scholarly journals Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9

2021 ◽  
Vol 11 (10) ◽  
pp. 4659
Author(s):  
Eun-Jung Kim ◽  
Gyu-Min Im ◽  
Chang-Soo Lee ◽  
Yun-Gon Kim ◽  
Byoung Joon Ko ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Calcium Binding ◽  
Nucleotide Sequences ◽  
Antibody Fragments ◽  
High Yield ◽  
Antigen Binding ◽  
Hybridoma Cell Culture ◽  
Inflammatory Protein ◽  
Inflammatory Processes ◽  
Yield And Purity

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.

scholarly journals Generation and diversification of recombinant monoclonal antibodies

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Keith F DeLuca ◽  
Jeanne E Mick ◽  
Amy Hodges Ide ◽  
Wanessa C Lima ◽  
Lori Sherman ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Low Cost ◽  
Antibody Fragments ◽  
Species Specificity ◽  
High Yield ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ethical Concerns ◽  
Commercial Sources ◽  
Kinetochore Function

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

scholarly journals Generation and diversification of recombinant monoclonal antibodies for studying mitosis

2021 ◽  
Author(s):  
Keith F. DeLuca ◽  
Jeanne E. Mick ◽  
Amy L. Hodges ◽  
Wanessa C. Lima ◽  
Lori Sherman ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Division ◽  
Low Cost ◽  
Mitotic Cell ◽  
Antibody Fragments ◽  
High Yield ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Ethical Concerns ◽  
Commercial Sources

AbstractAntibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitotic cell division; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

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

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.

scholarly journals Production of Recombinant Monoclonal Antibodies in the Egg White of Gene-Targeted Transgenic Chickens

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 38
Author(s):  
Takehiro Mukae ◽  
Sho Okumura ◽  
Takuma Watanobe ◽  
Kyoko Yoshii ◽  
Takahiro Tagami ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Capacity ◽  
Specific Binding ◽  
Egg White ◽  
Peptide Sequence ◽  
Efficient Production ◽  
Antigen Binding ◽  
Transgenic Chicken ◽  
Bioreactor System ◽  
Transgenic Chickens

Increased commercial demand for monoclonal antibodies (mAbs) has resulted in the urgent need to establish efficient production systems. We previously developed a transgenic chicken bioreactor system that effectively produced human cytokines in egg whites using genome-edited transgenic chickens. Here, we describe the application of this system to mAb production. The genes encoding the heavy and light chains of humanized anti-HER2 mAb, linked by a 2A peptide sequence, were integrated into the chicken ovalbumin gene locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized mAb in their eggs. The mAb expression level in the egg white was 1.4–1.9 mg/mL, as determined by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb obtained was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2-positive and -negative cells. These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and can serve as an alternative production system for commercial mAbs.

scholarly journals Novel idiotypic and antigen-binding characteristics in two anti-dinitrophenyl monoclonal antibodies.

1977 ◽  
Vol 146 (1) ◽  
pp. 282-286 ◽  
Author(s):  
N H Sigal
Keyword(s):  
Monoclonal Antibodies ◽  
Germ Line ◽  
Hypervariable Region ◽  
Weight Basis ◽  
Antigen Binding ◽  
The Novel ◽  
Maximum Frequency ◽  
Somatic Variant ◽  
Binding Characteristics ◽  
Cell Pool

Monoclonal anti-dinitrophenyl antibodies generated in the splenic focus system from B cells of adult BALB/c mice were studied for the presence or absence of murine anti-T15 (M anti-T15) reactivity and for their ability to bind phosphorylcholine (PC). Two foci of the 680 clones analyzed bound PC, and one of these antibodies reacted with M anti-T15 and anti-Fab on a 1:1 weight basis. The discovery of a clonotype reactive with M anti-15 but not with rabbit anti-T15 (R anti-T15) serum, the converse of the R anti-T15+, M anti-T15- clonotype identified in the PC-specific repertoire, points to the novel idiotypic relationships which may be found among homogeneous antibodies binding diverse antigens. The R anti-T15-, M anti-T15+ clonotype may represent a distinct set of hypervariable region sequences inserted into the T15 framework or may be a somatic variant of the T15 germ-line sequence. In addition, the maximum frequency with which this clonotype occurs within the B-cell pool is estimated.

Secreted modular calcium-binding protein 1 binds and activates thrombin to account for platelet hyperreactivity in diabetes

Blood ◽  
2021 ◽  
Vol 137 (12) ◽  
pp. 1641-1651
Author(s):  
Fredy Delgado Lagos ◽  
Amro Elgheznawy ◽  
Anastasia Kyselova ◽  
Dagmar Meyer zu Heringdorf ◽  
Corina Ratiu ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Monoclonal Antibodies ◽  
Platelet Function ◽  
Calcium Binding ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Matricellular Protein

Abstract Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/− mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/− mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and β3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-β signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223–deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.

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

1985 ◽  
Vol 148 (1) ◽  
pp. 163-173 ◽  
Author(s):  
Mary C. Byrne ◽  
Michele Sbaschnig-Agler ◽  
Dennis A. Aquino ◽  
Joseph R. Sclafani ◽  
Robert W. Ledeen
Keyword(s):  
High Yield ◽  
Neutral Glycosphingolipids ◽  
Yield And Purity
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