Broadening the Detection Spectrum of Small Analytes Using a Two-Antibody-Designed Hybrid Immunoassay

Abstract

The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities toward sulfonamides were examined for their binding to a panel of hapten conjugates. mAb-hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwich-ELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their maximum residue limit (MRL) concentration was demonstrated using the examples of turkey muscle and milk samples.

Novel haptens and monoclonal antibodies with subnanomolar affinity for a classical analytical target, ochratoxin A

“…Unlike the results reported by different research groups 40 – 42 , no signal was obtained when antisera from hapten OTA f were combined with the homologous enzyme tracer (HRP‒OTA f ). Poor or no binding between antibodies and enzyme tracers based on haptens without or with short spacer arm has been previously observed by our group and others for a variety of analytes 43 – 45 . Insufficient signal could be a consequence of either no binding because of steric hindrances or a result of enzyme inactivation due to the close proximity of the antibody to the label.…”

Rapid Multi-Residue Detection Methods for Pesticides and Veterinary Drugs

“…The monoclonal antibodies were labeled by a horseradish peroxidase. Due to the summation effect in formation of a two-monoclonal antibodies sandwich complex, simultaneous determination of 14 SAs was achieved in turkey muscle and milk samples [58]. methyl-parathion, fenpropathrin, carbofuran, thiacloprid, chlorothalonil, and carbendazim) [57].…”

Experimental Study on the Influence of Sulfonamide Drug Residues From Honey on Biochemical Parameters in Lab Rats

“…Excretion of sulfonamides is done in the kidneys by glomerular filtration in unchanged form or metabolites. The elimination of sulfonamides is variable over time, depending on the compound, pharmaceutical form and animal species [3,16,18,26]. At the same time, the proportion of metabolites and the non-transformed substance eliminated differs from species to species [15,28,31,35].…”