CYANOBACTERIAL TOXINS - Cyanobacteria - page 547

was allowed to interact with MC-LR and after a certain period of incubation (15 min at 37ºC), the
colorimetric assay of PP inhibition was performed. In order to interpret the percentage of inhibition
of PP by the toxin after interaction with MC-LR antiserum, a protective index (PI) is deduced by
applying a formula PI = (%Act AS -%Act NS )/%Inhib NS . %Act AS is the rate of p-nitrophenol production in
the protein phosphatase inhibition assay for PP1 inhibitors after incubation with MC-LR antiserum,
expressed as a percentage of the MC-free control value while %Act NS is the rate of p-nitrophenol
production in the protein phosphatase inhibition assay for PP1 inhibitors after incubation with
null serum substracted from the rate of p-nitrophenol production by the MC-free control value.
A PI value of nearly 1.00 was obtained for assays with MC-LY and nodularin where as the PI was
0.94 and 0.90 for MC-LA and MC-LR, respectively. In case of the rest, MC-Asp 3 -RR, MC-LF, MC-
LW and MC-YR the PI value ranged from 0.80 to 0.86. In case of non-cyanobacterial toxins such as
tautomycin, calyculin A and okadaic acid the PI value was lowest at 0.18, 0.07 and 0.02, respectively
suggesting that the MC-LR antiserum did not give protection. This method has an added advantage
as concentrations as low as 10 nM could be detected.
Further, a new PP-inhibition assay that enables the recognition of variants of MCs and nodularin
has been developed. In the fi rst step, PP-inhibition by different variants of MCs and nodularin
revealed IC 50 values of 2.2, 1.8, 9 and 175 nM for MC-LR, nodularin, MC-YR and MC-RR, respectively.
In the second step, taking MC-LR as standard ELISA assay was conducted that showed equivalence
in toxin responses. When these toxins were determined through PP inhibition assay by taking MC-
LR as standard, the ratio of values determined by PP2A inhibition to ELISA decreased in the order:
nodularin (2.23), MC-LR (1.1)>MC-YR (0.63)>Mc-RR (0.06). The differential sensitivity of PP2A
assay to various standards enabled the development of an indicative toxicity ranking (ITR) where
a ranking of 1 was assigned to ratios > or =0.8 or greater and 3 (the lowest) to values < or =0.2. By
this new method, analysis of samples and extracts of cyanobacterial mats from ponds and streams
in cold temperate locations has been conducted. The disadvantage with LC-MS is its dependence
on MC-LR as the standard commercial variable for the determination of all MC-variants whereas PP
inhibition and ELISA not only help in the determination of the variant of MC but also its stoichiometry
(Mountfort et al. , 2005).
A rapid and sensitive detection method for MCs in water samples was developed based on a
new fl uorescence immunochromatography assay system employing monoclonal antibodies of MC-
LR and compared with cyanobacterial toxins analyzed through HPLC. MC-LR was conjugated with
bovine serum albumin to produce monoclonal antibodies against MC-LR in BALB/c mice by an initial
injection of 0.2 ml of the conjugate and incomplete Freund's adjuvant followed by booster injections
of a similar kind. Hybridoma cell line (SP 2 /O-Ag14) was screened for production of anti-MC-LR
antibodies using an indirect ELISA. The positive hybridomas were cloned several times. Antibodies
so produced were purifi ed by membrane ultrafi ltration, ammonium sulfate precipitation with fi nal
step protein G column. Fluorescence coated toxin from water sample was mixed with monoclonal
antibodies against MC-LR. The intensity of fl uorescence of the conjugates was detected on the
detection zone that could be scanned by a Laser fl uorescence scanner. The concentration of MC-LR in
unknown sample was calculated from a standard curve. The monoclonal antibodies produced showed
fairly good binding ability with MC-RR, MC-YR as well as MC-LR. This is the reason why fl uorescence
immunochromatography could detect individual amounts of MC variants but helped in the estimation
of total MCs in a given sample. The performance of fl uorescence immunochromatography for the
detection of MCs compared very well with HPLC (with a correlation coeffi cient of 0.9929) (Pyo et
al ., 2005). A minimum detection limit (MDL) of 11 pg fi lament -1 of single fi laments of Planktothrix
( P . agardhii and P . rubescens ) was feasible by ELISA using antibodies raised against MC-LR. A