Plate:
Costar/Corning #3590 high binding capacity 8X12 RIA/EIA plate

Coating:
Goat polyclonal affinity purified a/hIgG antibody, 1 µg/ml in
0,1M Carbonate-Bicarbonate Buffer pH 9,5 containing 0,03%
5-Bromo-5-nitro-1,3-dioxane (BND), 100 µl/well, 18 hours
at +4°C.

Blocking:
1,375%(w/v) biotin-free casein in Phosphate Buffered Saline pH 7,3
containing 0,05% BND (PBS/BND) [prepared as Casein Buffer Concentrate
(CBC) diluted in PBS/BND (CBC/PBS)], 200 µl/well, 1 hour at room
temperature.

Wash:
BS/BND with 0,05% Tween-20, 5 times

"Analyte":
hIgG-biotin, diluted 2-fold in CBC/PBS starting from 10 ng/ml,
8-point calibrator in duplicates including 0 point, 200 µl/well, 2 hours at
room temperature onto ELISA shaker (approx 800 rpm).

Wash:
PBS/BND with 0,05% Tween-20, 4 times

Detection:
SA-GOD Conjugate diluted 1/125 from 1 mg/ml (8 µg/ml) in
CBC/PBS, 100 µl/well, 30 minutes at room temperature onto ELISA
shaker (approx 800 rpm).

Wash:
PBS/BND with 0,05% Tween-20, 6 times

Substrate mixture:
oxygenated 0,1M Na-Acetate/Citrate Buffer pH 5,5 containing
16 mg/ml D+Glucose
100 mg/ml TMB (as free base 1/100 diluted from 10 mg/ml stock solution
in DMSO) and
8 µg/ml HRP with specific activity 250 (purpurogallin) U/mg,
100 µl/well, 20 minutes at room temperature onto ELISA
shaker (approx 800 rpm) in darkness.

Stopping:
8,3% H2SO4, 50 µl/well

Reading:
450 nm vs. 620 nm reference filter