Analytical Techniques

MERHAB-LGL is charged with developing new techniques that can be used in both field and laboratory. There is no perfect technique for the analysis of cyanobacterial toxins and we currently use a number of different approaches for the measurement of cyanobacterial toxins:

Microcystin

• Protein phosphatase inhibition assay; an activity-based enzyme assay. This is the basic method described by Carmichael and An and provides a good estimate of the biological activity of microcystins present in the sample.
• ELISA; a structure based assay that uses antibodies against the microcystin molecule. We use several different commercially available kits as well as antibodies developed in house for our ELISA assays.
• HPLC with photodiode array detection; a rapid chromatographic assay that is specifically focused on the microcystin chromophore. It is useful to determining the different congeners present
• HPLC with mass spectral detection; a second rapid chromatographic assay that measures the molecular weight of the individual congeners.
• MALDI-TOF mass spectrometry; another mass spectroscopy techniques that does not require extraction of the toxins from the cells. It is commonly used to determine what congeners are present but not their quantitation.
• PCR; This is the polymerase chain reaction and specifical used to determine if potentially toxic organisms are present in a water body even if they are not producing toxins.
• LFI; A lateral flow immunoassay that uses a commercially available strip containing antibodies against microcystins similar to a "home pregnancy test"

Anatoxin-a

• HPLC- with fluorescent detection; This is the workhouse method of James et al. and readily detects both anatoxin-a as well as its degradation products.
• HPLC with mass spectral detection; a second chromatographic assay that measures the molecular weight of anatoxin-a to confirm its presence.
• ELISA; a structural based assay that uses antibodies prepared against an anatoxin-a conjugate to detect presence of the anatoxin ring system.

PSP-Toxins

• HPLC-ECOS with fluorescent detection; This is our workhouse method that used electrochemical oxidation of the saxitoxin ring system to form a fluorescent derivative.
• HPLC-PCRS with fluorescent detection; A second HPLC method that uses a post column reactor system to chemically form the fluorescent derivatives. This is the method currently used for detection of PSP toxins in shellfish.
• HPLC with mass spectral detection; a third chromatographic assay that measures the molecular weight of the PSP toxins.
• ELISA; a structure based assay that uses antibodies against the saxitoxin and neosaxitoxin. We use both commercially available kits as well as antibodies we are developing in house in our ELISA assays.
• LFI; A lateral flow immunoassay that uses a commercially available strip containing antibodies against saxitoxin similar to a "home pregnancy test". It also cross-reacts with many of the other PSP toxins.

Cylindrospermopsin

• HPLC with photodiode array detection; a rapid chromatographic assay that is specifically focused on the cylindrospermopsin chromophore. It is useful for rapid screening for the presence of cylindrospermopsin.
• HPLC with mass spectral detection; a second chromatographic assay that measures the molecular weight of cylindrospermopsin. It is less sensitive to interferences and used to confirm the presence of cylindrospermopsin in our screening program.

Anatoxin-a(S)

• Acetyl Cholinesterase inhibition assay; This is an activity based enzyme assay that provides a good estimate of the biological activity of anatoxin-a(S) that may be present in the sample. It is unfortunately also used to detect ACI-based pesticide activity and subject to interference from that class of compounds.