Studies on Natural Products Using Monoclonal Antibodies: A Review

An immunoblotting system (“eastern blotting”) was developed for small-molecule herbal medicines like glycosides, with no conjugation function to the membrane. Briefly, the crude extracts of herb medicines were developed by thin-layer chromatography (TLC). The small-molecule herbal medicines on TLC plates were transferred to polyvinylidene fluoride (PVDF) or polyethersulfone (PES) membranes by heating. Antigen components were divided into two categories based on their function, i.e., their membrane recognizing (aglycone part) and fixing (sugar moiety) abilities. This procedure allows for the staining of only target glycosides. Double eastern blotting was developed as a further staining system for two herb medicines using a set of MAbs and substrates.

Galactose and Sialo-galactose Modifications in Glycoproteins on the Surface of Bovine Milk Exosome Are Essential for Exosome Uptake in Non-bovine Species (OR34-07-19)

Objectives Exosomes are natural nanoparticles that can be found in most body fluids including milk. Bovine milk exosomes (BME) are bioavailable and transfer cargos such as RNAs, lipids and proteins across species boundaries. Previous competitor studies implicated galactose-modifications in exosome surface proteins in exosome transport. The objectiveof our study was to assess the importance of glycan modifications in BME surface glycoproteins for BME uptake and distribution in human intestinal cells (FHs cells) and mice. Methods BME were isolated from milk by differential centrifugation (Fig. 1). Putative glycan binding sites on the surfaces of BME were identified by LC/MS-MS and bioinformatics protocols and confirmed by eastern blotting. Surface glycans were altered using proteases, glycosidases and blocking of glycans with lectins for subsequent transport and distribution studies (Table 1). The uptake of BME by FHs cells was assessed using membrane and RNA cargo-labeled BME. The distribution of BME in mice was assessed using BME transfected with fluorophore (IRDye)-labeled synthetic miR-320a in C57BL/6 mice and MGAT-1 tamoxifen inducible conditional knockout (CKO) mice ages 5–8 weeks. One-way ANOVA and Bonferroni's multiple comparison were used for statistical analyses; P < 0.05 was considered significant. Results We identified 49 (N), 13 (O) and 13 (C) putative glycan binding sites on the BME surface, and confirmed the presence of mannose, galactose, N-acetylgalactose, fucose and neuraminate (Fig. 2). Galactose and N-acetylgalactosamine residues on the surface of BME were of particular importance for their uptake by FHs cells, whereas N-acetylglucosamine on the surface of FHs cells was more important than other glycans for BME uptake (Fig. 3). Enzymatic and genetic removal of glycans in BME and MGAT-1 CKO, respectively, caused a loss of BME accumulation in murine livers and pancreas (Fig. 4). Conclusions Distinct glycan features are essential for the uptake of BME in mice. The study laid a foundation to assess the importance of glycan modifications in infant formulas and the homing of drug-loaded exosomes to sites of disease. Funding Sources NIFA, NIH, Bill & Melinda Gates Foundation, PureTech Health, and USDA Hatch & Multistate. J.Z. is a consultant for PureTech. Supporting Tables, Images and/or Graphs