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Note:
This method provides a
general procedure for use with the majority of Serotec reagents.
In some cases specific recommendations are provided on product
datasheets, and these methods should always be used in conjunction
with product and batch specific information provided with each
vial. Please note that a certain level of technical skill and
immunological knowledge is required for the successful design
and implementation of these techniques - these are guidelines
only and may need to be adjusted for particular applications.
1. Coat microtitre plate wells with 100ml of appropriate coating antibody, at a concentration of between
1-10mg/ml
in carbonate/bicarbonate buffer. Cover plate and incubate overnight
at 4oC. Wash plate x4 in wash buffer.
2. Add 150ml
of blocking solution to each well. Incubate 60 minutes at 37oC.
Wash x4 in wash buffer.
3. Add 100ml
of appropriately diluted samples to relevant wells. Ensure that
appropriately diluted standards are included. Samples or standards
should preferably be run in triplicate. Incubate 90 minutes at
37oC. (Dilute samples and standards in PBS). Wash
x4 in wash buffer.
4. Add (Dilute antibody in PBS) 100ml
of appropriately diluted enzyme conjugated detection antibody
to each well. Incubate for 60 minutes at 37oC. Wash
x4 in wash buffer.
5 Add 200ml
of appropriate substrate solution to each well. Incubate at room
temperature (in the dark if required) for 30 minutes, or until
desired absorbance values are attained.
6. Read absorbance values at appropriate
wavelength.
Recommended buffers:
Coating buffer:
4.42g Na2CO3, 5.04g NaHCO3, 1 litre
distilled water pH 9.6.
PBS:
16.7g Na2HPO4, 5.7g NaH2PO4, 85g NaCl,
10 litres distilled water pH 7.4.
Blocking buffer:
PBS containing 1% w/v Bovine Serum Albumin
(BSA).
Wash buffer:
PBS containing 0.05% v/v Tween 20.
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