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Comparative Analysis of ELISA and MALDI‐TOF Mass Spectrometry Methods for Microcystins in Freshwater Samples
Author(s) -
Kadlec Kimberly Michele,
Turczyn Andrew,
Grant Jennifer
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1122.1
Subject(s) - chromatography , mass spectrometry , microcystin , chemistry , time of flight mass spectrometry , absorbance , matrix assisted laser desorption/ionization , analytical chemistry (journal) , desorption , cyanobacteria , ionization , ion , adsorption , biology , genetics , organic chemistry , bacteria
Health risks associated with freshwater cyanobacterial blooms have led to the development of a variety of analytical methods to detect the presence of microcystins, which are monocyclic heptapeptides that can cause acute and chronic hepatotoxicity when ingested by animals and humans. In this study of soluble microcystin levels in lake water, the sensitivity of detection of Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight mass spectrometry (MALDI‐TOF MS) was compared to that of Enzyme Linked Immunosorbent Assay (ELISA) methods. Lake water samples were collected monthly during the first week of each month, May through August in 2012, 2013 and 2014 and samples were frozen at −20 °C. Prior to analysis, lake water was centrifuged (5 min @ 10,000 × g ) to remove suspended solids. ELISA was performed using Microcystins (ADDA)‐DM ELISA Microtiter Plate, following the manufacturer's protocol (Abraxis Inc.). Absorbance was measured at 450 nm on a BioTek Epoch Microplate spectrophotometer. MALDI‐TOF MS was performed by applying a small volume of sample to a stainless steel MALDI plate, followed by an equal volume of α‐cyano‐4‐hydroxycinnamic acid, using the dried droplet method. Relative quantitation of microcystin content was performed using angiotensin II to create standards ranging from 0.06 μM to 5.0 μM in concentration. Data were acquired using a Bruker Microflex Linear Time of Flight MALDI‐TOF Mass Spectrometer. Spectra were acquired over the mass range of 0.6 to 4 kDa using 50 laser shots in the positive ion mode at a laser power of 25%. Calibration of the mass spectra was achieved using a calibration solution comprised of 1 μM bradykinin and 1 μM neurotensin. Semi‐quantitation with the ELISA kit showed seasonal fluctuations of microcystins peaking in July or August with concentrations up to 10‐fold greater than detected using the MALDI‐TOF MS method. The ELISA kit indicated microcystin levels less than 1.0 μg/L, the maximum concentration for drinking water set by the WHO, in 50% of the samples. MALDI‐TOF MS analysis of the same samples detected seasonal fluctuations of microcystins and indicated the presence of 18 different congeners of microcystin over the course of the entire study. The congener that was visualized most consistently was MC‐LAba (m/z 923), recorded at both locations at concentrations ranging from 0.25 – 0.47 μg/L in 70% of the samples. The congener present in the greatest concentration was found to be MC‐RR (m/z 1037), at 3.42 μg/L in August 2013. Our findings showed general agreement between the ELISA and MALDI‐TOF MS data. The primary drawback in the ELISA methodology is that ELISA cross‐reacts with non‐microcystin targets (e.g. nodularins, N‐hemi‐ADDA and ADDA), often causing an overestimation of the actual concentration of the target microcystins in the sample. MALDI‐TOF MS, in addition to requiring far less sample preparation, identified and quantified specific congeners.