5-[4-(amino)phenylsulfonamide]-5-oxopentanoic acid (SA2), coupled to ovalbumin (OVA), on an indirect ELISA format. The immunizing hapten has been designed to address selectivity against the common aminobenzenesulfonylamino moieties, using theoretical calculations and molecular modeling tools. Hapten SA1 has been synthesized in four steps from methyl 5-(4-amino-3-pyridinyl)-2-methyl-4-pentenoate through a Heck reaction, under Jeffery conditions, to avoid introduction of additional epitopes in the linker. The microplate immunoassay developed is able to reach the necessary detectability for the determination of the sulfonamide antibiotics most frequently used in the veterinary field, in compliance with the EC Regulation 2377/90. As an example, the IC(50) and LOD values accomplished for sulfapyridine are 2.86 +/- 0.24 and 0.13 +/- 0.03 microg L(-1), respectively. Studies performed with different types of milk samples demonstrate that direct and accurate measurements can be performed in this type of matrix without any previous sample cleanup method.

Sensitive immunoassay for simultaneous determination of tetrabromobisphenol A bis(2-hydroxyethyl) ether and tetrabromobisphenol A mono(hydroxyethyl) ether: An effective and reliable strategy to estimate the typical tetrabromobisphenol A derivative and byproduct in aquatic environments

“…3); the LOD (calculated, based on 90% B/B 0 (Adrian et al, 2009)) and IC 50 of this method were 0.702 and 9.868 ng/mL, respectively. With the help of enhancement through sample pretreatment, the ELISA we developed could be used for detection of trace TBBPA-DHEE in a variety of environmental samples.…”

“…The immunogens were prepared by coupling the hapten (M3) to BSA with the activated ester method, as previously reported (Adrian et al, 2009). In brief, 45 mg M3, 40 mg N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were dissolved in 2 mL anhydrous (DMF) and reacted for 8 h at 4 C under magnetically stirred conditions; the final supernate was then added into 1 mL BSA solution (17 mg/mL, carbonate buffer, pH 9.6), followed by continuous stirring overnight at 4 C. The immunogens (BSA conjugates) were purified using dialysis solution (0.01M PBS, pH 7.4) for 3 days.…”

Immunochemical detection of emerging organic contaminants in environmental waters

“…Furthermore, they explained the mechanism of specificity of this mAb by evaluating its conformational and electronic properties [20]. Adrian et al also synthesized haptens against the common aminobenzenesulfonylamino moieties with the aid of theoretical calculations and molecular modeling tools, based on the haptens, they generated pAbs that showed high sensitivity and excellent cross-reactivity to recognize 10 sulfonamides directly [21]. Other selected examples of the antibodies against EOCs are listed in Table 1.…”

Recent Advances and Applications of Biosensors in Novel Technology

“…Current advances in sample-preparation methods, detectors, and other apparatuses have enhanced the limits of detection (LODs). Instead, immunochemical methods with high-sample throughput ability could be used to achieve demands of pharmaceutical examinations [7].…”