Genetic information of single cells is extracted using reverse transcription PCR with suitable primers to focus on Ig-regions of interest. Modern methodologies are employed to screen and sort single B cells using fluorescent reagents with flow cytometry and antigens on magnetic beads to enrich promising cells. Monoclonal antibody production with single B cell technologies starts with blood samples from human donors. The resulting construct is then introduced into suitable mammalian cells that produce the desired antibody therapies. Biochemical amplification of the associated genetic material and subsequent sequencing yields bioinformatic data to inform the development of full-length human antibody constructs. This allows scientists to expose the antibody-bearing phages to immobilized antigens of interest – similar to receptors – and enrich conjugates with higher affinity. After introducing these genetic blueprints into bacterial cell cultures, the next generation of phages will present the antibodies on their surface, thus physically linking the amino acid sequence of antibodies with their respective DNA (=phage library). Phage display technology was developed in the early 1990s. 2 Using recombinant technology, the genetic code for antibodies (mostly of the variable region Fab) is fused in vitro to genes for surface peptides of bacteriophages. The method relies on suitable immune responses of the injected animal to the antigen and requires humanization (chimeric antibodies) of the antibodies to reduce immunogenicity, meaning side effects due to being recognized as foreign by the patient’s immune system. The resulting cell lines undergo characterization assays and are validated to produce the specific antibody of interest and then either cultured by the ascites method (transplantation into animal peritoneum) or cell culture in bioreactors. After injecting an animal with the antigen of interest, spleen cells are isolated and fused with myeloma cells to combine the properties of both cell types: antigen-specific Ab-secretion and being immortal. The monoclonal antibody production by hybridoma cells made monoclonal antibody treatments first feasible from the late 1970s on. If you want to learn more about antibody production in general, read our articles: Antibody production and In vitro antibody production. In the next paragraphs we will take a closer look at the technologies. In principle, three methods are currently used:Įach method has its advantages and disadvantages and is chosen based on the specific requirements of the desired monoclonal antibody. Human monoclonal antibodies are biotechnological tools with very large traded volumes, thus large-scale commercial manufacturing is employed. Monoclonal antibody production – technologies Selectively isolating and culturing lymphocytes has been elusive until 1975, when Köhler and Milstein found a way to immortalize B cells, thus being able to generate identical copies of antibody producing cells and culturing them for extended periods of time. Biologically speaking, each monoclonal antibody is secreted by one single B lymphocyte, hence the name “monoclonal”. In contrast to polyclonal antibodies that target many structural regions (epitopes) of an antigen, mAbs show high specificity for one single epitope. With thousands of ongoing in vivo studies and clinical trials, the widespread application in diagnostics (enzyme-linked immunosorbent assay, ELISA) and general use as research reagents, the worldwide demand for mAbs is ever increasing. immunotherapies, ADCC antibodies and antibody drug conjugates). against SARS-COV-2 “coronavirus”), and oncology (e.g. Over a hundred monoclonal antibodies (mAbs) are approved by the US FDA for human use in immunology (autoimmune diseases such as rheumatoid arthritis), infectious diseases (e.g. More about monoclonal antibodies Importance of monoclonal antibodies
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