Fig. 1

Establishment and characterization of recombinant positive and negative anti-polySia-hIgG. (A) Schematic of the biolayer interferometry (BLI) method used to analyze polySia–antibody interactions. Antibodies were immobilized onto a chip via their Fc region, and colominic acid (purified polySia derived from Escherichia coli K1 cell walls) was used as the analyte. (B) Chromatogram of the interaction. The positive antibody (pAb; black) and negative antibody (nAb; orange) were immobilized onto the chip, and polysialic acid (25 nM) was applied from 0–300 s. After 300 s, dissociation was analyzed in phosphate-buffered saline (PBS). (C) ELISA of mouse embryonic brain (MEB) lysate. Immobilized polySia in MEB was detected using pAb (black) or nAb (orange). As a control, immobilized polySia was digested with Endo-N and detected using pAb (light blue). (D) Western blot analysis of adult mouse brain lysate with and without Endo-N treatment. Left panel: pAb detects polySia. Right panel: nAb shows no detection. HSC70 was used as a loading control. (E) Immunohistochemistry of the hippocampus (HIP) in a mouse brain section (polySia: green; IBA1: red; DAPI: blue; scale bar: 200 μm). Left panel: pAb detects polySia-positive cells, particularly in the dentate gyrus, CA3, and CA1 regions of the HIP. Right panel: nAb fails to detect polySia. Abbreviations: polySia, polysialic acid; pAb, positive antibody; nAb, negative antibody; Endo-N, endo-N-acylneuraminidase;; DAPI, 4′,6-diamidino-2-phenylindole; IBA1, ionized calcium-binding adaptor molecule 1; HIP, hippocampus; BLI, biolayer interferometry; PBS, phosphate-buffered saline; MEB, mouse embryonic brain.