Protocols
Screen | T-dependent Humoral Response Screen |
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Common Name | |
Posted On | 2010-02-18 12:24 PM |
Author | Carrie N. Arnold |
Science Writer | Eva 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.
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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)
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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.
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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. |
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Alleles Identified | |
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bumble busy cellophane crab craw dew Finlay frazz frizz Gypsy Han honey Hulk kama kiwis Lemon lucky Orange riogrande screamer snowcock stinger swan walla waterfowl Worker |
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References | |