Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation

Abstract

The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

Exploring alternative hapten tethering sites for high-affinity anti-picoxystrobin antibody generation

“…It seems that PCa6-and PCo6-based immunogens generated antibodies with a binding pocket in which the pyridyl ring of the PC molecule played a determinant role, whereas antisera obtained by immunization with BSA-PCb6 did not accept antigens with the tethering site at the aryl ring (PCa6) or at the pharmacophore group (PCo6). Similar results have also been found with antisera generated against siteheterologous immunogens of azoxystrobin (unpublished results) and pyraclostrobin [24].…”

An innovative hapten and monoclonal antibody-based immunoassay for determining tebuconazole residues in aqueous samples

“…Finally, the atomic charge distribution of TEB and hapten1, hapten2, hapten3, hapten4 in the common structure can be seen from Figure 2. On the contrary, charge distribution could play a more important pole according to the description by the Mercader (Mercader, Agullo, Abad-Somovilla, & Abad-Fuentes, 2011). The total charge of the common chemical structure for TEB and four haptens was all negative, moreover, hapten1 is the closest to the sum atomic charge of TEB.…”

Structure–immunogenicity relationship of kresoxim-methyl regioisomeric haptens

“…In previous studies with other strobilurins, functionalization through the toxophore moiety, particularly at the methoxy group, also resulted in optimum affinity responses. [36][37][38] With the linker at such position, the characteristic aromatic rings of the molecule seem to be better 15 exposed to the immune system. In contrast, the a-ring was revealed as a less adequate site for derivatization -particularly in para position from the (methoxyimino)acetate group -if highaffinity antibodies are sought.…”

“…Synthesized compounds were purified by flash chromatography with a silica gel column (Merck 60, 230-400 mesh). Melting points and spectra were recorded as 65 in previous studies[8] 38. The most stable conformation of kresoxim-methyl was calculated with CAChe WorkSystem Pro software, version 7.5.0.85 (Fujitsu Ltd, Tokyo, Japan).…”