Protocols


Screen (pdf version)
ScreenOVA/Alum 2nd challenge
Posted On03/17/2017 3:20 PM
AuthorTakuma Misawa
Background

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

PBS (Gibco)

 

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

 

OVA-Alum solution

*** 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)

 

Lanset

 

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)

Method

OVA/Alum injection

Inject 200 μl OVA/Alum solution (containing 200 μg OVA) via intraperitoneal injection into the G3 mouse. Return animals to housing.

 

Serum preparation

  1. 7 days post-OVA/Alum immunization, collect 100 μl blood in 1.5 ml tubes.
  2. Centrifuge the blood to separate the serum.
  3. Dilute serum with ELISA blocking buffer (1/10 for OVA specific IgE; 1/100 for Total IgE)

 

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:

  1. Coat a 96-well plate with 100 μl OVA (20 μg/ml). Incubate plate at 4°C overnight.
  2. Wash plate 3 times with 300 μl of ELISA washing buffer per well.
  3. Add 350 μl of ELISA blocking buffer per well.
  4. Seal plate and incubate at least 2 hour.
  5. Wash plate 3 times with 300 μl of ELISA washing buffer per well.
  6. Reconstitute OVA specific IgE standard by addition of ELISA Blocking buffer. Allow the standard to reconstitute for 10-30 minutes.
  7. Add diluted serums to plate (100 μl for each wells). In addition, add reconstitute OVA specific IgE (5, 2.5, 1.25, 0.6, 0.3, 0.15, 0.075, and 0 ng/ml) to create standard curve.
  8. Seal plate and incubate at least 2 hour.
  9. Wash plate 3 times with 300 μl of ELISA washing buffer per well.
  10. Add 100 μl of ELISA blocking buffer containing HRP-conjugated goat-anti-mouse IgE (1/3000) per well.
  11. Seal plate and incubate 30 min at room temperature.
  12. Wash plate 6 times with 300 μl of ELISA washing buffer per well.
  13. Add 100 μl of TMB SureBlue reagent per well.
  14. Incubate for 1 to 5 minutes at room temperature.
  15. Stop the reaction by adding 100 μl of TMB Stop Solution per well.
  16. Read absorbance at 450 nm.