An improved competitive inhibition enzymatic immunoassay method for tetrodotoxin quantification

Tetrodotoxin (TTX) is a low molecular weight neurotoxin that blocks the pore region of voltage-gated sodium channels [1-3] and is found in a wide array of taxa [reviewed by [4]]. The diversity of species with TTX raises questions about the ecological functions and evolutionary implications of TTX [reviewed by [5]]. Further, TTX is of concern to human health as fugu and marine gastropods are commonly consumed in Asian countries [e.g. [6,7]]. Therefore, quantification of TTX is of high importance for multiple fields of research. Traditional approaches for quantifying TTX have limited efficiency and practicality. For example, one common method of quantifying TTX is High Performance Liquid Chromatography [HPLC; reviewed by [8,9]]. HPLC is an effective means of measuring TTX but is costly, time consuming, and requires special training and expensive equipment.A more efficient method of quantifying TTX is to use an immunoassay specific to tetrodotoxin. Several methods for Competitive Inhibition Enzymatic Immunoassays (CIEIA) or other competitive enzyme immunoassays (EIA) exist [e.g. [10-13]]. However, in our experience previously published immunoassay methods are not replicable without detailed knowledge of EIA procedures, contain errors, or report suboptimal concentrations of reagents. Here, we report a modified CIEIA procedure that employs a commercial monoclonal antibody specific to TTX for identification and quantification. Additionally, we report the repeatability between plates within a lab. This method is flexible and adaptable and could identify and quantify TTX in a range of medical or ecological studies using readily available and more affordable lab equipment and reagents.This assay is highly repeatable, sensitive, and an accurate means of quantifying TTX (Table 1). The minimum limit of detection was 10 ng/ml (13; Figure 1a), and the linear range of the standard curve was 10-500 ng/mL with r2 = 0.9759 (Figure 1b). In the linear range, the average intraplate CV f