Protocols


Screen (pdf version)
ScreenT-dependent Antibody Response to rSFV-bgal Screen
Posted On02/18/2010 12:24 PM
AuthorCarrie N. Arnold
Science WriterEva Marie Y. Moresco
Background
This screen is designed to identify genes required for the in vivo B cell response to a model antigen encoded by a recombinant Semliki Forest Virus vector. Mice are immunized on day 0, boosted on day 14, and serum is collected and analyzed for antigen-specific IgG on day 28. 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.
 
During a T cell-dependent humoral immune response, a subset of activated CD4+ cells -- follicular homing T cells or T-FH -- migrate to the T-B borders of secondary lymphoid organs, and interact with cognate antigen-specific conventional (B-2) B cells that have been activated by soluble antigen, and stimulate them through CD40-CD40 ligand (1) and other interactions to expand within T cell or B cell zones. B cells in the T cell zone develop into short-lived plasma cells that produce germline-encoded antigen-specific antibody. In contrast, B cells expanding in B cell zones generate regions called secondary follicles, precursors of the germinal center (GC) reaction. As the GC reaction progresses, it becomes polarized into separate “dark” and “light” zones. B cells proliferate and diversify via somatic hypermutation within the dark zone, and then migrate to the light zone, where B cells with high-affinity variant Ig receptors are selected to either re-enter the GC cycle or exit the GC reaction. Cells exit the GC reaction as either long-lived plasma cells or recirculating memory B cells. Long-lived plasma cells produce large amounts class-switched, high-affinity antibody. Memory B cells do not actively secrete antibody. Instead, these cells are programmed for expansion and differentiation into high-affinity plasma cells upon secondary encounter with antigen, the hallmark of the memory B cell response.
 
Single-round infectious recombinant viral vectors, including recombinant Semliki Forest Virus (rSFV) vaccine vectors (2-5), induce both cellular and humoral immune responses. SFV is an alphavirus genetically related to Sindbis virus and Venezuelan equine encephalitis virus, for which single-round replicon systems have also been developed (6-8). The vector used here contains the SFV translation-enhancer element upstream and in frame with an internal signal sequence and the target antigen-encoding sequence (9). This vector was shown to result in secretion of ten-fold more antigen compared to the same vector lacking the enhancer element and using the N-terminal CD5 signal sequence, as well as improved antibody titres in the sera of immunized mice (9). In the protocol outlined here, the rSFV vector encodes either the model antigen β-galactosidase (GAL) or ovalbumin (OVA) and is administered via intraperitoneal injection. Detection of GAL- or OVA-specific IgG two weeks after administration of a second dose of the vector is used as a readout for the class-swiched B cell response.  Most wild type mice mount a robust GAL-specific IgG response, and mutagenized mice that fail to do so may possess mutations in genes required for B or CD4+ T cell development or activation, isotype class switching, or terminal differentiation.  By contrast, wild type mice fail to produce detectable OVA-specific IgG responses; of the strains tested, only interferon (IFN)α receptor-deficient mice mount an OVA-specific IgG response.  Mutagenized mice that are able to respond may have mutations in the type I IFN signaling pathway or other genes that normally downregulate weak B cell responses.
 
Reagents and Solutions
β-galactosidase (Roche Applied Science, Indianapolis, IN)
 
ovalbumin (Sigma-Aldrich, St. Louis, MO)
 
HRP-conjugated goat-anti-mouse IgG (Southern Biotech, Birmingham, AL)
 
TMB SureBlue reagent (KPL, Inc., Gaithersburg, MD)
 
TMB Stop Solution (KPL, Inc., Gaithersburg, MD)
Method
Priming
1. On day 0, dilute in a total volume of 200 μl 0.9% saline and inject intraperitoneally into each G3 mouse:
a)       2x106 infectious units (IU) of rSFV vector encoding β-galactosidase (GAL), or
b)       107 IU of rSFV vector encoding ovalbumin (OVA).
 
Boosting (Same procedure as priming)
2. On day 14 after priming, dilute in a total volume of 200 μl 0.9% saline and inject intraperitoneally into each G3 mouse:
a)       2x106 infectious units (IU) of rSFV vector encoding β-galactosidase (GAL), or
b)       107 IU of rSFV vector encoding ovalbumin (OVA).
 
Measurement of antigen-specific IgG by ELISA
3. Prepare an ELISA plate. Coat a 96-well round bottom plate overnight at 4°C or for 2 hours at room temperature with:
a)       2 μg/mL β-galactosidase in PBS, or
b)       10 μg/mL ovalbumin in PBS.
 
4. On day 28 after priming, collect about 50 μl of blood from the orbital sinus of each mouse. For the detection of GAL-specific IgG, dilute 2 μL of sera in 1 mL cold 1% (w/v) milk in PBS (prepare serum dilutions in eppendorf tubes). For the detection of OVA-specific IgG, dilute 5 μL of serum in 250 μL of cold 1% (w/v) milk in PBS (prepare serum dilutions in separate eppendorf tubes).
 
5. Perform ELISA according to standard protocol:
a)       Snap plate to remove the coating antigen.
b)       Wash plate 3 times with 200 μL of PBS per well.
c)       Add 200 μL of 5% (w/v) milk in PBS 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 PBS per well.
f)        Add 100 μL of 1% (w/v) milk in PBS per well.
g)       Add 100 μL of diluted serum to appropriate well containing 100 uL of 1% (w/v) milk.  
h)       Make at least 4 serial dilutions at 1:2 from this well, discarding the last 100 μL.
i)        Wrap or cover plate and incubate for exactly 1 hour at 37°C.
j)        Wash plate 6 times with PBS + 0.05% Tween-20.
k)       Add 100 μL of 1% milk containing HRP-conjugated goat-anti-mouse IgG diluted 1:5,000 per well.
l)        Wrap or cover plate and incubate for exactly 1 hour at 37°C.
m)      Wash plate 6 times with PBS + 0.05% Tween-20.
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.
Critical Parameters and Troubleshooting

Sera from immunized mice are pooled and aliquoted to make a “positive control” stock of sera.  The positive control stock is diluted in the same way as experimental samples, and run on a column of uncoated wells and on a column of coated wells on every plate.  Running the positive control sera on mock-coated wells (i.e., wells coated with PBS alone) establishes the background for the plate.  Running the positive control sera on coated wells establishes the plate-to-plate variation for the assay.

Alleles Identified
agate
Amber
Amethyst
Atlas
Belaya
Beni
Brazos
bumble
busy
Cauca
cellophane
Chenab
Citrine
Coral
crab
Craw
Cumberland
Desna
dew
Dnieper
Draa
finlay
Fraser
frazz
frizz
Godavari
Gypsy
Han
honey
hulk
Ishim
Jackfruit
kama
lemon
Liard
Limpopo
lucky
Murray
Nectarine
Oka
Olenyok
Orange
Paraguay
Pechora
Platte
Purus
riogrande
Salween
screamer
Seine
Senegal
Sepik
snowcock
Songhua
stinger
swan
Tanana
Tarim
Tobol
Ural
Vaal
Virgin
walla
Waterfowl
Worker
Yalong
Yellow
Yuri
Zeya
References
Edit History
Stephen Lyon 10/07/2013 11:13 AM (current)
Eva Marie Y. Moresco 01/19/2011 4:41 PM