Rapid Immunochemical Methods for Anatoxin-a Monitoring in Environmental Water Samples.

Algal blooms that contaminate freshwater resources with cyanotoxins constitute, nowadays, a global concern. To deal with this problem, a variety of analytical methods, including immunochemical assays, are available for the main algal toxins, for example, microcystins, nodularins, and saxitoxins, with the remarkable exception of anatoxin-a. Now, for the first time, highly sensitive, enantioselective immunoassays for anatoxin-a have been validated using homemade monoclonal antibodies. Two competitive enzyme-linked immunosorbent assays were developed in different formats, with detection limits for (+)-anatoxin-a of 0.1 ng/mL. Excellent recovery values between 82 and 117%, and coefficients of variation below 20%, were observed using environmental water samples fortified between 0.5 and 500 ng/mL. In addition, a lateral-flow immunochromatographic assay was optimized for visual and instrumental reading of results. This test showed a visual detection limit for (+)-anatoxin-a of 4 ng/mL. Performance with a reader was validated in accordance with the European guidelines for semiquantitative rapid methods for small chemical contaminants. Thus, at a screening target concentration of 2 ng/mL, the probability of a blank sample to be classified as "suspect" was as low as 0.2%. Finally, the optimized direct enzyme immunoassay was validated by comparison with high-performance liquid chromatography-tandem mass spectroscopy data and showed a good correlation (r = 0.995) with a slope of 0.94. Moreover, environmental water samples containing more than 2 ng/mL of anatoxin-a were detected by the developed dipstick assay. These results provide supplementary and complementary strategies for monitoring the presence of anatoxin-a in water.

Synthetic Haptens and Monoclonal Antibodies to the Cyanotoxin Anatoxin-a.

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin-a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin-a, we have succeeded in generating the first-ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin.

Dispersive magnetic immunoaffinity extraction. Anatoxin-a determination.

Specific monoclonal antibodies were coupled with magnetic Sepharose-based beads and used, for the first time. The methodology was applied to preconcentrate anatoxin-a from water and the later determination by ion mobility spectrometry (IMS). Dispersive magnetic immunoaffinity (d-MagIA) extraction methodology provided a limit of detection of 0.02µgL-1 and a satisfactory precision with a relative standard deviation lower than 15%. Recoveries were evaluated at 0.5, 1.0 and 5.0µgL-1 anatoxin-a with quantitative values from 91 to 115%. Additionally, isobaric interferences with phenylalanine were completely avoided by the use of the developed d-MagIA extraction coupled to IMS determinations.

Monoclonal antibody-based immunoassays for cyprodinil residue analysis in QuEChERS-based fruit extracts.

Cyprodinil is among the most common agrochemical residues found in highly perishable fruits, such as strawberries. In the present study, high-affinity monoclonal antibodies to this anilinopyrimidine fungicide were raised for the first time with the aim to produce valuable immunochemical analytical assays. Cyprodinil bioconjugates and the generated novel monoclonal antibodies were employed for sensitive competitive immunoassay development in two different formats. The limits of detection of the optimized assays were 20 and 30 ng L(-1) for the indirect and direct assay, respectively. Influence over assay parameters of different physicochemical factors was studied. Strawberry samples were extracted following the recommended QuEChERS procedure for pesticide residues in food, and analyzed by the optimized immunoassays. Recoveries and coefficients of variation from fortified samples were within standard values. In addition, the obtained immunochemical results with naturally contaminated samples were statistically comparable, according to Deming regression analysis, to those of a reference chromatographic method.