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

Abstract

Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated 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. However, 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 potentially 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 standard cell culture medium containing 10% FBS served to investigate potential matrix effects. Inter-laboratory 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, sample concentration and/or MC spiking may be required for detection of low levels of MCs in human blood.AbbreviationsMeOH, methanol; SPE, solid phase extraction; MC, microcystin; LOD, limit of detection; FBS, fetal bovine serum; RT, room temperature; DMEM, Dulbecco’s modified Eagle medium; PPIA, protein phosphatase inhibition assay; HPLC, high performance liquid chromatography; LC–MS, liquid chromatography–mass spectrometry; ELISA, enzyme-linked immunosorbent assay; PP, polypropylene; LDPE, low-density polyethylene; HDPE, high-density polyethylene