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Despite increasing evidence that basophils can infiltrate into inflamed
tissues during allergic reactions, determination of the extent of infiltration
and elucidation of their role in allergic disease has been frustrated
by the lack of reliable means for detecting this cell type in tissues.
In the present study, we report on a new monoclonal antibody specific
for basophils and on the initial characterization of the antigen it recognizes.
Basophils were isolated from peripheral blood by Percoll density gradient
centrifugation and a positive-selection immunomagnetic procedure and injected
into mice to produce monoclonal antibodies. A hybridoma clone, designated
BB1, secreted antibody of the IgG2a isotype; this antibody bound selectively
to basophils on immunocytochemistry but did not react with any other cell
type or tissue structure, although it did stain a proportion of cells
from the basophilic cell line KU812F. In sections of mixed populations
of peripheral blood cells, similar numbers of cells stained with Alcian
blue dye and BB1 over a wide range of basophil purity. BB1 antibody was
effective in identifying basophils in sections of mixed cells or in tissues
after fixation with ethanol, Carnoy's solution, or formalin. Staining
of basophils with BB1 gave a granular appearance, although flow cytometry
indicated that some antigen was also present on the surface of the cell.
Activation of these cells with anti-IgE antibody or with the calcium ionophore
A23187 provoked release of the antigen in parallel with that of histamine.
BB1 antibody did not, by itself, stimulate histamine release. The molecular
mass of the antigen was determined on Hedrick-Smith gels to be 124+/-
11 kd. This new monoclonal antibody will be a valuable experimental tool
in future studies, allowing the reliable detection of basophils in tissues
of patients with allergic and chronic inflammatory disease; in addition,
the antigen it identifies has potential as a unique marker of basophil
activation. (Lab Invest 1999, 79:27-38).
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