A-1 should use an uncoated well; therefore no conjugate should bind, and any color cannot be the result of IgG bound to the well. (The Tween 20 detergent in the conjugate solution prevents the conjugate protein from binding to bare well surface.)

A good (optional) control would be an uncoated well to which no conjugate is added. This shows whether the A-1 well was properly rinsed, and whether the Tween worked.

Each plan should have at least duplicate positive controls. For these, 50 µl of PBST is the 'competitor', i.e. no IgG competition so maximal signal. Half of the absorbance of these wells is the 50% inhibition point. If the positive controls give A450 greater than 1.2, it will be better to use A450 = 0.5 as the comparison point, rather than half of the positive control value.

A good strategy is to do the standard and unknowns with 3-fold serial dilutions, in duplicate, to get a rough determination of the 50% inhibition point. It should then be possible to refine each case with triplicate 2-fold dilutions.

Under the conditions given, the 50% inhibition is expected at about 0.5 µg IgG/ml.