TY - JOUR
T1 - Quantitation of rare memory B cell populations by two independent and complementary approaches
AU - Amanna, Ian J.
AU - Slifka, Mark K.
N1 - Funding Information:
We thank the volunteers for their time and their participation in this research study; Dr. S. Crotty for the provision of reagents; Dr. S. Shiigi for his FC technical advice; and M. Lewis and E. Hammarlund for human blood collection and processing. This work was supported by National Institutes of Health contract N01 AI40029, PO1 AG023664, RR00334, and RR00163.
PY - 2006/12/20
Y1 - 2006/12/20
N2 - Current methodology for quantitation of memory B cells (MBC) in clinical samples is limited and often does not allow for detection of multiple MBC specificities in a single assay. Here we describe two independent approaches to antigen-specific MBC quantitation. First, a sensitive flow cytometry (FC) assay was developed for simultaneous quantitation of two prototypical B cell antigens, tetanus and diphtheria. Second, an ELISA-based MBC limiting dilution assay (LDA) was developed that provides quantitative analysis of up to 8-12 different MBC frequencies from a single blood sample. Cross-validation studies indicated that MBC numbers measured by FC correlated significantly with the frequencies obtained by LDA (R2 = 0.92, p = 0.0002). These two functionally distinct approaches will be useful for accurate quantitation of rare MBC populations specific for either simple antigens (e.g. tetanus and diphtheria) or complex antigens (e.g. vaccinia or other viruses), and is amenable for use in a variety of model systems.
AB - Current methodology for quantitation of memory B cells (MBC) in clinical samples is limited and often does not allow for detection of multiple MBC specificities in a single assay. Here we describe two independent approaches to antigen-specific MBC quantitation. First, a sensitive flow cytometry (FC) assay was developed for simultaneous quantitation of two prototypical B cell antigens, tetanus and diphtheria. Second, an ELISA-based MBC limiting dilution assay (LDA) was developed that provides quantitative analysis of up to 8-12 different MBC frequencies from a single blood sample. Cross-validation studies indicated that MBC numbers measured by FC correlated significantly with the frequencies obtained by LDA (R2 = 0.92, p = 0.0002). These two functionally distinct approaches will be useful for accurate quantitation of rare MBC populations specific for either simple antigens (e.g. tetanus and diphtheria) or complex antigens (e.g. vaccinia or other viruses), and is amenable for use in a variety of model systems.
KW - B cell memory
KW - Flow cytometric analysis
KW - Limiting dilution analysis
KW - Multiple species
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U2 - 10.1016/j.jim.2006.09.005
DO - 10.1016/j.jim.2006.09.005
M3 - Article
C2 - 17055526
AN - SCOPUS:33751319424
SN - 0022-1759
VL - 317
SP - 175
EP - 185
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -