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 this method 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 antigen, at a concentration of between

1-10mg/ml in carbonate/bicarbonate buffer.

2. Cover plate and incubate overnight at 4^oC. Wash plate x4 in wash buffer.

3. Add 150ml of blocking solution to each well. Incubate 60 minutes at 37^oC. Wash x4 in wash buffer.

4. Add 100ml of appropriately diluted antigen specific antibody to each well (dilute antibody in PBS).

Incubate for 90 minutes at 37^oC. Wash x4 in wash buffer.

5. Add 100ml of appropriately diluted enzyme conjugated secondary antibody to each well. Incubate

for 60 minutes at 37^oC (dilute in PBS). Wash x4 in wash buffer.

6. 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.

7. 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