Quantitation of CD38 expression using QuantiBRITE beads.
The QuantiBRITE bead method was compared with the CD4 biological calibration method for quantitation of CD38 expression on CD8+ T-lymphocytes of Multicenter AIDS Cohort Study participants. Results were expressed as CD38 antibodies bound per cell (ABCs) and were the same with the two methods provided two conditions were met. These were the use of repurified (> 95% of the monoclonal antibodies [mAbs] have 1 phycoerythrin [PE] molecule per mAb) CD38-PE for both methods and use of repurified CD4-PE to calculate the relative fluorescence intensity multiplier for the CD4 biological calibration method. Our results indicate that the prognostic significance of CD38 values obtained using the QuantiBRITE method can be interpreted using previously published reports (Liu et al.: J Acquir Immune Defic Syndr Hum Retrovirol 16:83-92, 1997 and 18:332-340, 1998). Sample preparation using NH4Cl and FACS lysing solution gave similar results for CD38 relative fluorescence intensity. Dilution into either phosphate-buffered saline with 2% fetal calf serum and 0.1% sodium azide or fixation in 1% paraformaldehyde for 1 or 24 h also gave similar results. In experiments using Raji cells, which express high levels of CD38, the valence of binding of the intact Leu 17 antibody was approximately 68% bivalent and approximately 32% monovalent. This emphasizes the complexity of determining antigen density from ABCs. We conclude that repurified PE conjugates of CD38, which can be consistently made, together with QuantiBRITE PE beads, provide a convenient and reliable method for quantitation of CD38 expression as ABCs.
Iyer
S B
SB
Becton Dickinson Immunocytometry Systems, San Jose, California, USA. sujata_iyer@bdis.com
Hultin
L E
LE
Zawadzki
J A
JA
Davis
K A
KA
Giorgi
J V
JV
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000163:Acquired Immunodeficiency Syndrome / Q000097:blood / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000032:analysis*; D015704:CD4 Antigens / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes / Q000737:chemistry*; D002138:Calibration; D015331:Cohort Studies; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins; D008863:Microspheres; D009244:NAD+ Nucleosidase / Q000032:analysis*; D012015:Reference Standards; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D015704:CD4 Antigens; D008562:Membrane Glycoproteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1