The Stability of Fluorescence-conjugated Antibodies under Temperature and Time Related Stressors
- Restrictions on Access:
- Open Access.
- Fluorescence-activated Cell Sorting utilizes the natural mechanics of the antibody-target interactions to quantify and classify a sample of cells by way of their expressed proteins. Monoclonal antibodies, generated to target a singular cellular marker, are conjugated to a fluorescent molecule known as a fluorochrome. Because the fluorochrome is bound to the antibody, it will only emit light if the antibody is bound to the protein marker. Thus, it is imperative that the antibody binds with high affinity and the covalent bond between the fluorochrome and antibody remains stable. We hypothesized the max fluorescence intensity as determined by flow cytometry would be decreased in fluorescence-conjugated antibodies as a result of temperature and time. We tested nine fluorescence-conjugated antibodies targeting cluster of differentiation 8 (CD8) against temperature stress by way of freeze-thaw cycles. A phenotyping master mix was also put through the freeze-thaw cycles to examine effects of temperature on a mixture of antibodies. A subset of antibodies was tested for effects of time, comparing results of four years of storage versus a few months. Experimental data revealed time had a pronounced impact on CD25-PE and CD8-APC. In terms of temperature stress, both individual antibody aliquots as well as the master mix experienced a decrease in overall intensity, but not to the severity predicted. PacBlue, PE-Cy7, PE-TxR, FITC, A700, and PerCP maintained viability over the duration of the freeze-thaw cycles. However, PacBlue, PE-Cy7, and PE-TxR demonstrated the least degree of destabilization with applied stress. As such, freezing aliquoted antibodies at -80 ̊ C may provide a useful method of prolonging shelf life and preventing the destabilization resulting from extended storage.
- Dissertation Note:
- B.S. Pennsylvania State University 2015.
- Technical Details:
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
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