Key result
The human regenerative antibody HIgM12 specifically targets polysialic acid attached to the neural cell adhesion molecule (PSA-NCAM) in central nervous system tissue, as demonstrated by a novel immunoprecipitation and Western blotting protocol.
This study provides a detailed protocol for detecting antigens using human IgM antibodies and identifies polysialic acid as the specific binding epitope for HIgM12.
Animal model protocol for IgM may aid MS/ALS research; leaves open human translation.
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.
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Watzlawik et al. (2016) studied this question. HIgM12 antibody protocol was evaluated. The human regenerative antibody HIgM12 specifically targets polysialic acid attached to the neural cell adhesion molecule (PSA-NCAM) in central nervous system tissue, as demonstrated by a novel immunoprecipitation and Western blotting protocol.
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