Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells.
The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.
Zuber
Bartek
B
Mabtech AB, Box 1233, SE-131 28 Nacka, Sweden. bartek@mabtech.com
Levitsky
Victor
V
Jönsson
Gun
G
Paulie
Staffan
S
Samarina
Arina
A
Grundström
Susanna
S
Metkar
Sunil
S
Norell
Håkan
H
Callender
Glenda G
GG
Froelich
Christopher
C
Ahlborg
Niklas
N
D000818:Animals; D000911:Antibodies, Monoclonal; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry / Q000276:immunology / Q000821:virology*; D014780:Viruses / Q000276:immunology*
D000911:Antibodies, Monoclonal; D018984:Epitopes, T-Lymphocyte; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin