Development and Evaluation of Monoclonal Antibodies for Paxilline.

Chris M Maragos

doi:10.3390/toxins7103903

Abstract

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage.

Highlights

The tremorgens are a class of fungal secondary metabolites, which, like the ergot alkaloids, contain a substituted indole-moiety
The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX
In this format an immobilized paxilline-bovine serum albumin (PAX-BSA) conjugate competed with free PAX for PAX antibodies

Summary

Introduction

The tremorgens are a class of fungal secondary metabolites (mycotoxins), which, like the ergot alkaloids, contain a substituted indole-moiety. Toxins 2015, 7 the penitrems (tremortins), janthitrems, lolitrems, aflatrem, paxilline, paspalines, paspalitrems, fumitremorgens, tryptoquivalines, verruculogen, and others [1]. They are produced by certain species of Aspergillus, Penicillium, Claviceps, and Acremonium [2,3,4,5]. Paxilline (PAX, Figure 1) was first isolated as a metabolite of Penicillium paxilli (P. paxilli) that caused sustained tremors and hypersensitivity to sound when given orally to one-day old cockerels and intra-peritoneally to mice [6]. Lolitrem B, a related tremorgen, inhibits such channels and this effect is believed to be the mechanism of action for the disease “ryegrass staggers” [10].

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