Comparison of two ELISA-based methods for the detection of microcystins in blood serum

Abstract

a b s t r a c tMicrocystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contam inated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC MS, GC MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC MS methodology would appear to allow detection of these MC levels. How ever, LC MS detection depends on the availability of respective MC congener standards and the levels of non covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC ELISA poten tially could detect even covalently bound MC, provided the MC antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda side chain moiety is present in nearly all of the MC congeners known to date, the anti Adda antibodies, when applied in Adda ELISAs, could represent a relatively simple and robust technique for the qualitative and quantitative determination of MC in human serum.The aim of the current study was to determine whether commercially available Adda ELISAs and their respective sample preparation methods would allow MC quantification in human serum. The Adda ELISA (polyclonal antibody) and the Adda ELISA (monoclonal antibody) kit for serum (Serum ELISA) were used for determination of the concentration dependent recovery of MCs in MC spiked serum.Human serum samples were spiked with varying concentrations of MCs (MC LR, YR, RR, LA, LW, LF and defined MC mixtures) and extracted using two different methods. MC spiked bovine serum and stan dard cell culture medium containing 10% FBS served to investigate potential matrix effects. Inter labora tory comparison was performed allowing identification of potential sources of error.The results suggest that both ELISAs are suitable tools for the analysis of MCs in human blood serum although both also displayed some weaknesses notably the time needed for sample preparation or the overestimation of some specific MC congener concentrations. Based on the ELISA detection ranges, sam ple concentration and/or MC spiking may be required for detection of low levels of MCs in human blood.

Pitfalls in microcystin extraction and recovery from human blood serum

“…Based on the detectability of the various MC cong eners in the ELISA and the reported coefficients of variation (CVs) for standards (CV < 10%) and samples (CV < 15%), expected theoretical range of MC recovery was set ranging between 80% and 120%. The latter is supported by our preliminary data, CVs and observations by the manufacturer and earlier reports [11,20,21].…”

“…6). A previous study dem onstrated promising MC recovery performance of high density polyethylene (HDPE) vials with respect to short term (hours) use [11]. Thus HDPE vials may be an alternative for the centrifugation step, which cannot be performed in the conical glass vials.…”

“…Due to lack of validated coefficients for each of the MC congeners used in this study, all MC congeners were prepared as if they had the same absorption coefficient as MC LR. Although some investigators used specific extinction coefficients (Wayne Carmichael, personal communica tion), using the same coefficient for the major MC congeners repre sents a widely accepted approach because the differences seem to be quite small (personal communication with Jussi Meriluoto and Linda Lawton) and thus will not impact the overall findings (for a more detailed discussion of this issue see [11,19]). The structural differences among the six MC congeners employed in this study are depicted in Fig.…”

Current Techniques for Detecting and Monitoring Algal Toxins and Causative Harmful Algal Blooms

“…Another preferred feature of ELISA compared to LC-MS is that ELISA could detect covalently bound algal toxin such as MCs, whereas LC-MS relies on the availability of congener standards and the proportion of non-covalently bound MC in the sample [47]. Its technical limitations are self-evident, such as the lack of selectivity and the occurrence of cross-reactivity due to the high variability of compounds that might be found in water samples.…”

“…One particular constrain is its lower sensitivity limited relatively to a narrow range of toxins. Consequently, it has the potential to give false negative for insensitive toxic congeners [45], false positive at low concentration [48] and overestimate some specific toxin congener concentration [47]. ELISA tends to give false positive detection at the low concentration range (0-0.15 µg/L), even though some good correlations were demonstrated between ELISA and HPLC for toxins in surface and drinking water [48,49].…”