The human eosinophil Charcot-Leyden crystal protein: biochemical characteristics and measurement by radioimmunoassay.

Abstract

A double-antibody radioimmunoassay (RIA) was developed to measure the protein comprising the human eosinophil Charcot-Leyden crystal (CLC). Critical requirements in the development of this assay were the production of large quantities of pure CLC, the solubilization of CLC at neutral pH, and an assessment of the purity of the CLC protein. CLC were prepared from eosinophil-rich leukocyte suspensions by homogenization of cells in hypotonic-buffered saline. Centrifuged eosinophil extracts formed CLC spontaneously at 4°C, but the yield of crystals was significantly augmented by slow freezing of extracts to −20°C and thawing. Lyophilization of crystals was required to obtain material soluble at neutral pH. Soluble CLC protein produced 3 dense bands on polyacrylamide gel electrophoresis (PAGE) at pH 8.9, and these bands gave reactions of identity in immunodiffusion analyses with rabbit anti-CLC antibody. PAGE in 1% sodium dodecyl sulfate revealed a single protein band of 13,000 daltons. Isoelectric focusing of soluble CLC protein yielded 2 major and 4 minor bands with pI’s between 5.7 and 5.1, indicating the slightly acidic nature of the CLC protein. In the RIA, CLC levels were measured by inhibition of the binding of 131I-CLC reacted with specific rabbit anti-CLC antibody. Burro anti-rabbit IgG was used as the second antibody to precipitate immune complexes. The maximum precipitability of 1 ng 131I-CLC by antibody was 97%; the RIA was sensitive to 0.25 ng. CLC protein is immunologically distinct from human eosinophil major basic protein as determined by RIA and Ouchterlony analysis. Serum levels of CLC for 10 normal healthy subjects averaged 37 ± 4.7 ng/ml compared with 8507 ± 2494 ng/ml for 10 patients with diseases associated with a peripheral blood eosinophilia greater than 30%. The RIA for CLC protein provides us with a tool to investigate the biological function(s) of this protein and the role of eosinophils in certain disease states.