Protocols


Screen (pdf version)
ScreenTotal IgE post immunization and OVA-specific IgE post immunization screen
Posted On03/17/2017 3:21 PM
AuthorTao Yue
Background

This screen is designed to identify genes regulating IgE production in vivo in response to aluminum hydroxide (alum)-emulsified ovalbumin (OVA) [OVA-alum]. Alum is an adjuvent usied in human vaccines. Mice are immunized on day 0, without any boosting step on day 14, and serum is collected and analyzed for antigen-specific IgE on day 14. ENU-mutatgenized G3 mice that produce a increased amount of OVA-specific IgE(normalized by the level of the total IgE), relative to wild type mice are identified as potential mutants.

Reagents and Solutions

0.9% saline

 

1 cc syringe

 

Alhydrogel (2%) (vaccine adjuvant: aluminum hydroxide gel; InvivoGen: vac-alu-250)

Shake well before use

 

OVA (Sigma-Aldrich: A5503)

Dilute the OVA in 0.9% saline to 1000ug/ml to make the OVA-Alum solution (below)

A solution of 10 ug/mL OVA in PBS will be needed for the ELISA

 

OVA-Alum solution

*** Always make OVA solution fresh the day of the injection.***

1. Mix equal parts the OVA solution and Alydrogel 2%. For example, for one mouse mix 100 μL OVA solution and 100 μL of the Alhydrogel 2% together. Mix well by pipetting up and down for at least 5 minutes to allow Alhydrogel 2% to effectively adsorb the antigen.

2. Centrifuge a sample of the mixture at 14, 000 × g for 10 min; check the supernatant for unbound protein by bicinchoninic acid assay (Pierce, Rockford, IL).

 

96-well round bottom plate

 

Serum separator tubes (Becton Dickinson (BD): 365956)

 

ELISA washing buffer

0.1% TWEEN 20 in PBS

 

ELISA Blocker Blocking buffer (Thermo, Cat#: N502)

 

TMB SureBlue reagent (KPL: 52-00-01)

 

TMB Stop Solution (KPL: 50-85-05)

Method

Priming

1. Inject 200 uL OVA+Alum (containing 100 ug OVA) via intraperitoneal (i.p.) injection into the G3 mouse. Return animals to housing.

 

Measurement of the total IgE and OVA-specific IgE by ELISA

3. Thirteen days post-OVA+Alum immunization, prepare an ELISA plate. Coat a 96-well round bottom plate overnight at 4°C or for 2 hours at room temperature with IgE capture antibody or 100 μL ovalbumin (10 μg/mL).

 

4. Fourteen days post-OVA+Alum immunization collect 100 μL blood in serum separator tubes.

 

5. Centrifuge the blood from step 4 to separate the serum and procede to step 6.

 

6. For the detection of the toal IgE and OVA-specific IgE: dilute 4 μL of the serum in 100 μL of cold ELISA blocking buffer (prepare serum dilutions in separate eppendorf tubes). Basal serum Ig levels are measured using unvaccinated animal serum.

 

7. Perform ELISA according to the standard protocol:

a) Snap plate to remove the coating antigen.

b) Wash plate 3 times with 200 μL of ELISA washing buffer per well

c) Add 200 μL of blocking buffer per well.

d) Wrap or cover plate and incubate at least 1 hour and up to 3 hours at 37°C.

e) Wash plate 3 times with 200 μL of the ELISA washing buffer per well.

f) Add 150 μL of cold blocking buffer per well in the first row and 100 μL of blocking buffer in PBS per well in the next four rows.

g) Perform 1:100 dilution of the serum for the total IgE measurement, and 1:25 dilution for OVA-specific IgE measurement in blocking buffer and mix well.

h) Wrap or cover plate and incubate for exactly 1 hour at 37°C.

i) Wash plate 6 times with PBS + 0.05% Tween-20.

j) Add 100 μL of 1blocking buffer containing HRP-conjugated goat-anti-mouse IgE (Southern Biotechnology) diluted 1:5,000 per well.

k) Wrap or cover plate and incubate for exactly 1 hour at 37°C.

l) Wash plate 6 times with PBS + 0.1% Tween-20.

m) After last wash has been removed, develop plate by adding 100 μL of room temperature TMB SureBlue reagent per well.

n) Incubate for 1 to 5 minutes at room temperature.

p) Stop the reaction by adding 100 μL of TMB Stop Solution per well.

q) Read absorbance at 450 nm.