Antibody binding specificity for kappa (Vκ) light chain-containing human (IgM) antibodies: Polysialic acid (PSA) attached to NCAM as a case study

Jens O. Watzlawik, Robert J. Kahoud, Bharath Wootla, Meghan M. Painter, Arthur E. Warrington, William A. Carey, Moses Rodriguez

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Original languageEnglish (US)
Article numbere54139
JournalJournal of Visualized Experiments
Volume2016
Issue number112
DOIs
StatePublished - Jun 29 2016

Fingerprint

Neural Cell Adhesion Molecules
Antibody Specificity
Cell adhesion
Neurology
Antibodies
Immunoglobulin M
Central Nervous System
Light
Molecules
Acids
Antigens
Animal Disease Models
Nerve Tissue
Central Nervous System Diseases
Amyotrophic Lateral Sclerosis
Tissue
Epitopes
Immunoprecipitation
Knockout Mice
Multiple Sclerosis

Keywords

  • Antigen
  • IgM antibody
  • Immunology
  • Immunotherapy
  • Issue 112
  • Multiple sclerosis (MS)
  • Neural cell adhesion molecule (NCAM)
  • Neurological disease
  • Polysialic acid (PSA)
  • Regeneration

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Watzlawik, J. O., Kahoud, R. J., Wootla, B., Painter, M. M., Warrington, A. E., Carey, W. A., & Rodriguez, M. (2016). Antibody binding specificity for kappa (Vκ) light chain-containing human (IgM) antibodies: Polysialic acid (PSA) attached to NCAM as a case study. Journal of Visualized Experiments, 2016(112), [e54139]. https://doi.org/10.3791/54139

Antibody binding specificity for kappa (Vκ) light chain-containing human (IgM) antibodies : Polysialic acid (PSA) attached to NCAM as a case study. / Watzlawik, Jens O.; Kahoud, Robert J.; Wootla, Bharath; Painter, Meghan M.; Warrington, Arthur E.; Carey, William A.; Rodriguez, Moses.

In: Journal of Visualized Experiments, Vol. 2016, No. 112, e54139, 29.06.2016.

Research output: Contribution to journalArticle

Watzlawik, Jens O. ; Kahoud, Robert J. ; Wootla, Bharath ; Painter, Meghan M. ; Warrington, Arthur E. ; Carey, William A. ; Rodriguez, Moses. / Antibody binding specificity for kappa (Vκ) light chain-containing human (IgM) antibodies : Polysialic acid (PSA) attached to NCAM as a case study. In: Journal of Visualized Experiments. 2016 ; Vol. 2016, No. 112.
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