Protocols


Screen (pdf version)
ScreenT-independent B Cell Response 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 the T-independent model antigen 2,4,6-trinitrophenyl (TNP)-Ficoll. Mice are immunized on day 0 and serum is collected and analyzed for TNP-specific IgM between days 5 and 7. ENU-mutagenized G3 mice that produce a reduced amount of TNP-specific IgM are identified as potential mutants.
 
B cell responses are classified as T-dependent (T-D) or T-independent (T-I) based on their requirement for T cell help in antibody production. Antigens that elicit a T-D antibody response are proteins that are processed and presented to cognate helper T cells in the context of MHC class II molecules. T-D antigens induce a long-lasting immune response, including the formation of memory B and T cells, and the production of high-affinity antibodies of multiple isotypes. The T-D B cell response is mediated by conventional (follicular B-2) B cells, the major B cell subset in the body.  The T-dependent Humoral Response Screen is employed to discover genes required for the in vivo T-dependent B cell response.
 
In contrast, T-I antigens do not require T cell help to elicit an immune response (1;2). These antigens are typically polysaccharides that cannot be presented via MHC molecules, and are of two types distinguished by their requirement for B cell receptor (BCR) recognition. T-I type 1 antigens are mitogenic stimuli such as LPS, CpG, or poly(I:C) that activate Toll-like receptors (TLR) to induce polyclonal B cell activation. Isotype switching and memory do not occur to T-I type 1 antigens. T-I type 2 antigens consist of highly repetitive structures, such as capsular polysaccharides from bacterial cell walls or repetitive antigenic epitopes from viral particles, that activate B cells by crosslinking surface-exposed BCR. These type 2 antigens elicit robust antigen-specific primary and memory responses. Splenocytes from mice immunized with T-I type 2 antigens respond to secondary challenge when adoptively transferred into naïve recipients (3;4), and polysaccharide vaccines confer long-term humoral protection in adult humans (5). The T-I immune response is mediated by the B-1 and marginal zone (MZ) B cell populations, which expand upon immunization in extrafollicular sites (6) and confer protective immunity by producing antigen-specific IgM and/or IgG3 without somatic hypermutation (7-11).  T-I memory B cells differ from conventional T-D memory B cells in their cell surface phenotype, and in their regulation by antigen-specific IgG antibodies (12)
 
Ficoll is a neutral, highly branched, high mass, hydrophilic polysaccharide. Conjugation with the hapten 2,4,6-trinitrophenyl generates TNP-Ficoll, a widely used T-I model antigen that induces TNP-specific IgM production when used to immunize mice. In this screen, mutagenized mice that fail to mount a TNP-specific IgM response may harbor mutations in genes required for B-1 or MZ B cell development, activation, costimulation, or homeostasis.

 

Reagents and Solutions
TNP(65)-AECM-Ficoll (BioSearch Technologies, Novato, CA)
 
TNP(30)-BSA (Bovine Serum Albumin) (BioSearch Technologies, Novato, CA)
 
HRP-conjugated goat-anti-mouse IgM (Southern Biotech, Birmingham, AL)
 
TMB SureBlue reagent (KPL, Inc., Gaithersburg, MD)
 
TMB Stop Solution (KPL, Inc., Gaithersburg, MD)
Method
Immunization
1. On day 0, inject intraperitoneally into each G3 mouse 25 μg TNP(65)-AECM-Ficoll in a total volume of 200 μl 0.9% saline.
 
Measurement of antigen-specific IgM by ELISA
2. Prepare an ELISA plate. Coat a 96-well round bottom plate overnight at 4°C or for 2 hours at room temperature with 5 μg/mL TNP(30)-BSA in PBS.
 
3. On day 5-7 after immunization, collect about 50 μl of blood from the orbital sinus of each mouse. Dilute 2 μL of sera in 1 mL cold 1% (w/v) milk in PBS (prepare serum dilutions in eppendorf tubes).
 
4. 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 IgM 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
acamar
Achird
Apple
Aquila
Bartle
bumble
busy
Caelum
Cassiopeia
cellophane
Centaur
Cetus
Coral
Corvus
crab
Crater
Delphinus
frazz
Gypsy
Han
honey
Jackfruit
Lepus
Nectarine
Octans
Orion
Pisces
queen
Salween
screamer
serpens
snowcock
stinger
Tanana
Tigris
Triangulum
Untied
Ural
Vulpecula
walla
Worker
References