Fluorescent derivatization of a protease antigen to track antigen uptake and processing in human cell lines.
BACKGROUND
We have devised a simple and efficient fluorescence-based method to track antigen uptake and processing in human B lymphoblastoid cells (B-LCL). Fluorescein labelled subtilisin was used to optimize antigen uptake conditions and identify processed peptides from human cell lines.


RESULTS
Fluorescein labelled subtilisin conjugates had 0.06 to 2 moles of fluorescein per subtilisin molecule. High performance liquid chromatography and mass spectrometry (NanoESI-LC/MS/MS) analysis identified fluorescein conjugated to K141, K256, and the N terminus. Conjugates retained antigenic specificity to subtilisin specific antibodies and could be processed by whole cell extracts into low molecular weight fragments at pH 5.2. Maximal antigen uptake and processing occurred when PMSF (phenylmethylsulfonyl fluoride) inhibited subtilisin conjugate was incubated with cells at 100-200 microg/ml for 16 to 24 hr. Once optimal uptake conditions were established, processed subtilisin peptides were isolated and identified from human cell lines.


CONCLUSIONS
Our studies show that FITC-conjugation provides an efficient tool to track the uptake and processing of this protease antigen and to facilitate identification of processed antigenic peptides from human cell lines.
Patil
Namrata S
NS
Genencor International Inc, Palo Alto, CA 94304, USA. npatil@genencor.com
Wong
David L
DL
Collier
Katherine D
KD
McDonald
Hugh C
HC
D000941:Antigens / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003713:Dendritic Cells; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D006684:HLA-DR Antigens / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D020860:Subtilisin / Q000276:immunology / Q000378:metabolism*
D000941:Antigens; D006684:HLA-DR Antigens; D010455:Peptides; D020860:Subtilisin; D016650:Fluorescein-5-isothiocyanate