Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening.
Rama Devudu PuligeddaRashmi SharmaFetweh H Al-SaleemDiana KouiavskaiaArul Balaji VeluChandana Devi KattalaGeorge C PrendergastDavid R LynchKonstantin ChumakovScott K DessainPublished in: mAbs (2019)
Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS™). In OCMS™, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS™ demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG "cap", as a universal assay for anti-viral mAbs. We produced and characterized OCMS™-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS™ to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS™ overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.
Keyphrases
- monoclonal antibody
- cell surface
- flow cytometry
- endothelial cells
- poor prognosis
- high throughput
- binding protein
- single cell
- cell therapy
- single molecule
- sars cov
- quantum dots
- induced apoptosis
- living cells
- induced pluripotent stem cells
- machine learning
- dna binding
- mass spectrometry
- pluripotent stem cells
- immune response
- long non coding rna
- bone marrow
- dendritic cells
- endoplasmic reticulum stress
- signaling pathway
- cell proliferation
- oxidative stress
- mesenchymal stem cells
- structural basis