Evaluation of ELISA-based method for total anabaenopeptins determination and comparative analysis with on-line SPE-UHPLC-HRMS in freshwater cyanobacterial blooms

Anabaenopeptins (APs) are bioactive cyanopeptides of emerging concern produced by cyanobacteria. The research for analytical development has recently gained in importance due to their abundance in toxic cyanobacterial blooms. A new commercial enzyme-linked immunosorbent assay kit for the determination of total APs (AP(tot) ELISA) has been released promising a rapid response with good cost efficiency for the routine monitoring of uncommon cyanopeptides. The present study explores the suitability of this new kit in comparison with a validated quantitative analytical method based on liquid chromatography coupled to mass spectrometry (LC MS). The validation results were comparable with both methods for accuracy, precision, and calibration. Method detection limits were more sensitive using LC-MS specifically evaluated at 0.011 and 0.013 mu g L-1 for AP-A and B respectively, compared to AP(tot) ELISA evaluated at 0.10 mu g L-1 for total of the two. For AP(tot) ELISA, results were independent from the matrix; however, a systematic signal response was measured in blanks, requiring a blank subtraction in data treatment. Cross-reactivity of AP(tot) ELISA was investigated by analyzing ten cyanopeptides selected for their abundance and diversity. Cyanopeptolin A (CP-A), nodularin-R (NOD), microcystin (MC)-RR, [Asp(3)]RR, and HilR showed cross-reactivity with an average overestimation going from 25 to 66%. Considering the contribution of cross-reactive cyanopeptides, thirteen lake samples out of fifteen showed higher concentrations using AP(tot) ELISA with overestimation values up to 2261% compared to LC-MS. In light of this study results, LC-MS should still be preconized for the study and monitoring of APs when sensitivity and specificity are needed.

Automated summary

Anabaenopeptins (APs) are bioactive cyanopeptides of concern produced by cyanobacteria. A new enzyme-linked immunosorbent assay kit has been developed for their detection, showing comparable results with liquid chromatography coupled to mass spectrometry (LC-MS) for accuracy and sensitivity. However, the ELISA method may overestimate results in certain cases due to cross-reactivity.