A quantitative luminescence assay for measuring cell uptake of aqueous-based microcapsules in vitro

We recently developed a system of microencapsulation consisting of aqueous-based polymers (e.g. alginate) and aqueous amines (e.g. spermine). We found that microencapsulation enhanced virus-specific protective immune responses. In addition, we found that microencapsulation may enhance virus-specific...

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Bibliographic Details
Published in:Journal of immunological methods 2000-04, Vol.237 (1), p.85-93
Main Authors: Brubaker, Jeffery O, Patil, Reena T, Speaker, Tully J, Offit, Paul A
Format: Article
Language:English
Subjects:
Animals
Antigen-Presenting Cells - immunology
Antigen-Presenting Cells - metabolism
Biological Transport, Active
Capsules - pharmacokinetics
Female
Horseradish peroxidase
Horseradish Peroxidase - pharmacokinetics
In Vitro Techniques
Luminescence
Luminescent Measurements
Macrophages, Peritoneal - immunology
Macrophages, Peritoneal - metabolism
Mice
Mice, Inbred BALB C
Microcapsules
Peritoneal exudate cells
Spermine
Water
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Summary:We recently developed a system of microencapsulation consisting of aqueous-based polymers (e.g. alginate) and aqueous amines (e.g. spermine). We found that microencapsulation enhanced virus-specific protective immune responses. In addition, we found that microencapsulation may enhance virus-specific immune responses by selecting for antigen-presenting cells (APC) that are more efficient at processing and presenting viral antigens than those involved after natural infection. To determine the intracellular trafficking patterns and fate of microcapsules within APC, we developed a luminescence assay that permits the determination of specific quantities of proteins introduced into cells by microcapsules. We found that the time-dependent uptake of horseradish peroxidase (HRP)-labeled microcapsules was accurately detected in lysates of peritoneal exudate cells using luminol. The amplitude of HRP-catalyzed chemiluminescence in cell lysates correlated with the capture efficiency and retention kinetics of HRP in three different microcapsule preparations. HRP was most efficiently captured and retained by linking biotinylated HRP to microcapsulses chemically modified at the amine moiety with egg avidin. This preparation yielded more accurate and sensitive quantitation of HRP contained within cells than preparations capturing HRP or HRP-conjugated goat antibody into the microcapsular matrix by ionic interactions.
ISSN:0022-1759
1872-7905
DOI:10.1016/S0022-1759(00)00140-X