Immunochemical approach to the detection of aminotriazoles using selective amino group protection by chromophores

Abstract

A selective approach for amino masking and hapten design for aminotriazole conjugates is described. Haptenic 1,2,4-triazole and 3-amino-l ,2,4-triazole compounds were conjugated to proteins through spacer arms on the C5 ring carbon, containing hydroxyl or mercapto groups. The amino groups present in several haptens were protected via sulfenylation, using a chromophoric protecting reagent, 2-nitrophenylsulfenyl chloride. Protection of haptens, conjugation to proteins, and deprotection on the proteins were followed by UV spectroscopy, monitoring the specific absorbance of the protecting group at 349 nm. Homoand heterobifunctional spacers allowing spacer, hapten, and multiple heterology were used for the protein conjugates for immunizing and coating. Antisera raised against aminotriazole conjugates in New Zealand white rabbits and Swiss Webster mice were found to be specific for amitrole. Inhibition tests resulted in 60% inhibition by amitrole, indicating low-affinity antibodies. Antibody binding to the various antigens synthesized was characterized in a qualitative immunoassay.

ELISA for the detection of the triazole fungicide myclobutanil.

“…A working dilution of 1:4000 for the purified antiserum appeared to be optimal, because the sensitivity of the detection was not significantly improved beyond this dilution factor. In our hands, an aminotriazole herbicide, amitrole, was found to be a poor immune epitope (Jung et al, 1991;Szekdcs et al, 1995). Although the sensitivity of ELISA systems against various pesticides often ranges in the low parts per billion level (Marco et al, 1993;Bekheit et al, 1993; Absorbances are expressed as percent of the control absorbance at pH 7.4.…”

“…The nitrile group appeared to be unusually resistant to chemical hydrolysis, probably due to steric hindrance. A molar ratio of over 100 hapten molecules per mole of protein was used in the conjugation reactions for all conjugates based on the results of Jung et al (1991). The hydrolysis using hydrogen peroxide and phase-transfer catalytic conditions resulted-although at a moderate yield-in the corresponding amide, and the acidic hydrolysis, using an exceedingly long reaction time, proceeded only to the amide stage.…”

“…Within the scope of the extending collaboration between our laboratories at Davis and Budapest (Hammock et al, 1990;Jung et al, 1991;Székács et al, 1995) and in a development project sponsored by the U.S.-Hungarian Joint Fund, we aim to develop ELISA systems for the environmental monitoring of pesticides and toxins. Driven by the lack of a specific detection system for myclobutanil and to continue our previous work on development of immunodetection systems for pesticides containing a triazole moiety, one of our present objectives was to develop an ELISA system for this target analyte.…”

Inmunoensayos rápidos para la determinación de residuos de plaguicidas organofosforados en aceite de oliva

“…El brazo espaciador óptimo debe contener entre 3 y 6 eslabones -átomos de carbono, generalmente-(272, 273), aunque los estudios realizados hasta el momento no han sido exhaustivos y se han descrito inmunoensayos con anticuerpos obtenidos utilizando brazos espaciadores más cortos (274,275). No es aconsejable que posea grupos funcionales o heteroátomos, ya que podrían inducir la producción de anticuerpos contra el propio espaciador, lo que no es deseable (276,277).…”

Desarrollo de un inmunoensayo multianalito, basado en anticuerpos monoclonales, para la determinación de plaguicidas N-metilcarbamatos en frutas y hortalizas.

“…Es un hecho bien documentado que el pH del tampón en el que tiene lugar la inmunorreacción competitiva influye tanto en la sensibilidad como en la señal máxima de los inmunoensayos para plaguicidas (Jung et al, 1991;li et al, 1991;Krämer et al, 1994;Lee et al, 1995;Manclús y Montoya, 1996 b,c;Wortberg et al, 1996;Abad y Montoya, 1997;Mercader y Montoya, 1999).…”