Methods for selecting low frequency antigen-specific single B ly

Details Methods for selecting low frequency antigen-specific single B ly Methods for selecting low frequency antigen-specific single B ly

1992-03-09

1993-05-25

Kepplinger, Esther L.

Chemistry: molecular biology and microbiology

Maintaining blood or sperm in a physiologically active state...

435 724, 436536, 436546, 436 63, 436172, 530403, G01N 3352, G01N 33533, G01N 33536

Patent

active

052139603

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 the difference between the two probes is that each is labeled with a different fluorochrome. 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 B cell markers, such as .gamma. chain and CD19, that are conjugated with different fluorochromes. Thus, the antigen-specific IgG-producing B cells of interest may be labeled with these unique reagents in three or four color FACS, which can sort the desired antigen-specific B cells at enhanced proportions. The differences in relative intensities between the antigen labels and the isotype labels (e.g., IgG labels) among the different antibodies of the single cells selected can be used to determine the relative antigen binding affinity of those antibodies. For example, the ratio of antigen label to IgG label can be calculated for each labeled B cell. The higher the ratio, the higher the relative affinity of the antibodies on the B cells for the antigen. 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.

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