A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity.
We demonstrate that the FATAL cytolysis assay can be adapted into a rapid and fluorometric antibody-dependent cellular cytotoxicity assay (RFADCC). The RFADCC relies on double-staining target cells with a membrane dye (PKH-26) and a viability dye (CFSE) prior to the addition of antibody and effector cells. We used the RFADCC to assess dose-dependent and envelope-specific anti-human immunodeficiency virus (HIV) ADCC responses mediated by monoclonal antibody-2G12 and human sera. Using the assay, we also detected early anti-simian immunodeficiency virus (SIV) ADCC responses in rhesus macaques infected with pathogenic SIV(mac251). Importantly, the RFADCC was further useful in monitoring anti-HIV and anti-SIV ADCC responses elicited by immunizing chimpanzees and rhesus macaques with replicating adenovirus-based AIDS vaccine candidates. In comparison to the standard chromium release assay, the RFADCC provides a higher cell killing readout and is advantageous in allowing use of viably frozen as well as fresh effector cells, thus facilitating assay standardization. The RFADCC is therefore a simple, reliable, and highly sensitive method that can be applied to assess the ADCC activity of monoclonal antibodies as well as ADCC responses elicited by HIV or SIV infection or by AIDS vaccine candidates.
Gómez-Román
V Raúl
VR
Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 41 Medlars Drive, Room D804, Bethesda, MD 20892-5065, USA.
Florese
Ruth H
RH
Patterson
L Jean
LJ
Peng
Bo
B
Venzon
David
D
Aldrich
Kris
K
Robert-Guroff
Marjorie
M
D016915:AIDS Vaccines / Q000008:administration & dosage / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000097:blood; D000920:Antibody-Dependent Cell Cytotoxicity; D002857:Chromium; D003601:Cytotoxicity Tests, Immunologic; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D015658:HIV Infections / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D008253:Macaca mulatta; D009930:Organic Chemicals; D002679:Pan troglodytes; D016097:Simian Acquired Immunodeficiency Syndrome / Q000276:immunology; D013388:Succinimides
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D016915:AIDS Vaccines; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26; D013388:Succinimides; D002857:Chromium