Secretion Assay
Description
The rapid increase
in the number of recombinant proteins and monoclonal antibodies
used therapeutically has created a demand for methods
that accelerate and simplify generation of high producing, stable
cell lines. The effort required to plate cells, obtain detectable
levels of secretion, assay supernatants and recover positive
clones is labor intensive and slow; the entire process can take
weeks to months. The gel microdrop (GMD)
assay format expedites detection, isolation, and analysis
of individual cells for bioprocessing and research by providing
an efficient, flow cytometric alternative to limited dilution
cloning. In this system, a derivatized gel matrix surrounding
each cell functions analogously to an antibody-coated microtiter
well, capturing the protein of interest within the immediate
surroundings of the cell. Because GMDs are small (20-50 µm diameter),
encapsulated cells can be processed
in bulk culture and then individually isolated using a fluorescence
activated cell sorter (FACS). Using
FACS, the fluorescence intensity of
each GMD can be quantified and high secretors isolated and recovered
based on the level of secretion. Any secreted molecule for which
there is an appropriate antibody pair can be captured and quantified.
Isolation of rare cells present in the population at frequencies
of 0.1% and a two to five-fold increase
in the protein production have been achieved. Productivity
is confirmed by analyzing the culture supernatant using
a standard immunoassay. In addition, the secretion assay can
be used ro rapidly isolate the small population of secretor
cells after trancfection and to access the productivity of cells
during fermentation. This high throughput screening method improves
productivity and shortens biotechnology product development
timelines.
General
Secretion Assay Protocol
Encapsulate cells
in CelBioGel™ Encapsulation Matrix.
Add streptavidin.
Add biotinylated capture antibody.
Secretion incubation, typically 15 minutes.
Add fluorescently labeled detection antibody.
Isolate and recover the sub-population of interest using FACS.
The complete
assay takes approximately 4 hours.
GMD Secretion Assay
Advantages
| Recover
individual, viable cells based on the amount of a specific,
secreted protein. |
|
Reduce/eliminate
Limiting Dilution Cloning
|
| Improve
yields |
|
Reduce
development timelines and material and labor costs
|
The
GMD Secretion Assay isolates and recovers individual producer
cells in one day.
