Congener-Independent Immunoassay for Microcystins and Nodularins

Abstract

Cyanobacteria (blue-green algae) (e.g., Microcystis and Nodularia spp.) capable of producing toxic peptides are found in fresh and brackish water worldwide. These toxins include the microcystin (MC) heptapeptides (>60 congeners) and the nodularin pentapeptides (ca. 5 congeners). Cyanobacterial cyclic peptide toxins are harmful to man, other mammals, birds, and fish. Acute exposure to high concentrations of these toxins causes liver damage, while subchronic or chronic exposure may promote liver tumor formation. The detection of cyclic peptide cyanobacterial toxins in surface and drinking waters has been hampered by the low limits of detection required and that the present routine detection is restricted to a few of the congeners. The unusual -amino acid ADDA (4E,6E-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) is present in most (>80%) of the known toxic penta-and heptapeptide toxin congeners. Here, we report the synthesis of two ADDA-haptens, the raising of antibodies to ADDA, and the development of a competitive indirect ELISA for the detection of microcystins and nodularins utilizing these antibodies. The assay has a limit of quantitation of 0.02-0.07 ng/mL (depending on which congeners are present), lower than the WHO-proposed guideline (1 ng/mL) for drinking water, irrespective of the sample matrix (raw water, drinking water, or pure toxin in PBS). This new ELISA is robust, can be performed without sample preconcentration, detects toxins in freshwater samples at lower concentrations than does the protein phosphatase inhibition assay, and shows very good cross-reactivity with all cyanobacterial cyclic peptide toxin congeners tested to date (MC-LR, -RR, -YR, -LW, -LF, 3-desmethyl-MC-LR, 3-desmethyl-MC-RR, and nodularin).

Guidance values for microcystins in water and cyanobacterial supplement products (blue-green algal supplements): a reasonable or misguided approach?

“…Although the providers of Aphanizomenon flos-aquae based BGAS state that they screen out MC-levels of more than 1.0 Ag/g dw in their products , independent investigations into the MC contamination of BGAS products have demonstrated toxin concentrations of up to 35 Ag/g dw (Gilroy et al, 2000;Lawrence et al, 2001). Although samples with toxin contaminations of more than 10 Ag MC-LR equivalents/g dw are the exception, 8 of 13 blue-green algae products from the German and Swiss markets tested recently have shown more than 1.0 Ag MC-LR equiv./g DW in concurrent analyses carried out with an Adda-ELISA (Fischer et al, 2001) and cPPA (Heresztyn and Nicholson, 2001). Our own studies ) and the study of Lawrence et al (2001) have shown differences in detectable toxin amounts when employing ELISA, PPA and LC-MS/MS.…”

Occurrence and elimination of cyanobacterial toxins in drinking water treatment plants

“…The ELISA method was performed as described by Fischer et al (2001). Briefly, ELISA plates (NUNC MaxiSorp, Denmark) were coated overnight with OVA-ADDA-hemiglutaryl (OVA-ADDA-HG) in 0.05 M sodium carbonate buffer pH 9.6 (50 Al/well, 2.5 Ag/ml) at 20 jC.…”

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants

“…The detection range (20 -80% inhibition of PP1) of the colorimetric PPA used is 0.9-3.2 mg MC-LR/l with a derived IC 50 of 1.7 mg MC-LR/l. As a structural biological test system, an ADDA-ELISA established by Fischer et al (2001) was used. The limits of quantitation and detection of the ELISA were 0.2 and 0.05 mg/l, respectively.…”