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

doi:10.1038/nprot.2007.4100

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Biological techniques

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

Stefan Wirtz, Clemens Neufert, Benno Weigmann, Markus F Neurath

Stefan Wirtz

Clemens Neufert

Benno Weigmann

Markus F Neurath

DOI:

10.1038/nprot.2007.4100

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

This protocol was used in our Nature Immunology paper

Keywords

--- - 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

Anzelon, A. N., Wu, H. & Rickert, R. C. Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function. Nat Immunol 4, 287-294 (2003).
Sanchez, Y. et al. Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund complete adjuvant alum, and liposomes. Infect Immun 30, 728-733 (1980).
Engel, P. et al. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39-50 (1995).
Gallagher, E. D., Xu, S., Moomaw, C., Slaughter, C. A. & Cobb, M. H. Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation. J Biol Chem 277, 45785-45792 (2002).
Gallagher, E. D., Gutowski, S., Sternweis, P. C. & Cobb, M. H. RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity. J Biol Chem 279, 1872-1877 (2004).
Rush, J. S., Hasbold, J. & Hodgkin, P. D. Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation. J Immunol 168, 2676-2682 (2002).

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).

Version History

CURRENT STATUS: Posted

Version 1

Posted 14 Mar, 2007

Metrics

Comments: 0

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Subject Areas

Biological techniques

Associated Publications

Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production
by Gallagher, E. et al.
Nature Immunology (22 January, 2007)

Method Article

Biological techniques

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

Stefan Wirtz, Clemens Neufert, Benno Weigmann, Markus F Neurath

Stefan Wirtz

Clemens Neufert

Benno Weigmann

Markus F Neurath

Version History

CURRENT STATUS: Posted

Version 1

Posted 14 Mar, 2007

MetricsComments: 0HTML Views: ...

Subject Areas

Biological techniques

DOI:

10.1038/nprot.2007.4100

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

This protocol was used in our Nature Immunology paper

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).

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.

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

Anzelon, A. N., Wu, H. & Rickert, R. C. Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function. Nat Immunol 4, 287-294 (2003).
Sanchez, Y. et al. Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund complete adjuvant alum, and liposomes. Infect Immun 30, 728-733 (1980).
Engel, P. et al. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3, 39-50 (1995).
Gallagher, E. D., Xu, S., Moomaw, C., Slaughter, C. A. & Cobb, M. H. Binding of JNK/SAPK to MEKK1 is regulated by phosphorylation. J Biol Chem 277, 45785-45792 (2002).
Gallagher, E. D., Gutowski, S., Sternweis, P. C. & Cobb, M. H. RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity. J Biol Chem 279, 1872-1877 (2004).
Rush, J. S., Hasbold, J. & Hodgkin, P. D. Cross-linking surface Ig delays CD40 ligand- and IL-4-induced B cell Ig class switching and reveals evidence for independent regulation of B cell proliferation and differentiation. J Immunol 168, 2676-2682 (2002).

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).

Associated Publications

Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production
by Gallagher, E. et al.
Nature Immunology (22 January, 2007)

This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.

Method Article

Biological techniques

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

Stefan Wirtz, Clemens Neufert, Benno Weigmann, Markus F Neurath

Stefan Wirtz

Clemens Neufert

Benno Weigmann

Markus F Neurath

DOI:

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Abstract

Keywords

--- - Mouse - Intestinal inflammation - Crohn's disease - Ulcerative colitis

Reagents

Procedure

Timing

References

Acknowledgements

Comments (0)

Version History

CURRENT STATUS: Posted

Version 1

Metrics

Comments: 0

HTML Views: ...

Subject Areas

Biological techniques

Associated Publications

Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production

by Gallagher, E. et al.

Nature Immunology (22 January, 2007)

Method Article

Biological techniques

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

Stefan Wirtz, Clemens Neufert, Benno Weigmann, Markus F Neurath

Stefan Wirtz

Clemens Neufert

Benno Weigmann

Markus F Neurath

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CURRENT STATUS: Posted

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License

License:

This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

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Abstract

Reagents

Procedure

Timing

References

Acknowledgements

Comments (0)

Related Articles

Associated Publications

Kinase MEKK1 is required for CD40-dependent activation of the kinases Jnk and p38, germinal center formation, B cell proliferation and antibody production

by Gallagher, E. et al.

Nature Immunology (22 January, 2007)

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