A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity (ADCC) activity in HIV-1 infected individuals.
Enhanced green fluorescent protein (EGFP) was stably expressed in CEM-NKr cell, a natural killer (NK) resistant human T-lymphoblastoid cell line, as EGFP-CEM-NKr cells. The cells pulsed with HIV-1 gp120 were then used as target cells for the measurement of antibody dependent cell mediated-cytotoxicity (ADCC) by flow cytometry. Compromised EGFP-CEM-NKr target cells stained with propidium iodide (PI) showed dual (green-red) fluorescent. Kinetic studies demonstrated that the sum of ADCC activity measured at 1-h and again at 2-h incubations by this flow cytometric method was comparable to the activity at 6 h by the standard chromium (51Cr) release assay (CRA). ADCC activity of HIV-1 seropositive sera measured by this new technique correlated strongly with that of CRA (Pearson's correlation coefficient of 0.832; p-value < 0.001 and intraclass correlation coefficient of 0.903; p-value < 0.001). The EGFP-CEM-NKr stable cell line provides a novel method to measure ADCC activity to HIV-1 gp120 by flow cytometry without pre-staining or pre-labeling target cells.
Kantakamalakul
Wannee
W
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. siwkk@mahidol.ac.th
Pattanapanyasat
Kovit
K
Jongrakthaitae
Surat
S
Assawadarachai
Vatcharain
V
Ampol
Silawun
S
Sutthent
Ruengpung
R
D000915:Antibody Affinity; D000920:Antibody-Dependent Cell Cytotoxicity; D002460:Cell Line; D002857:Chromium / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D015483:HIV Antibodies / Q000032:analysis*; D015699:HIV Envelope Protein gp120 / Q000032:analysis; D015658:HIV Infections / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology*; D007700:Kinetics; D013997:Time Factors; D014162:Transfection
D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; C485184:enhanced green fluorescent protein; D002857:Chromium; D049452:Green Fluorescent Proteins