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A study on the temperature dependency and time course of the cold capture antibody secretion assay

Paper ID Volume ID Publish Year Pages File Format Full-Text
24153 43501 2009 4 PDF Available
Title
A study on the temperature dependency and time course of the cold capture antibody secretion assay
Abstract

The cold capture assay as described by Brezinsky et al. [Brezinsky, S.C.G., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W., Thill, G., 2003. A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J. Immunol. Methods 277, 141–155] stands out as the most simple of single cell secretion assays which can be used to sort for high productivity in recombinant cell lines. At low temperatures the process of protein release from transport vesicles is assumed to be delayed as both vesicle fusion and product release is slowed, so that secreted proteins can be stained on the cell surface using a fluorescent antibody. Typically, the fluorescent signal obtained correlates to the cell specific production rate of the analysed cell. In the present study we compared staining of human antibody producing CHO cells performed at different temperatures and we observed the fluorescent signal over 24 h. We found that the staining temperature did not influence signal intensity. The fluorescent signal was stable for 24 h at 4 °C, decreased to 80% at room temperature (21 °C), while it decreased significantly already after 2 h at 37 °C. Initially, the fluorescent signal was observed on the cell surface, however, at later stages it was found in compartments in the cytoplasm. Finally we compared differences in signal stability depending on whether the antibody used for staining bound to the light or heavy chain of the product and on whether the fluorescent label was a relatively stable protein (phycoerythrin) or a pH-dependent small molecule (FITC). Our results indicate that the secreted product is trapped by the staining antibody on the cell surface at all temperatures. Subsequently these aggregates are endocytosed by the cells, a process which is slowed down at low temperatures.

Keywords
Flow cytometric cell sorting; High production rates; Single cell secretion assay; Recombinant CHO cells
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A study on the temperature dependency and time course of the cold capture antibody secretion assay
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 141, Issues 1–2, 20 April 2009, Pages 80–83
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us