Protocols


Screen (pdf version)
ScreenT-independent B Cell Response Screen
Posted On02/18/2010 12:24 PM
AuthorJin Huk Choi, Samantha Kuwada Teixeira
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 200 μl (40 μg in sterile PBS) of NP(49)-AECM-Ficoll intraperitoneally into each G3 mouse.

 

Measurement of antigen-specific IgM by ELISA

2. Prepare an ELISA plate. Coat a 96-well plate overnight at 4°C 5 μg/mL NP(8)-BSA in PBS.

 

3. On day 6 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 IgM 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.

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
Apple
Bartle
bumble
busy
cellophane
crab
Crater
frazz
Gypsy
han
honey
kiwis
queen
screamer
serpens
snowcock
stinger
Tigris
Untied
walla
Worker
References
Edit History
Anne Murray 11/30/2016 2:16 PM (current)
Eva Marie Y. Moresco 01/19/2011 4:41 PM