Environmental Engineering Reference
In-Depth Information
e.g., a bacterium or a virus) is then treated with a second antibody which recognises and binds to a
different region of the analyte. This type of ELISA is called 'sandwich immunoassay' which is a non-
competitive format. This second antibody is covalently attached to an enzyme. After the addition of
the second antibody, the plate is washed to clear unbound substances.
In contrast to 'large' analytes, immunoassays to detect small chemicals (e.g., pesticides), rely
almost entirely on competitive ELISAs, of which there are two types. In the direct competitive
format (i.e., capture assay) the antibody is immobilised on the solid phase (the microtiter plate).
Sample analyte and a specially synthesised analyte-enzyme conjugate (the so-called 'label') are
added which then compete for the limited antibody on the coated surface. In the indirect competitive
format, instead of the antibody, a coating antigen is immobilised consisting of an analyte-protein
conjugate. Depending on the assay format, either analyte-enzyme conjugate (direct format) or anti-
body (indirect format) binding to the surface-immobilised reagent occurs in inverse proportion to
the amount of free analyte present in the sample. For further details, the reader is referred to Knopp
and Riessner (2004).
The presence of the indicator enzyme enables quantitative analysis because it transforms for
example, a colourless reactant into a coloured product (Nelson and Cox 2008). Since one enzyme
molecule can catalyse the same reaction many times, numerous molecules of coloured product are
created for each analyte molecule (Harris 2007). Hence, the presence of the enzyme amplifi es the
signal until the reaction is stopped, normally with a dilute acid. The higher the concentration of
analyte in the original unknown sample, the more enzyme is bound and the greater the extent
of the enzyme-catalysed colour-based reaction. Alternately, the enzyme can convert a non-fl uores-
cent reactant into a fl uorescent product. Both colorimetric and fl uorescent enzyme-linked immuno-
sorbent assays are sensitive to less than a nanogram of analyte (Harris 2007).
1.5.1.7 Field testing kits
Analytical techniques such as gas and liquid chromatography are very sensitive and reliable, but are
simply not practical for fi eld use. These techniques are time consuming and expensive, and must
be performed by highly trained operators who can analyse and interpret the results correctly. Field
test kits have however been developed to detect the presence of organophosphorus and carbamate
compounds in water samples (e.g., in drinking and surface water) and in residues prepared as a dry
extract (i.e., evaporated down from a solvent extract). In principle, these kits should be adaptable
to environmental samples such as plant matter, soil and wildlife tissues. The test kits are qualitative
and colorimetric, meaning that the presence or absence of compounds is determined on the basis of
the appearance or lack of a distinct colour. The principle behind the tests is based on the inhibition
of cholinesterase activity by organophosphates or carbamates. The reduction of the catalytic activity
is dependent on the concentration of the carbamate in contact with the enzyme and can be detected
visually or with inexpensive colorimeters.
A popular test kit (the Organophosphate/Carbamate Screen Kit) developed by Abraxis LLC
(www.abraxiskits.com) has now been evaluated by the US EPA under the Environmental Testing
Verifi cation Program (ETV). The kit utilises acetyl cholinesterase (AChE), which in the absence of
an inhibitor (i.e., carbofuran), hydrolyses acetylthiocholine (ATC) which reacts with 5,5'-dithiobis-
2-nitrobenzoic acid (DTNB) to generate a yellow colour (read visually or at 405 nm with a spec-
trometer). Each test kit comes with specifi c instructions regarding the volumes required and the
incubation times needed. The best results are obtained when each sample is prepared in an identical
manner. Specifi c volumes of sample are introduced into assay tubes, an oxidiser is added and incu-
bated for fi ve minutes, this is followed by a neutraliser, then by the AChE, which is incubated for
between 15 and 30 minutes. Finally a stop solution is added to terminate the reaction and 'fi x' the
colour before it is read.
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