Antibody discovery via multiplexed single cell characterization

The secreted immunoglobulin footprint of single hybridoma cells, containing ~ 10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of ea...

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Bibliographic Details
Published in:Journal of immunological methods 2009-02, Vol.341 (1), p.135-145
Main Authors: Harriman, William D., Collarini, Ellen J., Sperinde, Gizette V., Strandh, Magnus, Fatholahi, Marjan M., Dutta, April, Lee, Yunji, Mettler, Shelley E., Keyt, Bruce A., Ellsworth, Stote L., Kauvar, Lawrence M.
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antibody Affinity - immunology
Antibody Specificity - immunology
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
Fundamental immunology
Hybridomas
Hybridomas - cytology
Hybridomas - immunology
Hybridomas - metabolism
Immunoassay - methods
Immunoglobulins - immunology
Immunoglobulins - metabolism
Mice
Mice, Inbred BALB C
Microspheres
Molecular immunology
Multiplexed
Single cell assay
Specificity
Techniques
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Summary:The secreted immunoglobulin footprint of single hybridoma cells, containing ~ 10 fg of antibody purified in situ, has been probed for 9 properties concurrently by use of detection labels comprising 280 nm combinatorially colored fluorescent latex beads functionalized with proteins. Specificity of each individual hybridoma cell's product has thereby been assessed in a primary screen. Varying the density of antigen on beads to modulate the avidity of the interaction between bead and secreted antibody footprint allowed rank ordering by affinity in the same primary screen. As more criteria were added to the selection process, the frequency of positive cells went down; in some cases, the favorable cell was present at < 1/50,000. Recovery of the cell of interest was accomplished by plating the cells in a viscous medium on top of a membrane. After collecting the antibody footprint on a capture surface beneath the membrane, the immobilized cells were transferred to an incubator while the footprints were analyzed to locate the hybridoma cells of interest. The desired cells were then cloned by picking them from the corresponding locations on the membrane.
ISSN:0022-1759
1872-7905
DOI:10.1016/j.jim.2008.11.009