Methods and compositions for inducible extracellular membrane capture of monoclonal immunoglobulins secreted by hybridomas
AssigneeLankenau Institute for Medical Research
InventorsScott K. Dessain, Rama Devudu Puligedda
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Abstract
Compositions and methods are provided for making and using a cell having expressed on its outer plasma membrane surface an Anchor designed to form a first complex with a selected Linker. The Linker is designed to form a second complex with a target protein that is not recognized by the Anchor. These cells can be primary cells or immortalized cells, and can function as fusion partners to create hybridoma cells.
Claims18
A method of identifying an immortalized cell that secretes an antibody or fragment thereof among a heterogeneous population of immortalized cells, the method comprising:
- (a) providing the population of immortalized cells having expressed on their outer plasma membrane surfaces an Anchor, wherein the Anchor comprises i) an extracellular region comprising an antibody or fragment thereof capable of binding a selected Linker to form a first complex, and ii) a transmembrane amino acid sequence that immobilizes the Anchor on the outer plasma membrane surfaces, wherein said Anchor cannot bind the antibody or fragment thereof;
- with
- (b) contacting the immortalized cells of (a) with an effective amount of the Linker, wherein the Linker is a soluble antibody or antibody fragment comprising a variable domain that binds and forms a second complex with a conserved domain of the antibody or fragment thereof, wherein the contacting permits the antibody or fragment thereof to be linked to the surface of the immortalized cell that secretes it upon formation of the first and second complexes;
- and
- (c) identifying the existence, antigen specificity, antigen binding affinity, titer, amount, or biological activity of the antibody or fragment thereof secreted by the immortalized cells and bound thereto by the cell-bound multiple immune complexes formed by Anchor-Linker-antibody or fragment thereof.
A method of detecting or measuring a characteristic of a target protein secreted by an immortalized cell comprising:
- (a) contacting
- i. a population of immortalized cells, wherein each cell of the population has expressed on its outer plasma membrane surface an Anchor, wherein the Anchor comprises i) an extracellular region comprising an antibody or fragment thereof capable of binding a selected Linker to form a first complex, and ii) a transmembrane amino acid sequence that immobilizes the Anchor on the outer plasma membrane surfaces, wherein said Anchor cannot bind the target protein;
- with
- ii. the Linker designed to form the first complex and to form a second complex with the target protein that is not recognized by the Anchor,
- iii. the contacting permitting the Linker to adhere to the outer plasma membrane of the cells by binding in the first complex with the Anchor;
- (b) maintaining the cells of (a) in the presence of the Linker under conditions in which the cells secrete the target protein and the target protein binds to the cell-adhered first complex via formation of the second complex;
- and
- (c) identifying the existence, antigen specificity, antigen binding affinity, titer, amount, or biological activity of the target protein bound onto the cell by the first and second complexes.
Dependencies are inferred from the claim text.