Labeling of Fluorescent Probes to Albumin Microspheres and B16 Melanoma Extra-Cellular Antigen.
Fluorescent coupling of bovine serum albumin (BSA) and extracellular antigen of melanoma (ECA) using modified carbodiimide method was evaluated enabling BSA to serve as model protein to evaluate the release profiles of different microsphere formulations and uptake in B16 melanoma cells. Properties of the fluorescent labeled BSA (FBSA) including stability, transition temperature and fluorescent intensity were evaluated. It was found that FBSA produced using this method showed comparable transition temperature and fluorescent intensity compared to the commercially available FITC-labeled BSA and demonstrated stability of the fluorescent-protein linkage after overnight treatment with both trypsin and human plasma using fluorescent microscope. This study showed that the modified carbodiimide labeling method can serve as an alternative method for fluorescent labeling of target protein at reasonable cost particularly when sufficient amount of target protein is required for microsphere formulation screening.
Lai
Yin Hwa
YH
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Mercer University, 3001 Mercer University Drive, Atlanta, GA 30341, USA. yinhwalai@gmail.com.
D'Souza
Martin J
MJ
D000818:Animals; D002152:Calorimetry, Differential Scanning; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008546:Melanoma, Experimental / Q000276:immunology / Q000378:metabolism*; D058950:Melanoma-Specific Antigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008863:Microspheres; D010587:Phagocytosis; D055550:Protein Stability; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D058950:Melanoma-Specific Antigens; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate; D005022:Ethyldimethylaminopropyl Carbodiimide