Enzyme-Linked Immunosorbent Assay (ELISA)

Immunoassays are based on the binding interaction between a highly specific antibody and the analytes of interest. The most common of these assays is the ELISA kit using antibodies raised to specific cyanotoxins. The toxins are detected by the modification in the colour reaction with the intensity of the colour being inversely related to the amount of toxin.

ELISAs can offer rapid results with a relatively low investment in capital equipment. As these assays do not identify specific cyanotoxin variants of a toxin class and give an indication of total toxin concentration - total

Setup for solid-phase extraction

Figure 14.2 Setup for solid-phase extraction (SPE) for concentrating trace amounts of cyanotoxins dissolved in water. I. Filtered water sample in stoppered glass bottle; 2. PTFE tubing carrying water under vacuum; 3. PTFE tubing; 4. SPE cartridge with sorbent; 5. PTFE stopcocks used to stop and start flow; 6. water flowing to waste while cyanotoxins are adsorbed on the cartridge; 7. vacuum manifold system with removable rack; 8: reservoir used to introduce solvents for conditioning and eluting the cartridge (syringe); 9. pressure gauge with needle valve; 10. concentrated sample eluted into a sample collection tube or vial; II. vacuum line connection.

microcystins, for example - they are often used as a screening method. It is recommended to confirm toxin content and to routinely check for false negatives using instrumental methods (HPLC, LC/MS; Gaget et al., 2017). Where such methods are not available, periodic shipping of a few selected samples to a support laboratory elsewhere may be an option.

ELISA kits are very popular for a rapid, straightforward detection of most classes of cyanotoxins, although an individual kit is required for each class of cyanotoxin and even different kits may be necessary to cover the variants within one class. The kit-based formats provide a straightforward guidance on how to perform, calibrate and interpret the results. Multiple samples can be evaluated at one time, and results can typically be reported in less than a day. As with all biochemical test kits, care has to be taken with storage, since ambient or elevated temperature during prolonged transportation can reduce their reliability. The 96-well plate format allows samples to be read and quantified in a plate reader, facilitating the analysis of many samples and the calibration in a short space of time.

Kits often come with a removable strip format so that not all wells need to be used at the time of analysis thus increasing the cost-effectiveness of the assay. If only a few samples are to be assayed, it is advisable to confirm the format before making a purchase.

ELISA kits with sensitivity in the range of the WHO lifetime cyanotoxin guideline values are commercially available for almost all classes of cyano- toxins (see below). However, it is important to remember that cell-bound toxins need to be extracted prior to performing the ELISA. Also, care has to be taken to quench oxidants used in water treatment (chlorine or chlorine dioxide; see above) and to ensure the pH of the sample is appropriate for the specifications of the assay. Filtration or centrifugation may be required to remove particulates, and dilution may prove necessary to ensure that the quantification is in the approved range given in the instructions. Where the cost of these kits is a problem and access to producing antibodies is available, an option may be to produce antibodies in-house or to have this provided through co-operation with a an external institution (university, company, etc.), as demonstrated in the case study described in Box 15.1 in Chapter 15.

High-Performance Liquid Chromatography (HPLC)

HPLC has become well established for the routine analysis of environmental pollutants. These systems consist of a solid-phase chromatography column through which analytes dissolved in liquid solvents are pumped and separated due to differences in the interaction of individual analytes with the solid phase. The flow then passes through a detector, for example, UV absorbance or fluorescence detectors, with the absorption proportional to the amount of analyte, with data collected on a computer. Most systems now include an autosampler to allow a set of samples to be loaded and automatically analysed. The number of samples that can be analysed in a given space of time depends primarily on the duration of a single sample run. For example, the run time for microcystins with a conventional HPLC is around 1 h per sample. Analyses with fewer target compounds, that is, less structural variants such as cylindrospermopsin or anatoxin-a, generally require shorter run times and hence allow a higher sample throughput. The separation of the analytes can be achieved by isocratic elution; this is when the solvent composition remains the same throughout the analysis. Isocratic elution is suitable for analyses that target only a few analytes and with a limited matrix interference, that is, with relatively low amounts of other, nontarget compounds. To allow for better separation of target analytes, gradient elution is commonly applied, where the proportions of the solvents change over the run time. This allows a wide range of analytes to be separated, such as multiple variants of microcystins. To ensure that analytes and contaminants are not carried over to the next sample, a washing step with 100% solvent is often included in the analytical run.

The most common detector used on HPLC systems is the photodiode array (PDA), which will provide an adsorption spectrum (200-600 nm) for the compounds being analysed. This is useful for the analysis of cyanotox- ins as many of them have characteristic UV absorption spectra (Figure 14.4 and 14.5), thus providing an indication even of cyanotoxins in the sample for which no standard reference material is available (see Box 14.2).

BOX 14.2: REFERENCE MATERIAL FOR CALIBRATION

According to IUPAC, reference material is defined as “a substance or mixture of substances, the composition of which is known within specified limits [...] to be used for the calibration of an apparatus”. For cyanobacterial toxins to be used as reference material for establishing a calibration curve for the quantification of these cyanotoxins, two criteria need to be fulfilled:

  • 1. Purity defines the share of an individual compound of the total material. Purity is generally expressed in gravimetric percent that should be at least 95% in reference material.
  • 2. Amount is generally defined in gravimetric units, and ideally with the specified limits, that is, a range of amount that should be as narrow as possible.

In this sense, not all cyanotoxins that are commercially available are reference materials. Hence, these compounds cannot be used directly to establish calibration curves. In particular, the nominal amount in a vial may deviate considerably from the true amount. In consequence, this means that a calibration curve established with such a “standard” would introduce a systematic error to all subsequent analytical quantifications.

The true amount hence needs to be determined. This can be done either by weighing with a sufficiently precise and sensitive balance or by spec- trophotometric analysis (ISO, 2005). For the latter, wavelength-specific extinction coefficients need to be available, which is the case for some but not all cyanotoxin variants. Extinction coefficients are specific for solvents and temperature; that is, a compound dissolved in water cannot be quantified by using an extinction coefficient established for the compound dissolved in methanol.

For cyanobacterial toxins sold as certified reference material, the purity as well as the amount is well defined, and its can be used directly as standard for calibration.

The typical capital investment required for an HPLC is around $30,000 USD with relatively modest costs for maintenance, including replacement UV lamps and columns.

Training of staff and adoption of a standard protocol is easily achievable, while interpretation of samples and cyanotoxin identification (especially unknowns from their spectra alone) requires more time to develop confidence. This applies equally to UPLC (ultra-performance liquid chromatography) discussed below.

Ultra-Performance Liquid Chromatography (UPLC)

UPLC offers a considerable advantage over conventional HPLC as it allows very rapid separation of analytes (run times of around 10 min) and a greatly reduced solvent usage, typically 0.3 mL/min compared to 1 mL/min for conventional HPLC systems. For example, these systems can achieve the separation of multiple microcystin variants in run times little over 10min, thus providing high throughput of samples, substantial saving and results on the same day for samples with short extraction times (e.g., bloom material and filter discs with cells). For samples requiring longer extraction (SPE of water samples or tissue samples), it can yield results within 24 h.

UPLC systems are highly reliable with the response factor for micro- cystins in the UV detector changing little over time. The capital investment should typically be around $50 000 USD for the complete system at relatively low levels of maintenance, with the main component that needs replacement being the UV lamp (it is useful to have a spare in stock).

 
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