Generic anti-sulfonamide immunoaffinity columns made using sulfamethazine-specific polyclonal antibodies

Abstract

Reusable ( /100 times) immunoaffinity columns (IACs) with high capacity ( /50 mg sulfonamide/ml gel) were made with affinity purified anti-sulfonamide polyclonal antibodies (pAbs). The specificity and capacity of these IACs for sulfonamide N 1 -analogues and N 4 -derivatives were investigated in competitive and non-competitive environments. Even though antibody affinity toward various sulfonamides differed by as much as 500-fold when measured by competitive enzyme-linked immunosorbent assay (ELISA), we demonstrated that a generic immunoaffinity application was still possible as long as the saturation limit of the column was not exceeded. Anti-sulfamethazine pAbs could equally recover a mixture of sulfonamide compounds under non-competitive conditions by immunoaffinity chromatography; however, under saturating and competitive conditions, IACs could only recover sulfonamides closely resembling the immunogen. This study also presents evidence that ELISA can serve to screen pAbs for use in generic immunoaffinity chromatography of sulfonamides.

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

“…The almost negligible matrix effect caused by the milk samples has been attributed to the excellent features of the antibodies produced, being also crucial the fact of shaking the microtiter plates during the competition step, which minimized the formation of fat/protein layers in the bottom of the microtiter wells, improving assay reproducibility and accuracy − . Thus, although several monoclonal , , , polyclonal − , and recombinant antibodies , against sulfonamide antibiotics with different recognition patterns versus the different congeners have been reported, on only a very few occasions has direct performance of these antibodies in complex biological samples been demonstrated. Thus, for tissue immunochemical analysis, several procedures involving different extraction/cleanup methods have been reported (i.e., refs and ).…”

Immunochemical Assays for Direct Sulfonamide Antibiotic Detection In Milk and Hair Samples Using Antibody Derivatized Magnetic Nanoparticles

“…Attending to it, the main objective of this work was the establishment of a rapid and reliable immunochemical method for the detection of sulfonamides in complex biological samples while avoiding sample preparation methods. Several monoclonal (29)(30)(31)(32), polyclonal (33)(34)(35), and recombinant (36)(37)(38) antibodies against sulfonamide antibiotics with different recognition patterns versus the different congeners have been reported. However, in very few occasions, direct performance of these antibodies in complex biological samples has been demonstrated.…”

“…Several monoclonal – , polyclonal – , and recombinant – antibodies against sulfonamide antibiotics with different recognition patterns versus the different congeners have been reported. However, in very few occasions, direct performance of these antibodies in complex biological samples has been demonstrated.…”

A high-throughput screening (HTS) immunochemical method for the analysis of stanozolol metabolites in cattle urine samples

“…Even though 3 OH-St was poorly recognized by ELISA with the same antibody, this metabolite was retained by the immunosorbent. The lower selectivity of the immunosorbents under non-competitive conditions in respect to the ELISA has already been reported by Grant and Sporns [39], which on the other side is very useful since often extraction of structurally related substances is desired. The explanation of this behaviour is that the As145 was raised with the hapten 5 which was designed to detect simultaneously St, 16bOH-St and NorSt.…”