Bio-Rad Mouse Typer Isotyping Kit - Manual (Page 7)

4. Monoclonal Antibody Sample: To insure proper typing of
culture media samples, do not dilute when applying to
microplates. When using ascites fluid, dilute at least 1:1,000 with
PBS-tween before testing. Serial dilutions may be necessary to
establish the working dilution of ascites fluid required for the
best signal-to-noise ratio in the typing immunoassay.
5. GAR-HRP Antibody Conjugate: Bio-Rad’s affinity purified
antibodies should be used at recommended dilutions. These
products give excellent signal-to-noise ratios while using less
reagent. More antibody may be used, but this could result in
higher backgrounds with minimal increase in detection
sensitivity.
6. Background: Nonspecific background reactions are usually
the result of low-purity second antibody and/or using
excessive conjugate antibody concentrations. Always wash
plates thoroughly, especially after the conjugate incubation.
Tween 20 is essential in all wash steps after blocking. At a
0.05% concentration, it will not disrupt antigen-antibody
interactions.
For further assistance in determining sub-isotyping of
monoclonal antibodies, contact Bio-Rad Technical Services (in the
USA 1-800-424-6723) or your local technical representative.
2.3 Procedure
Before starting the assay, read through the entire protocol.
1. Adsorb antigen (Ag) to the microplate by adding 100 µl Ag
solution to all wells (0.1-1.0 µg Ag/well). Cover the plate and
incubate at room temperature for at least 1 hour.
Note: If the Ag coated plates are not used immediately, cover
and store at 4 °C. Antigen solution should contain 0.01%
thimerosal. Plates can be stored for up to 1 month.
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