Biology Reference
In-Depth Information
During recent years, recombinant antibody technology has replaced the conventional methods of
production of polyclonal and monoclonal antibodies specifi c for MCs. This method is very simpler
and cost effective than the conventional methods used for antibody production. The existence of
antibody variable domains in fi lamentous phages proved to be advantageous in the selection and
enrichment of recombinant antibody fragments against a range of heptens including MCs from large
antibody libraries (McCafferty
et al
., 1990; McElhiney
et al
., 2000). Antibody fragments so selected
can be expressed in a bacterial host such as
E
.
coli
so that large amounts of antibodies are produced
than those of existing methods. This has been termed as phage display technology. By the application
of this technique, recombinant phage antibody clones have been isolated against MC-LR from a
naïve human semi-synthetic phage display library (McElhiney
et al
., 2000). Such selected phage
antibodies were expressed as soluble single-chain antibody fragments (scAbs). Characterization
by competitive ELISA, cross-reactivity of the most sensitive scAb clone as determined by ELISA of
related toxins and the quantitative determination of the contents of MCs in cyanobacterial extracts
enabled the detection of MC-LR, MC-RR, MC-LW and MC-LF in micromolar range. The isolation
of scAb with the help of Griffi n 1 phage display library proved to be 800 times more sensitive for
MC-LR since it could detect the toxin below 1 µg L
-1
concentration. Further, immobilized anti-MC-LR
scAb was used in immunoaffi nity columns for concentrating MC-LR from large volumes of water
for subsequent analysis by HPLC. The recovery rate was found to be 94% (McElhiney
et al
., 2002).
Thus this technology offers a large scale, low-cost, rapid, specifi c and sensitive assay method.
iii) PP Inhibition
:
PP1 and PP2A are severely inhibited by MCs. The activity of PPs can be determined
by the release of phosphate from phosphorylated protein substrates. Since MCs inhibit phosphatase
activity, the extent of release of phosphate can be measured by this assay. Assay systems involving
P
32
-labelled substrate (MacKintosh
et al
., 1990; Yoshizawa
et al
., 1990; Honakanen
et al
., 1990, 1994;
Holmes and Boland, 1993; MacKintosh and MacKintosh, 1994; Chaivimol
et al
.,
1994; Ash
et al
., 1995)
and P
33
-labelled
substrate (Sahin
et al
., 1995) have been described that measure the radiolabelled
phosphate produced. MCs from environmental samples have been determined by using P
32
-labelled
substrate (Sim and Mudge, 1993; Jones and Orr, 1994; Lambert
et al
., 1994; Lam
et al
., 1995). Though
the detection limits are around 0.1 µg L
-1
or lower than this (Lambert
et al
., 1994; Xu
et al
., 2000) due
to cumbersome experimental procedures, costly nature of chemicals and precautions to be taken
in handling the chemicals, it did not fi nd favour in routine analysis. Alternatively, a colorimetric
assay using p-nitrophenol (An and Carmichael, 1994; Ward
et al
., 1997; Liu
et al
., 2002; Akin-Oriola
and Lawton, 2005) was developed. PP-inhibition assay is generally carried out by using a modifi ed
colorimetric method described by these workers in which the substrate p-nitrophenylphosphate
(uncoloured) is allowed to react with PP1 (purifi ed) and the resultant product p-nitrophenol (yellow
colour) formed is assayed using a microtitre plate reader (Dynex MRX II with Revelation software
version 4.02). Though not specifi c for MCs, PP inhibition would greatly help as a convincing screen
for the presence of hepatotoxins in any given sample. One important precaution to be kept in view
is the endogenous phosphatase activity of the extracts.
A colorimetric immuno-protein phosphatase inhibition assay was developed for the identifi cation
of MCs and nodularin from laboratory cultures and cyanobacterial bloom samples. Polyclonal
antibodies raised against MC-LR were used in conjunction with protein phosphatase inhibition
which was possible to distinguish seven variants of MCs (MC-LR, D-Asp
3
-RR, -LA, -LF, -LY, -LW
and -YR) one from the other as well as nodularin on the one hand and okadaic acid, calyculin A
and tautomycin on the other (Metcalf
et al
., 2001). This technique is based on the ability of MC-LR
antiserum to protect PP1 from the inhibitory action of cyanobacterial toxin. MC-LR antiserum
