# Determination of Cyanotoxins and Prymnesins in Water, Fish Tissue, and Other Matrices: A Review
## 4 Sample Preparation and Analytical Methods for Cyanotoxins and Prymnesins Detection
### Analytical Methods  
#### 4.2.1 Immunological Assays  
Cyanotoxins can be detected through recognition and binding to specific antibodies, either monoclonal or polyclonal. For example, ELISA kits are commercially available for the detection of MCs in water based upon either the common \"adda\" moiety present or specific recognition of a single MC congener [72; 73; 49; 74]. Depending upon the antibody and the procedure employed, these kits can achieve a detection limit (DLs) as low as 4 ng/L, with an upper quantitation limit (due to saturation) of 5 ug/L for MC-LR [73]. While ELISA is frequently employed for the detection of MCs, particularly in drinking water, ELISA kits have also been made commercially available for the detection of ANA-a, CYN, and STX [75; 76]. The most significant advantage of ELISA methods is that they do not require expensive and high-upkeep analytical instrumentation to be maintained, as they typically rely only on colorimetric assays for quantification.  
However, detection methods based on ELISA have some limitations of varying severity by target compound. The measurement of a variety of MCs by ELISA is possible because the antibody assay is broadly cross-reactive over different MC congeners; however, this cross-reactivity is not uniform, and in some cases, MCs may be measured with greater or lesser responses relative to MC-LR [20]. Beyond that, only a single signal for a general MC concentration will be obtained, even for a sample that might contain a variety of MC congeners of varying potential toxicity levels. As a result, ELISA-based measurements should be considered to be semi-quantitative in that the measured concentrations are influenced by a number of variables that may or may not be known at the time of analysis. In addition to this limitation, cross-reactivity of the assay with other compounds in the sample and matrix may lead to over- or underestimation of the concentration of toxins, for both MCs and other cyanotoxins. This was observed in one paper, mentioned in Table 3, where ELISA kits targeting the \"adda\" moiety common to most MCs were found to be generating false positive results when compared with LC-MS/MS and an alternative, monoclonal antibody specific to MC-LR [77]. Conversely, it has also been demonstrated that ELISA-based screening may fail to measure protein-bound or glutathione-conjugated species in tissue matrices, perhaps due to the molecules being inaccessible to the antibody. When ELISA extracts were compared with a chemical oxidation/derivatization technique for measuring total MCs in tissue, a significant enhancement in the measured concentrations was observed in the latter [78]. These types of issues are not specific only to ELISA, but rather a general complication for any measurement technique. The sequestration/transformation of toxins through metabolic/removal processes may make them unavailable for many types of assays.  
ELISA-based techniques can also be sensitive to the presence of solvents such as methanol and acetonitrile that are often used for tissue extraction. Commercially available ELISA kits typically recommend <5-10% solvent, but this value may be as low as <2.5% for ANA-a and as high as <20% for CYN and STX, based on standard protocols included in the various ELISA kits; therefore, in many studies relying on ELISA to quantify toxins from tissue extracts (see Table 3), the organic extracts are evaporated to dryness under nitrogen at 30-60 \({}^{\circ}\)C and reconstituted in DI water or an appropriate diluent prior to analysis [20]. Low pH can also affect ELISA performance, which could be an issue for STX extracted under acidic conditions. In many of the reviewed studies, pH was adjusted with 0.1 N NaOH to improve compatibility with the ELISA assay.