Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 6)

Blocking solution, 1% BSA-PBS.
30 ml Add 0.3 g BSA to 30 ml PBS. Adjust pH
to 7.2.
Antibody conjugate Dilute GAR-HRP conjugate 1:3,000 by
solution, 10 ml adding 3.3 µl to 10 ml of PBS-Tween.
Peroxidase substrate Mix 9 ml solution A with 1 ml solution B.
solution, 10 ml
Prepare fresh prior to use and use
immediately.
Color stopping (2% oxalic acid)
solution, 50 ml Add 1 g oxalic acid dihydrate to 50 ml
distilled, deionized water.
Note: If stock solutions of PBS, PBS-tween, and blocking solution
are made, include the bacteriostat thimerosal at a concentration of
0.01%. Avoid the use of sodium azide, as it is an inhibitor of peroxi-
dase activity.
2.2 General Recommendations
1. Assay Incubation Temperature: All incubation steps are
performed at room temperature (23-25 °C), with the
microplate covered to prevent evaporation. For convenience,
any step may be carried out overnight at 4 °C. Incubation
times can be decreased to 0.5 hr if done at 37 °C.
2. Reagent Purity: All reagents should be ACS or EIA Grade.
Chemical impurities, as well as poor water quality, can cause
enzyme inhibition and/or increased backgrounds.
3. Antigen Adsorption: Coat the immunoassay microtitration
plates with 0.1 to 1.0 mg antigen per well. The optimal con-
centration should be determined empirically prior to sub-
typing. Antigen adsorption is a function of concentration,
diluent, type of assay plate, and purity of sample.
4 Whole cell
antigens also can be used for sub-isotyping. 4
4
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