an epidemiological study of listeriosis in dairy cattle

The plate was incubated at 37ºC overnight. The plate was washed 6 times in PBS
Tween 20, standing for 5 minutes at the last wash. 100µl volumes of blocking buffer
(1% Fetal Calf Serum in PBS) were added to each well and the plate was left at room
temperature for 4-5 hours. The plate was then washed 6 times in PBS Tween and 100µl
of serum diluted 1:50 in PBS were added to each well and incubated at room
temperature for 4-5 hours. The plate was then washed again 6 times and 100µl of
Alkaline phosphatase conjugated Rabbit α Bovine IgG (Sigma, Dorset, UK) diluted
1:5000 was added to each well. The plate was incubated at 4 0 C overnight, washed 6
times and 100µl phosphatase substrate (Sigma, Dorset, UK) (1mg/ml in carbonate-
bicarbonate buffer) was added to each well. After standing for 15-20 minutes the
reading was taken at 405nm absorbance using an ELISA reader (Dynex Tech.,
Guernsey UK).
c) Optimisation of the assay and specificity:
Determination of the optimal antigen and serum: The optimal working dilution of
antigen and positive control serum was worked out using chequerboard titration in
which different two fold dilutions of the antigen were tested against goat hyper-
immune serum. 100μl of coating buffer was put in to each well 100μl of the antigen
was added to the first row of the plate and doubling dilutions were made from 1:2 to
1:256. 100μl hyper-immune serum were put into the first column of the plate and
doubling dilutions made from 1:2 to 1:4096.
Determination of coating conditions: Three different temperatures, 4 0 C, room
temperature and 37 0 C, were used to optimise coating. 100μl of LLO in carbonate
bicarbonate buffer at 1:50 dilution was used to coat 3 identical plates. The ELISA was
done as described earlier.
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