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

4.2.3 -
High-throughput AFP assay
The assay was applied to all 866 isolates from the Antarctic lake environments.
Of these, 187 (21.6%) proved to be positive for AFP activity by this assay. Activit\
ww as
demonstrated by a reduction in transparency of the protein extracts when compared with
a 30% sucrose solution without protein extract. The samples could not be quantified or
qualified for level of activity as with a `SPLAT' assay, however, protein extracts were
marked as either active or non-active. Those with reduced transparency (increased
opacity) or colouration which were difficult to confirm were classed as positive, and
those extracts with refraction equal or less than the negative control (30% sucrose) were
classed as negative. The 187 bacteria strains, which were identified as positive using the
HTAP assay, are shown in table 4.1. These isolates were returned to England and
analysed using the `SPLAT' assay to confirm AFP activity within the bacteria. Of the
187 bacteria identified as AFP active using the HTAP assay, only 12 were Gram positive.
4.2.4 -
SPLAT assay
The 187 bacterial isolates from which positive protein extracts (using the HTAP
assay) were extracted, were brought back to Colworth (Unilever) and assessed for
activity using the `SPLAT' assay. Nineteen of the bacterial isolates were confirmed to
have some level of AFP activity within their total cellular extracts (Table 4.2), which
equates to 10.2% of those isolates showing a positive protein extract using the HTAP
assay. Ten of these 19 isolates showed a SPLAT score of 4 or 5 (Section 2.10.2).
indicating high AFP activity, comparable with pure fish AFP III protein (1 mg mU 1) or
total cellular protein extracts of Marinomonasprotea
(Mills, 1999). The rest of the
samples had an activity of 3. Samples with an activity of less than 3 were considered to
have insufficient activity for further analysis. AFP active bacteria with a score >3 were
isolated from Ace Lake, Triple Lake, Pendant Lake, Pendant Ice cores and
Oval Lake
(Table 4.2 and Fig. 4.5). All AFP active bacteria, as confirmed by the SPLAT assay of
total cellular protein extracts, were Gram negative.
The concentrations of protein extracts, prepared from each putative AFP active
isolate, were assessed using the `SPLAT' analysis and were calculated using the BioRad
Protein Kits (BioRad, see Chapter 2 section 2.10.1). Figure 4.6 shows that there was no
direct relationship between total cellular protein concentration used for anale sis and
122