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.

2. Cover plate and incubate overnight at room temperature. 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 50ml of appropriately diluted biotinylated antibody to each well (Dilute antibody in PBS). Add

50ml of diluted standards/samples to each well (we recommend the use of duplicate or triplicate samples and standards). Incubate for 2 hours at 37^oC.

5. Wash x4 in wash buffer.

6. Add 100ml of appropriately diluted enzyme conjugated to streptavidin to each well (Dilute in PBS).

Incubate for 60 minutes at 37^oC. Wash x4 in wash buffer.

7. 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.

8. 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.