Combined immunomagnetic cell sorting and ELISPOT assay for the phenotypic characterization of specific antibody-forming cells.
A combination of immunomagnetic cell sorting and ELISPOT techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (ASC). Cell suspensions containing putative ASC were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. After separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of ASC by an ELISPOT assay. As a model system, the expression of selected cell differentiation markers by human circulating ASC has been evaluated after parenteral tetanus vaccination and during the course of a Leishmania infection. Prior treatment of blood MNC with beads coated with antibodies to CD38, HLA-DR or CD19 permitted the isolation of virtually all blood ASC. Further, prior immunomagnetic removal of T (CD2+) cells from blood MNC, followed by isolation of CD38+ cells facilitated the detection of Leishmania major-specific ASC in all six patients examined, whereas parasite-specific ASC among unfractionated blood mononuclear cells could only be detected in 3 out of these six patients. Simple and rapid, this approach provides not only accurate estimates of the frequency of ASC within a given B cell population or subpopulation, but can also efficiently enrich functional ASC from complex cell suspensions and thus should be particularly useful in situations where ASC are present at low frequencies.
Lakew
M
M
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
Nordström
I
I
Czerkinsky
C
C
Quiding-Järbrink
M
M
D000328:Adult; D000818:Animals; D000918:Antibody Specificity; D000921:Antibody-Producing Cells / Q000145:classification*; D016175:B-Lymphocyte Subsets / Q000145:classification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis; D018189:Immunomagnetic Separation / Q000379:methods*; D016130:Immunophenotyping; D018320:Leishmania major / Q000276:immunology; D016773:Leishmaniasis, Cutaneous / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000145:classification; D051379:Mice; D013745:Tetanus Toxoid / Q000276:immunology
D007074:Immunoglobulin G; D013745:Tetanus Toxoid