|Screen||OVA/Alum 2nd challenge|
|Posted On||03/17/2017 3:20 PM|
This screen is designed to identify genes regulating IgE production in vivo in response to aluminum hydroxide (alum)-emulsified ovalbumin (OVA) [OVA-alum]. Serum is collected and analyzed for antigen-specific and total IgE on day 7 after OVA-alum challenge. ENU-mutatgenized G3 mice that either produce increased or reduced amount of OVA-specific and total IgE, relative to wild type mice are identified as potential mutants.
|Reagents and Solutions|
Alhydrogel (Alum; InvivoGen, vac-alu-250)
Shake well before use
OVA (InvivoGen, vac-pova-100)
Dissolve the OVA power in PBS (2 mg/ml) to make the OVA-Alum solution (below)
A solution of 20 μg/ml OVA in PBS will be needed for the ELISA
*** Always make OVA solution fresh the day of the injection***
Mix equal parts the OVA solution and Alum. For example, for one mouse mix 100 μl OVA solution (2 mg/ml) and 100 μl of the Alum together. Mix well by pipetting up and down for at least 5 minutes.
Goat anti-IgE antibody HRP conjugated (SouthernBiotech, 1110-05)
1 ml Syringes (BD)
96 wells plate (Thermo)
1.5 ml tubes (Fisher)
ELISA washing buffer (0.1% TWEEN 20 in PBS)
ELISA blocking buffer (0.1% Tween 20+1% BSA in PBS)
TMB SureBlue reagent (KPL, 52-00-01)
TMB Stop Solution (KPL, 50-85-05)
Recombinant OVA-specific IgE (Biolegend, CST-090514JI-UTS)
Mouse IgE ELISA Ready-SET-Go! (eBiocience, 88-50460-77)
Inject 200 μl OVA/Alum solution (containing 200 μg OVA) via intraperitoneal injection into the G3 mouse. Return animals to housing.
Measurement of the IgE by ELISA
For total IgE, follow instruction attached to Mouse IgE ELISA Ready-SET-Go! Kit.
Protocol for OVA-specific IgE by ELISA is described below: