title: A quantitative luminescence assay for measuring cell uptake of aqueous-based microcapsules in vitro.
abstract: 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.
authors.affiliation: Division of Immunologic and Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
authors.forename: J O
authors.initials: JO
authors.lastname: Brubaker
authors.forename: R T
authors.initials: RT
authors.lastname: Patil
authors.forename: T J
authors.initials: TJ
authors.lastname: Speaker
authors.forename: P A
authors.initials: PA
authors.lastname: Offit
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism; D001693:Biological Transport, Active; D002214:Capsules / Q000493:pharmacokinetics*; D005260:Female; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D013096:Spermine; D014867:Water
chemical_list: D002214:Capsules; D014867:Water; D013096:Spermine; D006735:Horseradish Peroxidase