Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

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.

Conserved brain myelination networks are altered in Alzheimer's and other neurodegenerative diseases

“…Pathway enrichment analysis of the most significant DEGs in the AD vs. PSP TCX analysis (3,094 genes, q< 0.05) implicated 66 enriched GO and MetaCore pathways with an FDR<0.05, including established pathways such oxidative phosphorylation, where a systematic downregulation in AD TCX of genes in this pathway is observed (Supplementary_Table.11, Supplementary_Fig.5), replicating prior observations[21, 28]. In the smaller AD vs. nTau analysis (572 genes) “Protein folding and maturation_POMC processing” was a significant MetaCore pathway, also detected in the AD vs. PSP analysis.…”

“…Myelination patterns were assessed in a subset of AD, PSP and control TCX samples (4 each) using established immunohistochemistry methods[26, 27]. We evaluated immunocytochemical patterns for the myelin and oligodendrocyte proteins using rat oligodendrocyte-enriched cultures[28].…”

Focal clusters of peri-synaptic matrix contribute to activity-dependent plasticity and memory in mice

“…IP protocol was adapted from Watzlawik et al 2016 61 . Manufacturer’s guidelines were followed for the use of protein L/Sepharose beads (Protein L Sepharos.…”