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Science Immunology Jul 2022Despite being the most abundantly secreted immunoglobulin isotype, the pattern of reactivity of immunoglobulin A (IgA) antibodies toward each individual's own gut...
Despite being the most abundantly secreted immunoglobulin isotype, the pattern of reactivity of immunoglobulin A (IgA) antibodies toward each individual's own gut commensal bacteria still remains elusive. By colonizing germ-free mice with defined commensal bacteria, we found that the binding specificity of bulk fecal and serum IgA toward resident gut bacteria resolves well at the species level and has modest strain-level specificity. IgA hybridomas generated from lamina propria B cells of gnotobiotic mice showed that most IgA clones recognized a single bacterial species, whereas a small portion displayed cross-reactivity. Orally administered hybridoma-produced IgAs still retained bacterial antigen binding capability, implying the potential for a new class of therapeutic antibodies. Species-specific IgAs had a range of strain specificities. Given the distinctive bacterial species and strain composition found in each individual's gut, our findings suggest the IgA antibody repertoire is shaped uniquely to bind "self" gut bacteria.
Topics: Animals; B-Lymphocytes; Clone Cells; Gastrointestinal Microbiome; Hybridomas; Immunoglobulin A; Mice
PubMed: 35857580
DOI: 10.1126/sciimmunol.abg3208 -
ACS Nano Nov 2017Pathogen-activated antibody-secreting cells (ASCs) produce and secrete antigen-specific antibodies. ASCs are detectable in the peripheral blood as early as 3 days after...
Pathogen-activated antibody-secreting cells (ASCs) produce and secrete antigen-specific antibodies. ASCs are detectable in the peripheral blood as early as 3 days after antigen exposure, which makes ASCs a potential biomarker for early disease detection. Here, we present a magnetic capture and detection (MCD) assay for sensitive, on-site detection of ASCs. In this approach, ASCs are enriched through magnetic capture, and secreted antibodies are magnetically detected by a miniaturized nuclear magnetic resonance (μNMR) system. This approach is based entirely on magnetics, which supports high contrast against biological background and simplifies assay procedures. We advanced the MCD system by (i) synthesizing magnetic nanoparticles with high magnetic moments for both cell capture and antibody detection, (ii) developing a miniaturized magnetic device for high-yield cell capture, and (iii) optimizing the μNMR assay for antibody detection. Antibody responses targeting hemolysin E (HlyE) can accurately identify individuals with acute enteric fever. As a proof-of-concept, we applied MCD to detect antibodies produced by HlyE-specific hybridoma cells. The MCD achieved high sensitivity in detecting antibodies secreted from as few as 5 hybridoma cells (50 cells/mL). Importantly, the assay could be performed with whole blood with minimal sample processing.
Topics: Acute Disease; Antibodies, Bacterial; Antibody-Producing Cells; Antigens, Bacterial; Bacterial Infections; Biosensing Techniques; Cell Separation; Hemolysin Proteins; Humans; Hybridomas; Iron; Lab-On-A-Chip Devices; Magnetite Nanoparticles; Oxides; Proof of Concept Study; Salmonella paratyphi A; Salmonella typhi; Typhoid Fever; Zinc
PubMed: 29121461
DOI: 10.1021/acsnano.7b06074 -
Veterinary Immunology and... Nov 1989The requirement for monoclonal antibodies derived from species other than rats and mice is becoming increasingly realised in veterinary, as well as human, medicine. This... (Review)
Review
The requirement for monoclonal antibodies derived from species other than rats and mice is becoming increasingly realised in veterinary, as well as human, medicine. This paper reviews current knowledge of the production of inter-species hybridomas (heterohybridomas) by the fusion of rodent myeloma cell lines with lymphocytes from species of veterinary importance. To date a number of monoclonal immunoglobulins derived from sheep, cattle, pig, rabbit, mink and primate species have been produced to a variety of different bacterial, viral and nematode pathogens as well as to blood group and MHC determinants and to hormones. The technique opens up a number of possibilities for the future; some of these applications are discussed in relation to the antibodies produced thus far.
Topics: Animals; Antibodies, Monoclonal; Cattle; Hybridomas; Mink; Primates; Rabbits; Sheep; Veterinary Medicine
PubMed: 2694587
DOI: 10.1016/0165-2427(89)90105-0 -
British Medical Journal (Clinical... Oct 1981
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Humans; Hybridomas; Immunization; Interferons
PubMed: 6170381
DOI: 10.1136/bmj.283.6300.1143 -
Analytical Biochemistry Nov 2022By using EDTPA-modified zirconia particles that selectively adsorb immunoglobulins in a column, we developed a chromatography separation system for efficient...
By using EDTPA-modified zirconia particles that selectively adsorb immunoglobulins in a column, we developed a chromatography separation system for efficient concentrating and purifying of IgM from hybridoma culture supernatants. Hybridoma culture supernatants containing IgMs were diluted 3-fold with 10 mM phosphate buffer (pH 7.0) and passed through the column. During this process, zirconia particles selectively adsorbed these IgMs, and most of the contaminating proteins flowed out into the flow-through. The adsorbed IgMs were easily eluted with a small volume of 400 mM phosphate buffer (pH 8.0), and high-concentration IgM solutions were prepared. Subsequent simple processing using a Capto™ Core 400 cartridge column provided highly purified IgM. The operation is easy, and the activity of IgM is maintained because the purification process is performed using only neutral ranges of phosphate buffers. Here, we showed that anti-globoside and anti-CDw75 IgM purified by this method can be used to stain cervical cancer and Burkitt lymphoma cells that specifically express these respective tumor-associated carbohydrate antigens.
Topics: Antibodies, Monoclonal; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Hybridomas; Immunoglobulin M; Phosphates; Zirconium
PubMed: 36122604
DOI: 10.1016/j.ab.2022.114900 -
Scientific Reports Sep 2023The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of...
The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.
Topics: Animals; Mice; Antibodies, Monoclonal; Hybridomas; Reproducibility of Results; Immunosuppressive Agents; Databases, Nucleic Acid
PubMed: 37758930
DOI: 10.1038/s41598-023-43233-4 -
Proceedings of the National Academy of... Feb 2014
Topics: Antibodies; History, 20th Century; History, 21st Century; Hybridomas; Molecular Biology
PubMed: 24532658
DOI: 10.1073/pnas.1401334111 -
Asian Pacific Journal of Allergy and... Jun 2012The hybridoma technique is the standard method for production of monoclonal antibodies of interest. However, the newly formed hybridomas and cells at low density often...
BACKGROUND
The hybridoma technique is the standard method for production of monoclonal antibodies of interest. However, the newly formed hybridomas and cells at low density often grow poorly or die. This is the major obstacle to production of monoclonal antibodies.
OBJECTIVE
The aim of this study was to establish a method for preparation of conditioned medium in the absence of mitogen for promoting the growth of hybridomas after cell fusion and during single cell cloning.
METHODS
Culture supernatants were obtained from the cultures of BW5147 mouse thymoma cells without mitogen stimulation. Novel conditioned mediums were investigated for their ability to support hybridoma single-cell cloning and hybridoma production.
RESULTS
We demonstrated that these conditioned mediums could support hybridoma single-cell growth, both for stable and newly generated hybridomas, at a level equal to the commercial conditioned medium BM-Condimed H1. The conditioned medium was most effective at a final concentration of 20% with the basal medium supplemented with 10% fetal calf serum. The novel conditioned medium could also be effectively employed for generation of hybridomas secreting various monoclonal antibodies. Interestingly, fibroblast overgrowth in the post-fusion wells was markedly reduced when using the novel conditioned medium, as compared to the commercial BM-Condimed H1, likely due to the absence of mitogen in the conditioned medium.
CONCLUSION
We describe the method for production of a novel conditioned medium for hybridoma technology. This method is simple, needing no special technology or sophisticated equipment. The novel conditioned medium is, therefore, recommended for use in place of expensive commercial conditioned medium for hybridoma technology, especially in resource-limited countries.
Topics: Animals; Antibodies, Monoclonal; Cell Fusion; Clone Cells; Culture Media, Conditioned; Fibroblasts; Hybridomas; Mice; Mitogens; Single-Cell Analysis; Thymoma; Thymus Neoplasms
PubMed: 22830290
DOI: No ID Found -
Virologica Sinica Aug 2022• Class-switch recombination was mimicked in hybridomas by controllable expression of activation-induced cytidine deaminase. • IgG antibodies were generated through...
• Class-switch recombination was mimicked in hybridomas by controllable expression of activation-induced cytidine deaminase. • IgG antibodies were generated through this system in an anti-Flu B IgM hybridoma 7G1. • IgG1 and IgG2a subtypes of 7G1 present improved antiviral activity and
Topics: Antibodies; Cytidine Deaminase; Hybridomas; Influenza B virus
PubMed: 35331970
DOI: 10.1016/j.virs.2022.03.009 -
Protein & Cell Apr 2010The study of antibodies has been a focal point in modern biology and medicine since the early 1900s. However, progress in therapeutic antibody development was slow and... (Review)
Review
The study of antibodies has been a focal point in modern biology and medicine since the early 1900s. However, progress in therapeutic antibody development was slow and intermittent until recently. The first antibody therapy, murine-derived murononab OKT3 for acute organ rejection, was approved by the US Food and Drug Administration (FDA) in 1986, more than a decade after César Milstein and Georges Köhler developed methods for the isolation of mouse monoclonal antibodies from hybridoma cells in 1975. As a result of the scientific, technological, and clinical breakthroughs in the 1980s and 1990s, the pace of therapeutic antibody discovery and development accelerated. Antibodies are becoming a major drug modality with more than two dozen therapeutic antibodies in the clinic and hundreds more in development. Despite the progress, need for improvement exists at every level. Antibody therapeutics provides fertile ground for protein scientists to fulfill the dream of personalized medicine through basic scientific discovery and technological innovation.
Topics: Animals; Antibodies, Monoclonal; Biotechnology; Humans; Hybridomas; Immunotherapy; United States; United States Food and Drug Administration
PubMed: 21203944
DOI: 10.1007/s13238-010-0052-8