This protocol was used in our Nature Immunology paper
Method Article
Immunizations and enzyme-linked immunosorbent assay (ELISA) for antibody isotype determination
https://doi.org/10.1038/nprot.2007.4100
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This protocol was used in our Nature Immunology paper
--- - Mouse - Intestinal inflammation - Crohn's disease - Ulcerative colitis
Keyhole limpet hemocyanin (KLH) and DNP-KLH (Calbiochem-Novabiochem)
Alum (Serva)
Sterile 1 ml syringes and 30 gage needles
96-well MaxiSorp™ ELISA plates (Nunc)
DNP-ovalbumin (Sigma)
PBS + 0.05% Tween 20 (Sigma)
PBS + 2.5% bovine serum albumin (Sigma)
Alkaline phosphatase-labeled mouse anti-IgM and subclass IgG-specific antibodies (Southern Biotech Associates)
Alkaline phosphatase substrate solution (freshly made; Bio-Rad)
Immunizations of mice
1 Perform precipitation of keyhole limpet hemocyanin (KLH) or DNP-KLH in alum as described (2).
2 Inject 100 µg of KLH or DNP-KLH precipitated in alum in a total volume of 300 µl (volume adjusted with PBS) into the peritoneal cavity. Collect serum at d 0, d 7, and d 14 and make serial dilutions in PBS.
ELISA for antibody isotype determination
3 Coat 96-well MaxiSorpTM ELISA plates with 50 µl/well of DNP-ovalbumin (5 µg/ml in PBS), incubate overnight at 4 ˚C (3).
4 Wash with PBS + 0.05% Tween 20, block for 3 h with PBS + 2.5% bovine serum albumin. Wash 3x with PBS + 0.05% Tween 20.
5 Add diluted serum in triplicates, incubate overnight at 4 ˚C. Wash 3x with PBS + 0.05% Tween 20.
6 Add alkaline phospatase-labeled mouse IgM- and subclass IgG-specific antibodies (working dilutions 1:250 – 1:500 in PBS + 2.5% bovine serum albumin). Incubate at 25 ˚C for 1 h. Wash 5x with PBS + 0.05% Tween 20.
7 Add 100 µl per well of alkaline phosphatase substrate solution. Read optical density of each well at 405 nm at 10 min and 20 min after substrate addition.
For ELISA, 2 days
Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).
CFI
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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