Antibody generation and immunoassay development in diverse formats for pyrimethanil specific and sensitive analysis

Abstract

Immunochemical techniques are complementary tools to modern analytical requirements. These methods rely on the production of immunoreagents with adequate binding properties. In the present study, a rationally-designed functionalized derivative of pyrimethanil -a modern anilinopyrimidine fungicidewas synthesized in order to generate for the first time high-affinity and selective antibodies to this 10 xenobiotic. A single coupling procedure -based on hapten activation using N,N'-disuccinimidyl carbonate and purification of the active ester -was followed to prepare both immunizing and assay conjugates. Polyclonal antibodies were produced and characterized by enzyme-linked immunosorbent assay (ELISA) in four alternative formats: one indirect and three direct competitive procedures. The selected immunoassay displayed a limit of detection of 0.024 µg L −1 , far lower than the official maximum 15 residue limits and close similar to the sensitivity of regular instrumental assays. This ELISA was shown to be robust to buffer changes and tolerant to the presence of little amounts of methanol, ethanol and acetonitrile. Finally, the developed assay was applied to the analysis of pyrimethanil in carrot juice samples, and a limit of quantification of 0.040 mg L −1 was determined.

Fungicide Residues Exposure and β-amyloid Aggregation in a Mouse Model of Alzheimer’s Disease

“…Brain tissue sections from 10-month-old J20 mice were incubated with 2 mM of these fungicides, either alone or in cocktail, for 1.5 h at RT. Primary antibodies against cypro (Esteve-Turrillas et al 2012), mepa (Esteve-Turrillas et al 2013), and pyri (Mercader et al 2012) were used to detect them (Figure 2A, column 3). Amyloid aggregates were revealed using a 6E10 antibody conjugated to Alexa488 (Figure 2A, column 2).…”

“…Sections were then washed in PBS and incubated 1.5 h at RT with a solution containing 2 mM of cypro, or mepa, or pyri or a cocktail of the three fungicides (2 mM each). Sections were incubated 1 h at RT with a blocking solution (3% goat serum, 0.3% Triton X100 in PBS) and then 2 h at RT with a mouse anti-b-amyloid 1-16 conjugated to AlexaFluor ® 488 (1:1,000, clone 6E10, Ozyme; referred to as 6E10-Alexa488), followed with the primary antibodies recognizing cypro (Esteve-Turrillas et al 2012), mepa (Esteve-Turrillas et al 2013), or pyri (Mercader et al 2012). Then, the sections were incubated with a goat anti-mouse Cy3 antibody (Jackson ImmunoResearch) for 2 h at RT, and sections were counterstained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI; 1:50,000 in PBS) (Molecular Probes).…”

Mepanipyrim haptens and antibodies with nanomolar affinity

“…1). 11,12 The aim of the present study was the 60 generation of high-affinity and selective antibodies to mepanipyrim. To that purpose, two derivatives carrying the same functionalized six-carbon aliphatic chain at two different tethering sites of the mepanipyrim framework were synthesized.…”

Development of immunocolloidal strip for rapid detection of pyrimethanil

“…In the growing season, harvesting period, and post-harvest storage period of vegetables and fruits, it is necessary to spray fungicides to inhibit the growth of pathogenic fungi, prevent fungal diseases of plants, and prevent fruit from rot. Pyrimethanil (PYR) belongs to the class of aniline pyrimidine bactericides (Esteve-Turrillas, Mercader, Agulló, Abad-Somovilla, & Abad-Fuentes, 2015), which can inhibit fungal secretion of hydrolase, thereby inhibiting the degradation and digestion of infected tissues by fungi (Mercader, Esteve-Turrillas, Consuelo, Antonio, & Antonio, 2012). Pyrimethanil is a broad-spectrum fungicide and can effectively control grey mold on fruits, vegetables, and ornamental plants (Esteve-Turrillas, Mercader, Agulló, Abad-Somovilla, & Abad-Fuentes 2017;Esteve-Turrillas et al, 2015).…”

“…In recent years, there have been reports on the development of pyrimethanil ELISA. Among them, Mercader et al developed an ELISA based on a pyridoxamine polyclonal antibody with a detection limit of 0.024 ng/mL (Mercader, Esteve-Turrillas, Agulló, Abad-Somovilla, & Abad-Fuentes, 2012). Esteve-Turrillas has developed an ELISA based on a pyridoxamine monoclonal antibody with an IC50 value of less than 0.3 ng/mL (Esteve-Turrillas et al, 2015).…”