Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot.
Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN. Mabs Pf-164 and Pf-344 reacted with intracellular PFN in peripheral blood mononuclear cells (PBMC) from cynomolgus and rhesus macaques by flow cytometry and stained PFN in rhesus lymphoid tissue by immunohistochemistry (IHC). Moreover, PFN capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays utilizing mAbs Pf-164/Pf-80 for capture and mAb Pf-344 for detection were used to quantify PFN release by mitogen-stimulated cynomolgus and rhesus PBMC. The PFN ELISpot was further used to quantify antigen-specific CD8+ T cells by ex vivo stimulation of PBMC from cynomolgus macaques immunized against SIV/HIV-1. These macaque PFN-reactive mAbs and immunoassays will be valuable new tools for investigation of cytotoxic T lymphocyte (CTL) responses in non-human primate models of infectious diseases as well as for vaccine development.
Zuber
Bartek
B
Mabtech, Box 1233, SE-131 28 Nacka, Sweden. bartek@mabtech.com
Quigley
Máire F
MF
Critchfield
J William
JW
Shacklett
Barbara L
BL
Abel
Kristina
K
Miller
Christopher J
CJ
Mörner
Andreas
A
Paulie
Staffan
S
Ahlborg
Niklas
N
Sandberg
Johan K
JK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D008251:Macaca / Q000276:immunology / Q000821:virology*; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D018449:SAIDS Vaccines / Q000276:immunology; D016097:Simian Acquired Immunodeficiency Syndrome / Q000276:immunology*; D015302:Simian Immunodeficiency Virus / Q000276:immunology*; D013154:Spleen / Q000737:chemistry / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D018449:SAIDS Vaccines; D054353:Perforin