1) Coat a 96 well plate (Immulon 2; Dynatech Cat # 011-010-3455) with 100 ul/well of a 10 ug/ml (or 0.01 OD280's/ml) solution of antigen in carbonate-bicarbonate buffer, pH 9.6. Incubate overnight at 4 C.
2) Remove antigen and block unreacted sites by filling wells completely with PBS-T and incubating at 37 C for 1 hr.
3) Wash plate three times (ELISA plate washer should be used) with PBS-T and empty wells. Add 100 ul of primary antibody diluted in PBS-T containing 1% BSA. In controls, use PBS-T/1% BSA alone or control antibody at same dilution or ug/ml concentration. Incubate overnight at 4 C.
4) Wash five times with PBS-T and empty wells. Add 100 ul of secondary antibody diluted (usually 1/1000) in PBS-T containing 1% BSA or 1% normal goat serum. Incubate for 1 hr at 37 C.
5) Wash five times with PBS-T and empty wells. Add 100 ul of ABTS/H2O2 (mix 1 ul of H2O2 with 1 ml of ABTS immediately prior to use) and incubate 10 - 20 or up to 60 min at RT on a shaker platform. Read plate at 405 - 414 nm.
Carbonate-Bicarbonate Coating Buffer, pH 9.6 (500 ml):
Na2CO3 0.795 gm NaHCO3 1.46 gm Thimerosol 0.05 gm
PBS-T (1000 ml):
NaCl 8 gm KH2PO4 0.2 gm Na2HPO4 1.15 gm KCl 0.2 gm Tween 20 ml Thimerosol 0.1 gm
ABTS (80 ml)\:
citric acid monohydrate 0.49 gm Na2HPO4 0.47 gm ABTS 44 mg
Store solution in a brown bottle; discard solution when no longer clear.
ABTS is: 2, 2'azino-di-(3-ethylbenzthiozoline sulfonic acid); Sigma#A-1888.