Methods for generation of monoclonal antibodies to the very small drug hapten, 5-benzimidazolecarboxylic acid

Abstract

Drug-specific monoclonal antibodies (MAbs) were produced against the very small drug hapten (162.15 Da), 5-benzimidazolecarboxylic acid, an analogue of 2-(4-Thiazolyl)benzimidazole (TBZ) but lacking the thiol group. TBZ is widely used as a broad-spectrum anthelmintic in various animal species and humans and also as a food preservative and agricultural fungicide. The anti-5-benzimidazolecarboxylic acid antibodies produced have potential use for extraction and/or detection of protein-bound residue forms of TBZ.Three in vivo immunisation regimes (with combinations of two related small drug haptens and two different adjuvants/ carrier molecules) and an in vitro immunisation procedure using a combination of three related unconjugated small drug haptens were investigated. Specificity for the hapten immunogen/s was initially determined using two different ELISA procedures. BIACORE analysis, in conjunction with drug binding inhibition studies, was used to confirm the specificity of a small number of selected clones.In vivo immunisation with a drug molecule conjugated to a lipopeptide/T-cell epitope, which acts both as a carrier molecule and an adjuvant was the most useful of the methods tested for the production of specific MAbs to a typically very small hapten with low immunogenic properties.

Review of methodology for the determination of benzimidazole residues in biological matrices

“…They developed an indirect ELISA based on HRP for determination of TBZ residues in fruit juices. Moran et al [229] produced mouse monoclonal antibodies raised against the novel immunogen 5-benzimidazole-carboxylic acid conjugated to the lipopeptide Pam 3 Cyst-T H . The antibodies were produced for development of ELISAs for determination of protein-bound residues of TBZ in tissue, although no applications were described.…”

Amide-containing neoepitopes: the key factor in the preparation of hapten-specific antibodies and a strategy to overcome

“…Following the typical strategy, BSA and OVA were pretreated by EDA through the EDC-induced reactions before coupling haptens to carrier proteins ( 34 – 36 ) ( Figure 1A ). Converting carboxyl groups to cationized groups ( ) was considered helpful in preventing crosslinking of carrier proteins and improving the hapten coupling efficiency.…”

Influence of Carrier Proteins on the Immunologic Response to Haptenic Antitetrodotoxin Vaccine

“…It is considered that proteins of large molecular size and originating from outside the mammalian order are strongly immunogenic and could be preferred as CPs of haptenic vaccine. Among the proteins studied, both TTH and LPH are excellent immunogenic agents themselves (26)(27)(28) and have been effectively used as carriers of other haptenic poisons (29,30); TT and DT have been clinically used as active immunizing agents for a long time; BSA has the advantage of being easily available in pure form and proven to be an effective carrier (4,31,32), whereas some reports showed it being a weak carrier in other poison conjugates (33,34).…”