Don Rio
SOLID PHASE RADIOIMMUNE ASSAY FOR
IgG
1) Apply antigen; (usually 0.1-1 mg) into polyvinyl chloride well of microtiter plate
(96-well) in 50 ml final volume in PBS, pH 7.4 (Costar vinyl assay plates, cat.
no. 2596).
2) Allow solution to evaporate (RT overnight, or 37°C incubator for 1-2 h).
3) Apply 150 ml 5% BSA, PBS, pH 7.4. (PBS = 0.01M NaPO4 pH 7.4, 0.1 M
NaCl).
4) Replace tray lid; place at 37°C for two hours.
5) Remove BSA-PBS blocking solution.
6) Apply antibody in 50 µl final volume, diluting in PBS, pH 7.4 if necessary.
Unconcentrated hybridoma; supernatants are used without dilution.
7) Replace tray lid; place at 37 °C for one hour.
8) Remove antibody; solution.
9) Wash three times with 1% BSA, PBS, pH 7.4; use about 100-150 µl per wash and
discard wash solutions between applications.
10) Apply 105 cpm [125I] - protein A or appropriate [125I] labeled second antibody in
50 µl final volume, using PBS, pH 7.4 as diluent.
11) Replace tray lid; place at 37 °C for one hour.
12) Remove [125I] solution.
13) Wash three times with 100-150 µl PBS, pH 7.4.
14) Aspirate any residual liquid in microtiter wells and wipe the top of the plate dry.
15) Place plate without lid between two glass plates with X-ray film and screens in
place and use binder clips to clamp.
16) Autoradiograph with XAR-5 or equivalent film being certain the well bottom is
against the film; use one intensifying screen; place at -70 °C for 16 hrs or
overnight.
PBS: 10 mM NaPO4, pH 7.4
0.1 M NaCl
The pH is very important as the reactions are not efficient below pH 7.0 and are less
efficient at pH 7.0. Therefore, adjust the pH of the PBS and the PBS containing BSA. Store
the BSA solutions at -20 °C.
NOTE: It is necessary to adjust pH of BSA/PBS solutions as pH drops when BSA is
dissolved in PBS.