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Molecular Pathology : MP Jun 2000Monoclonal antibodies are essential tools for many molecular immunology investigations. In particular, when used in combination with techniques such as epitope mapping... (Review)
Review
Monoclonal antibodies are essential tools for many molecular immunology investigations. In particular, when used in combination with techniques such as epitope mapping and molecular modelling, monoclonal antibodies enable the antigenic profiling and visualisation of macromolecular surfaces. In addition, monoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents. Furthermore, the continuous culture of hybridoma cells that produce these antibodies offers the potential of an unlimited supply of reagent. In essence, when compared with the rather limited supply of polyclonal antibody reagents, the feature of a continuous supply enables the standardisation of both the reagent and the assay technique. Clearly, polyclonal and monoclonal antibodies have their advantages and disadvantages in terms of generation, cost, and overall applications. Ultimately, monoclonal antibodies are only produced when necessary because their production is time consuming and frustrating, although greatly rewarding (at least most of the time!). This is especially apparent when a monoclonal antibody can be applied successfully in a routine pathology laboratory or can aid in the clinical diagnosis and treatment of patients. In this article, the generation and application of monoclonal antibodies are demystified to enable greater understanding and hopefully formulate novel ideas for clinicians and scientists alike.
Topics: Antibodies, Monoclonal; Humans; Hybridomas; Neoplasms
PubMed: 10897328
DOI: 10.1136/mp.53.3.111 -
Nature Protocols May 2013We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the...
We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.
Topics: Animals; Antibodies; Flow Cytometry; Fluorescent Dyes; Hybridomas; Mice; Microfluidic Analytical Techniques; Microfluidics; Single-Cell Analysis
PubMed: 23558786
DOI: 10.1038/nprot.2013.046 -
Nature Sep 2023Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response. However, the specificity of microbiome-induced T cells has not been...
Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.
Topics: Animals; Mice; Antigens, Surface; Bacteria; Firmicutes; Gastrointestinal Microbiome; T-Lymphocytes, Regulatory; Th17 Cells; T-Lymphocytes; Symbiosis; Germ-Free Life; Receptors, Antigen, T-Cell; Hybridomas; Cell Separation
PubMed: 37587342
DOI: 10.1038/s41586-023-06431-8 -
International Immunopharmacology Aug 2020The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved... (Review)
Review
The advancements in technology and manufacturing processes have allowed the development of new derivatives, biosimilar or advanced improved versions for approved antibodies each year for treatment regimen. There are more than 700 antibody-based molecules that are in different stages of phase I/II/ III clinical trials targeting new unique targets. To date, approximately more than 80 monoclonal antibodies (mAbs) have been approved. A total of 7 novel antibody therapeutics had been granted the first approval either in the United States or European Union in the year 2019, representing approximately 20% of the total number of approved drugs. Most of these licenced mAbs or their derivatives are either of hybridoma origin or their improvised engineered versions. Even with the recent development of high throughput mAb generation technologies, hybridoma is the most favoured method due to its indigenous nature to preserve natural cognate antibody pairing information and preserves innate functions of immune cells. The recent advent of antibody engineering technology has superseded the species level barriers and has shown success in isolation of hybridoma across phylogenetically distinct species. This has led to the isolation of monoclonal antibodies against human targets that are conserved and non-immunogenic in the rodent. In this review, we have discussed in detail about hybridoma technology, its expansion towards different animal species, the importance of antibodies isolated from different animal sources that are useful in biological applications, advantages, and limitations. This review also summarizes the challenges and recent progress associated with hybridoma development, and how it has been overcome in these years to provide new insights for the isolation of mAbs.
Topics: Animals; Antibodies, Monoclonal; Humans; Hybridomas
PubMed: 32473573
DOI: 10.1016/j.intimp.2020.106639 -
ImmunoHorizons Dec 2023mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B...
mAbs are highly indispensable tools for diagnostic, prophylactic, and therapeutic applications. The first technique, hybridoma technology, was based on fusion of B lymphocytes with myeloma cells, which resulted in generation of single mAbs against a specific Ag. Along with hybridoma technology, several novel and alternative methods have been developed to improve mAb generation, ranging from electrofusion to the discovery of completely novel technologies such as B cell immortalization; phage, yeast, bacterial, ribosome, and mammalian display systems; DNA/RNA encoded Abs; single B cell technology; transgenic animals; and artificial intelligence/machine learning. This commentary outlines the evolution, methodology, advantages, and limitations of various mAb production techniques. Furthermore, with the advent of next-generation Ab technologies such as single-chain variable fragments, nanobodies, bispecific Abs, Fc-engineered Abs, Ab biosimilars, Ab mimetics, and Ab-drug conjugates, the healthcare and pharmaceutical sectors have become resourceful to develop highly specific mAb treatments against various diseases such as cancer and autoimmune and infectious diseases.
Topics: Animals; Antibodies, Monoclonal; Artificial Intelligence; Biosimilar Pharmaceuticals; Hybridomas; Animals, Genetically Modified; Mammals
PubMed: 38149884
DOI: 10.4049/immunohorizons.2300102 -
MAbs 2022Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional...
Despite their common use in research, monoclonal antibodies are currently not systematically sequenced. This can lead to issues with reproducibility and the occasional loss of antibodies with loss of cell lines. Hybridoma cell lines have been the primary means of generating monoclonal antibodies from immunized animals, including mice, rats, rabbits, and alpacas. Excluding therapeutic antibodies, few hybridoma-derived antibody sequences are known. Sanger sequencing has been "the gold standard" for antibody gene sequencing, but this method relies on the availability of species-specific degenerate primer sets for amplification of light and heavy antibody genes and it requires lengthy and expensive cDNA preparation. Here, we leveraged recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing to develop Nanopore Antibody sequencing (NAb-seq): a three-day, species-independent, and cost-effective workflow to characterize paired full-length immunoglobulin light- and heavy-chain genes from hybridoma cell lines. When compared to Sanger sequencing of two hybridoma cell lines, long-read ONT sequencing was highly accurate, reliable, and amenable to high throughput. We further show that the method is applicable to single cells, allowing efficient antibody discovery in rare populations such as memory B cells. In summary, NAb-seq promises to accelerate identification and validation of hybridoma antibodies as well as antibodies from single B cells used in research, diagnostics, and therapeutics.
Topics: Animals; Antibodies, Monoclonal; Cell Line; Cost-Benefit Analysis; High-Throughput Nucleotide Sequencing; Hybridomas; Mice; Rabbits; Rats; Reproducibility of Results
PubMed: 35965451
DOI: 10.1080/19420862.2022.2106621 -
Frontiers in Cellular and Infection... 2023The rise of antimicrobial-resistant bacterial infections is a crucial health concern in the 21st century. In particular, antibiotic-resistant causes difficult-to-treat...
The rise of antimicrobial-resistant bacterial infections is a crucial health concern in the 21st century. In particular, antibiotic-resistant causes difficult-to-treat infections associated with high morbidity and mortality. Unfortunately, the number of effective therapeutic interventions against antimicrobial-resistant infections continues to decline. Therefore, discovery and development of alternative treatments are necessary. Here, we present pre-clinical efficacy studies on an anti- therapeutic monoclonal antibody. Using hybridoma technology, we generated a monoclonal antibody and characterized its binding to using ELISA and fluorescence correlation spectroscopy. We also characterized its function and against . The anti- antibody (WVDC-5244) bound clinical strains of various serotypes , even in the presence of alginate exopolysaccharide. In addition, WVDC-5244 induced opsonophagocytic killing of in J774.1 murine macrophage, and complement-mediated killing. In a mouse model of acute pneumonia, prophylactic administration of WVDC-5244 resulted in an improvement of clinical disease manifestations and reduction of burden in the respiratory tract compared to the control groups. This study provides promising pre-clinical efficacy data on a new monoclonal antibody with therapeutic potential for infections.
Topics: Mice; Animals; Pseudomonas aeruginosa; Pneumonia; Antibodies, Monoclonal; Hybridomas; Complement System Proteins; Pseudomonas Infections
PubMed: 37124031
DOI: 10.3389/fcimb.2023.1117844 -
Frontiers in Immunology 2022The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of...
Discovery and characterization of SARS-CoV-2 reactive and neutralizing antibodies from humanized CAMouse mice through rapid hybridoma screening and high-throughput single-cell V(D)J sequencing.
The coronavirus disease 2019 pandemic has caused more than 532 million infections and 6.3 million deaths to date. The reactive and neutralizing fully human antibodies of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are effective detection tools and therapeutic measures. During SARS-CoV-2 infection, a large number of SARS-CoV-2 reactive and neutralizing antibodies will be produced. Most SARS-CoV-2 reactive and neutralizing fully human antibodies are isolated from human and frequently encoded by convergent heavy-chain variable genes. However, SARS-CoV-2 viruses can mutate rapidly during replication and the resistant variants of neutralizing antibodies easily survive and evade the immune response, especially in the face of such focused antibody responses in humans. Therefore, additional tools are needed to develop different kinds of fully human antibodies to compensate for current deficiency. In this study, we utilized antibody humanized CAMouse mice to develop a rapid antibody discovery method and examine the antibody repertoire of SARS-CoV-2 RBD-reactive hybridoma cells derived from CAMouse mice by using high-throughput single-cell V(D)J sequencing analysis. CAMouse mice were immunized by 28-day rapid immunization method. After electrofusion and semi-solid medium screening on day 12 post-electrofusion, 171 hybridoma clones were generated based on the results of SARS-CoV-2 RBD binding activity assay. A rather obvious preferential usage of IGHV6-1 family was found in these hybridoma clones derived from CAMouse mice, which was significantly different from the antibodies found in patients with COVID-19. After further virus neutralization screening and antibody competition assays, we generated a noncompeting two-antibody cocktail, which showed a potent prophylactic protective efficacy against SARS-CoV-2 in cynomolgus macaques. These results indicate that humanized CAMouse mice not only provide a valuable platform to obtain fully human reactive and neutralizing antibodies but also have a different antibody repertoire from humans. Thus, humanized CAMouse mice can be used as a good complementary tool in discovery of fully human therapeutic and diagnostic antibodies.
Topics: Animals; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; COVID-19; Humans; Hybridomas; Mice; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 36211410
DOI: 10.3389/fimmu.2022.992787 -
Nature Communications Aug 2016Hybridomas, fusions of primary mouse B cells and myelomas, are stable, rapidly-proliferating cell lines widely utilized for antibody screening and production. Antibody...
Hybridomas, fusions of primary mouse B cells and myelomas, are stable, rapidly-proliferating cell lines widely utilized for antibody screening and production. Antibody specificity of a hybridoma clone is determined by the immunoglobulin sequence of the primary B cell. Here we report a platform for rapid reprogramming of hybridoma antibody specificity by immunogenomic engineering. Here we use CRISPR-Cas9 to generate double-stranded breaks in immunoglobulin loci, enabling deletion of the native variable light chain and replacement of the endogenous variable heavy chain with a fluorescent reporter protein (mRuby). New antibody genes are introduced by Cas9-targeting of mRuby for replacement with a donor construct encoding a light chain and a variable heavy chain, resulting in full-length antibody expression. Since hybridomas surface express and secrete antibodies, reprogrammed cells are isolated using flow cytometry and cell culture supernatant is used for antibody production. Plug-and-(dis)play hybridomas can be reprogrammed with only a single transfection and screening step.
Topics: Animals; Antibodies; Antibody Specificity; Antigens; Cell Line; Gene Editing; Genetic Engineering; Hybridomas; Mice; Reproducibility of Results
PubMed: 27531490
DOI: 10.1038/ncomms12535 -
BMC Research Notes Apr 2020The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion,...
OBJECTIVE
The amoeba Dictyostelium discoideum has been a valuable model organism to study numerous facets of eukaryotic cell biology, such as cell motility, cell adhesion, macropinocytosis and phagocytosis, host-pathogen interactions and multicellular development. However, the relative small size of the Dictyostelium community hampers the production and distribution of reagents and tools, such as antibodies, by commercial vendors.
RESULTS
For the past 5 years, our laboratory has worked to promote an increased use of recombinant antibodies (rAbs) by academic laboratories. Here we report our efforts to ensure that Dictyostelium researchers have access to rAbs. Using hybridoma sequencing and phage display techniques, we generated a panel of recombinant antibodies against D. discoideum antigens, providing a useful and reliable set of reagents for labelling and characterization of proteins and subcellular compartments in D. discoideum, accessible to the entire Dictyostelium community.
Topics: Antibodies; Dictyostelium; Hybridomas; Models, Biological; Recombinant Proteins
PubMed: 32276653
DOI: 10.1186/s13104-020-05048-8