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Chloroplast DNA and Phylogenetic Relationships
Published in S. K. Dutta, DNA Systematics, 2019
Early molecular studies on the genus Nicotiana were quite limited in scope, but did at least point to the potential of chloroplast DNA comparisons for illuminating relationships.58–60 More recently, Kung et al.61 used the restriction enzyme Sma I to analyze the parentage of the amphidiploids N. tabacum and N. rustica and to define the Australian species of Nicotiana as a distinct, probably monophyletic lineage within the genus. Scowcroft62 found a single EcoRI restriction site polymorphism that divided nine Australian populations of N. debneyi into two groups. This polymorphism proved crucial in following the assortment genomes following intraspecific somatic fusion.63
Human Monoclonal Antibodies and Immune Modulation in Viral Hepatitis, Schistosomiasis, and HTLV Infection
Published in Thomas F. Kresina, Immune Modulating Agents, 2020
Thomas F. Kresina, Garry A. Neil, Steven K. H. Foung
By using similar somatic fusion methodology, a human monoclonal antibody specific for hepatitis C virus (HCV) has been produced by hypoosmolar electrofusion of in vitro stimulated peripheral blood B cells from a patient with chronic HCV infection and chronic active hepatitis with end stage liver disease [52]. The hybridoma clone, termed JRA1, secreted an IgM lambda monoclonal antibody reactive to synthetic HCV peptides. The antibody was positive on both first- and second-generation hepatitis C virus antibody analysis. Additional laboratories have generated monoclonal antibodies to the hepatitis C virus [53–57], Siemoneit et al. [53] have described the generation of two monoclonal antibodies to the 22-kDa nucleocapsid core protein. Epstein-Barr virus transformed peripheral blood lymphocytes were fused with the heteromyeloma cell line K6H6/B5, resulting in heterohybridomas that secreted an IgG1 /kappa monoclonal antibody and an IgM/kappa monoclonal antibody. Epitope mapping studies resulted in the identification of amino acids 24–39 of the core protein as the recognition sequence for the IgG1 antibody. Additional studies by this group [54] have provided human monoclonal antibodies to envelope protein El of the hepatitis C virus. In these studies, the investigators screened the serum of anti-hepatitis C virus-positive blood donors for antibodies to the El glycoprotein. Thirty-two percent of the donors had positive findings for serum antibody to the El glycoprotein, specifically the EP3 domain comprising amino acid residues 314–330. Four anti-EP3-producing heterohybridomas secreting either IgM kappa or IgG1 kappa and lambda human monoclonal antibodies were generated. The IgG1 kappa antibody was further able to detect an envelope protein of 24–35 kDa on Western blot.
Design and characterization of mouse IgG1 and IgG2a bispecific antibodies for use in syngeneic models
Published in mAbs, 2020
Feng Wang, Jordan C. Tsai, Jonathan H. Davis, Bryant Chau, Jia Dong, Sean M. West, Jason M. Hogan, Matthew L. Wheeler, Christine Bee, Winse Morishige, Thomas Cayton, Donata David-brown, Chengyue Zhang, Alexander Kozhich, Tim Sproul, Gavin Dollinger, Arvind Rajpal, Pavel Strop
One of the primary challenges of generating IgG-like bispecific antibodies is the heterogeneity associated with combining four unique peptide chains, two heavy (HC) and two light chains (LC). Bispecific antibodies can be produced by the somatic fusion of two hybridoma cell lines resulting in quadromas capable of secreting whole IgG molecules.2 The limitation of this approach is the expression of 10 possible combinations of HC and LC, only one of which is correct. Various strategies have been devised to reduce the number of potential HC and LC mis-pairings, thereby increasing the likelihood of forming the correct pair. These technologies include, but are not limited to, species-restricted pairing,3 knobs-into-holes,4 SEEDbodies,5 electrostatic steering,6,7 CrossMabs,8 the Azymetric platform,9 DuetMab,10 and antigen-binding fragment (Fab)-arm exchange,7,11 as has been extensively reviewed.1,12 However, all of these technologies focus on producing human antibodies, which limit their use in preclinical mouse models such as immune-compromised settings, and studies that require longer dosing durations.
Rapid single B cell antibody discovery using nanopens and structured light
Published in mAbs, 2019
Aaron Winters, Karyn McFadden, John Bergen, Julius Landas, Kelly A. Berry, Anthony Gonzalez, Hossein Salimi-Moosavi, Christopher M. Murawsky, Philip Tagari, Chadwick T. King
Tool antibodies for use in LBAs are most frequently derived from in vivo immunization followed by either immortalization as hybridomas9,10 or as immune cell libraries for display technologies such as phage or yeast.11 Despite these methodologies being important and reliable discovery engines, they have notable process limitations. Hybridomas require immortalization of antibody-secreting cells (ASCs) via somatic fusion to a myeloma cell line. Even with optimized electrofusion protocols, only 1 of 5000 input B cells survives fusion, becomes immortalized, and secretes antibody.12 Additionally, hybridoma methods generally require extensive cell culture, which is labor intensive and dependent on mitosis, further slowing development timelines. Display technologies, which start from antigen-experienced IgG messenger RNA (mRNA) pools, suffer primarily from the loss of the cognate VH and VL pairing of the input repertoire.11,13,14 This can result in the identification of low-affinity antibodies, and usually necessitates multiple rounds of time-consuming affinity maturation to generate potent molecules.15 Finally, hybridoma and display technologies both require substantial laboratory space, aseptic and benchtop liquid handling automation, and multiple trained operators to perform antibody discovery campaigns at commercial scales.