This screen is designed to identify genes required for the in vivo B cell response to aluminum hydroxide (alum)-emulsified ovalbumin (OVA) [OVA-alum]. Alum is an adjuvent usied in human vaccines. Mice are immunized on day 0,  and serum is collected and analyzed for antigen-specific IgG on day 14. ENU-mutatgenized G3 mice that produce a reduced amount of IgG to a strong antigen or a detectable amount of IgG to a weak antigen (see below) relative to wild type mice are identified as potential mutants.

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.***

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 30 minutes to allow Alhydrogel 2% to effectively adsorb the antigen.

96-well round bottom/flat bottom plates

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

1% (w/v) BSA in PBS

ELISA washing buffer

0.1% TWEEN 20 in PBS

TMB SureBlue reagent (KPL: 52-00-01)

TMB Stop Solution (KPL: 50-85-05)

Priming

1. On day 0, inject 200 uL OVA+Alum mixture (containing 100 ug OVA and 1 mg of alum per mouse) via intramuscular injection into the G3 mouse. Return animals to housing.

Measurement of antigen-specific IgG by ELISA

2. Prepare an ELISA plate. Coat a 96-well plate overnight at 4°C with 10 μg/mL OVA in PBS.

3. On day 14 after immunization, collect about 50 μl of blood from the sub-mandibular vein of each mouse. 

4. Perform ELISA according to standard protocol:

a)       Snap plate to remove the coating antigen.

b)       Wash plate 4 times with 200 μL of 0.05% PBST per well.

c)       Add 200 μL of 1% (w/v) BSA in PBS per well.

d)       Wrap or cover plate and incubate at least 30 minutes at room temperature.

e)       Snap plate to remove the blocking buffer.

f)        Add 150 μL of 1% (w/v) BSA in PBS to well of rows A, C, E, G.

g)       Add 100 μL of 1% (w/v) BSA in PBS to well of rows B, D, F, H. 

h)       Add 3ul of sera to wells containing 150 ul of 1% (w/v) BSA in PBS (1:50 dilution) and mix well, then transfer 50 ul of volume from rows A, C, E, G to B, D, F, H, respectively, to make 1:150 dilution.  Mix well and discard the last 50 ul of the volume (prepare serum dilutions in 96 well plates)    

i)        Wrap or cover plate and incubate for 2 hour at room temperature.

j)        Wash plate 8 times with 0.05% PBST.

k)       Add 100 μL of HRP-conjugated goat-anti-mouse IgG diluted 1:4,000 in PBS per well.

l)        Wrap or cover plate and incubate for 1 hour at room temperature.

m)      Wash plate 8 times with 0.05% PBST.

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

o)       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.