Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-02-17
1994-07-05
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 2, 435808, 436536, 436546, 436172, 436800, 530403, G01N 33533, G01N 33536, G01N 2164
Patent
active
053266960
ABSTRACT:
Disclosed are immunofluorescence staining methods which increase the likelihood that antibodies expressed by a single B cell selected and sorted by fluorescence activated cell sorting are specific for the antigen of interest, and which also allow selection of B cells expressing antibodies of high affinity for the antigen of interest. The selection for B cells expressing antibodies to specific antigens is increased by labeling B cells with at least two antigen probes, where each antigen probe includes the antigen of interest and is labeled with a different fluorochrome. The positive selection is preferably combined with a negative selection step, in which autofluorescent cells and sticky cells are excluded out. The specificity of sorting of the desired B cells can be further enhanced by staining those antigen-specific B cells which produce the immunoglobulin isotype (typically IgG), with targeting molecules reactive with a B cell marker, such as .gamma. chain and CD19, that are conjugated with a different fluorochrome. Thus, the antigen-specific IgG-producing B cells of interest may be labeled with these unique reagents in four color FACS (including one for negative selection), which can sort the desired antigen-specific B cells at enhanced proportions. After sorting the B cells with FACS, those B cells with high affinity are preferred for analysis by the single cell-PCR procedure to amplify and clone the V.sub.H and V.sub.L segments of interest.
REFERENCES:
patent: 4510244 (1985-04-01), Parks
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patent: 4918000 (1990-04-01), Schubert
patent: 5213960 (1993-05-01), Chang
Muirhead, K. A., et al. Bio/Technology 3:337 1985.
Ault, K. A., et al., Ch 34 in Manual of Clinical Laboratory Immunology, 3rd ed., N. R. Rose ed. ASM 1986 pp. 247-253.
Kepplinger Esther L.
Mirabel Eric P.
Parsons Nancy J.
Tanox Biosystems, Inc.
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