Procedure for isolation of gangliosides in high yield and purity: Simultaneous isolation of neutral glycosphingolipids

The calcium-binding protein S100A9 regulates inflammatory processes and the immune response. It is overexpressed in a variety of inflammatory and oncologic conditions. In this study, we produced a recombinant human S100A9 (hS100A9) antigen with high yield and purity and used it to generate a hybridoma cell culture-based monoclonal anti-hS100A9 antibody. We selected five anti-hS100A9 antibodies from cell supernatants that showed high antigen binding efficiency and identified the nucleotide sequences of three antibodies: two with high effective concentration values and one with the lowest value. The antigen and antibody development procedures described herein are useful for producing large amounts of monoclonal antibodies against hS100A9 and other antigens of interest. The nucleotide sequences of the anti-hS100A9 monoclonal antibody revealed herein will be helpful in the generation of recombinant antibodies or antibody fragments against hS100A9.

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Defined MSC exosome with high yield and purity to improve regenerative activity

Journal of Tissue Engineering ◽

10.1177/20417314211008626 ◽

2021 ◽

Vol 12 ◽

pp. 204173142110086

Author(s):

Jun Yong Kim ◽

Won-Kyu Rhim ◽

Yong-In Yoo ◽

Da-Seul Kim ◽

Kyoung-Won Ko ◽

...

Keyword(s):

Cell Culture ◽

Culture Medium ◽

Density Lipoprotein ◽

High Yield ◽

Human Umbilical Cord ◽

Cell Culture Medium ◽

Human Coronary Artery ◽

Isolation Methods ◽

Regenerative Activity ◽

Yield And Purity

Exosomes derived from mesenchymal stem cells (MSCs) have been studied as vital components of regenerative medicine. Typically, various isolation methods of exosomes from cell culture medium have been developed to increase the isolation yield of exosomes. Moreover, the exosome-depletion process of serum has been considered to result in clinically active and highly purified exosomes from the cell culture medium. Our aim was to compare isolation methods, ultracentrifuge (UC)-based conventional method, and tangential flow filtration (TFF) system-based method for separation with high yield, and the bioactivity of the exosome according to the purity of MSC-derived exosome was determined by the ratio of Fetal bovine serum (FBS)-derived exosome to MSC-derived exosome depending on exosome depletion processes of FBS. The TFF-based isolation yield of exosome derived from human umbilical cord MSC (UCMSC) increased two orders (92.5 times) compared to UC-based isolation method. Moreover, by optimizing the process of depleting FBS-derived exosome, the purity of UCMSC-derived exosome, evaluated using the expression level of MSC exosome surface marker (CD73), was about 15.6 times enhanced and the concentration of low-density lipoprotein-cholesterol (LDL-c), known as impurities resulting from FBS, proved to be negligibly detected. The wound healing and angiogenic effects of highly purified UCMSC-derived exosomes were improved about 23.1% and 71.4%, respectively, with human coronary artery endothelial cells (HCAEC). It suggests that the defined MSC exosome with high yield and purity could increase regenerative activity.

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Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research

Journal of Clinical Medicine ◽

10.3390/jcm8111995 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1995 ◽

Cited By ~ 5

Author(s):

Moon ◽

Shin ◽

Kim ◽

Lee ◽

Mankhong ◽

...

Keyword(s):

Human Plasma ◽

Extracellular Vesicles ◽

Proteinase K ◽

High Yield ◽

Clinical Samples ◽

Diagnostic Biomarkers ◽

Micro Rnas ◽

Biomarker Research ◽

Protein Rich ◽

Yield And Purity

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

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Epimerization-free C-term Activation of Peptide Fragments by Ball-Milling

10.26434/chemrxiv.12593825.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yves Yeboue ◽

Marion Jean ◽

Gilles Subra ◽

Jean Martinez ◽

Frédéric Lamaty ◽

...

Keyword(s):

Amino Acids ◽

Ball Milling ◽

High Yield ◽

Coupling Agents ◽

Peptide Fragment ◽

Peptide Fragments ◽

Solution Synthesis ◽

High Yields ◽

Synthesis Approach ◽

Yield And Purity

Ball-milling enabled to perform [2+1], [2+2], and [2+3] peptide couplings with high yields and, if any, very low epimerization. Very good results were obtained with peptide fragments containing highly epimerization-prone and/or highly hindered amino acids at C-term such as phenylglycine, cysteine and valine. Ball-milling was clearly identified as the key element to obtain both high yield and purity along with low epimerization. Indeed, the ball-milling conditions proved to be superior to the classical solution synthesis approach on a various array of widely used coupling agents. These results open avenues for the development of highly efficient, convergent and flexible peptide synthesis strategies based on peptide fragment couplings mediated by ball-milling.

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Epimerization-free C-term Activation of Peptide Fragments by Ball-Milling

10.26434/chemrxiv.12593825 ◽

2020 ◽

Author(s):

Yves Yeboue ◽

Marion Jean ◽

Gilles Subra ◽

Jean Martinez ◽

Frédéric Lamaty ◽

...

Keyword(s):

Amino Acids ◽

Ball Milling ◽

High Yield ◽

Coupling Agents ◽

Peptide Fragment ◽

Peptide Fragments ◽

Solution Synthesis ◽

High Yields ◽

Synthesis Approach ◽

Yield And Purity

Ball-milling enabled to perform [2+1], [2+2], and [2+3] peptide couplings with high yields and, if any, very low epimerization. Very good results were obtained with peptide fragments containing highly epimerization-prone and/or highly hindered amino acids at C-term such as phenylglycine, cysteine and valine. Ball-milling was clearly identified as the key element to obtain both high yield and purity along with low epimerization. Indeed, the ball-milling conditions proved to be superior to the classical solution synthesis approach on a various array of widely used coupling agents. These results open avenues for the development of highly efficient, convergent and flexible peptide synthesis strategies based on peptide fragment couplings mediated by ball-milling.

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