Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1992-06-26
1993-10-26
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435962, 436536, 436546, 436172, G01N 2164, G01N 3358, G01N 33536
Patent
active
052565424
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 antigen-specific single B cells are to be used in a procedure which amplifies and selects their V.sub.H and V.sub.L sequences. 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 positive selection achieved using antigen probes with two different colors is preferably combined with a negative selection step, in which autofluorescent cells and sticky cells exhibiting fluorescence for the third irrelevant surface marker 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. 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.
REFERENCES:
patent: 4510244 (1985-04-01), Parks et al.
patent: 4727023 (1988-02-01), Wang et al.
patent: 4918000 (1990-04-01), Schubert
Ault, K. A. "Flow Cytometric Evaluation of Normal and Neoplastic B Cells" in Manual of Clinical Laboratory Immunology 3rd ed N. R. Rose ed 1986 American Society for Microbiology, Washington, D.C. pp. 247-253.
Kepplinger Esther L.
Mirabel Eric P.
Parsons Nancy J.
Tanox Biosystems, Inc.
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