ý.,,: V. ý ýý . - Nottingham eTheses - University of Nottingham

non-AFP active peptides from inhibiting ice-crystal growth (Knight et al.. 1995) and,
secondly, to prevent false positive results.
4.1.1 -
Disadvantages of the SPLAT assay
The `SPLAT' assay, although not as accurate as the `Colworth RI assay-' (which
employs computer analysis of photomicrographs of ice crystals to ascertain quantitative
measurements of recrystallisation),
is still a relatively rapid means of qualifying the
recrystallisation inhibition activity of a protein sample. However, the equipment required
is both expensive and cumbersome to transport. Samples also need to be supercooled to
-70°C in dry-ice cooled solvent, which for field conditions such as Antarctica where drv-
ice has to be made in situ, means the transportation of CO2 cylinders which
is both
dangerous and expensive. The protocol is also labour intensive and time consuming. All
of which make the `SPLAT' assay unfavourable for rapid field analysis in `difficult to get
to' environments such as Antarctica.
4.1.2 -
Development of the high-throughput AFP analysis protocol (HTAP)
A protocol was required which would overcome the disadvantages of the
`SPLAT' assay. The new protocol had to be cheaper, more easily transportable and able
to analyse large numbers of bacterial protein extracts in a short period of time in order to
make the most of the valuable field sampling time.
4.1.2.1 -
Premise of methodology
As it was still necessary to record a sample's RI activity, the new assay was based
on the `SPLAT' assay. Whereas the SPLAT assay uses crossed polarized microscopy to
qualify samples for gradated AFP activity, the new assay utilised direct visual
identification of activity by increasing the volume of the sample and observing the
refraction of light through the frozen sample. A supercooled sample has very small
crystals. When a non-AFP active solution is recrystallised, the lack of ice-growth
inhibition means that the crystals increase in size (Fig. 4.1 a) as opposed to solutions
containing AFPs which show a limited increase in crystal size (Fig. 4.1 b). The highly
dense, small crystals resulting from AFP activity, cause a high level of light refraction so
that they appear opaque as opposed to the transparent non-AFP active solutions.
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