Immunizations and enzyme-linked immunosorbent assay (ELISA) for antibody isotype determination

doi:10.1038/nprot.2007.4100

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

Biological techniques

--- - 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 MaxiSorp^TM 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

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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).

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Immunizations and enzyme-linked immunosorbent assay (ELISA) for antibody isotype determination

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