Further purification and characterization of serum proteins used to detect cystic fibrosis genotypes by isoelectric focusing.

Sera from cystic fibrosis (CF) homozygotes and obligate heterozygotes contain a CF factor (gamma CF factor) not found by isoelectric focusing in thin-layer polyacrylamide gels in most normal control sera. In addition, sera from most obligate heterozygotes lack another protein (bland B, C, or D) that is commonly found in sera from most normal and cystic fibrosis individuals. A standardized, biophysical assay is described that employs isoelectric focusing for the detection of both CF homozygotes and heterozygotes based on the analysis of whole serum for the presence of the gamma CF factor and bands B, C, and D. Results of analyzing sera from selected CF patients by isoelectric focusing indicated that there is a general correlation between the amount of the gamma CF factor and the clinical severity of the disease. Partial purification and characterization of the gamma CF factor and protein bands B, C, and D was accomplished by using DEAE-cellulose chromatography, Sephadex G-200 gel filtration, sequential molecular filtration through a series of Amicon Diaflo ultrafiltration membranes, affinity chromatography, and cellulose acetate electrophoresis. The gamma CF factor is a cationic protein with a pI of 8.46+/-0.05, has gamma electrophoretic mobility, a molecular weight between 3,500 and 10,000, and apparently exists in CF serum in 2 forms (free in solution and complexed to IgG). Bands B, C, and D are cationic proteins with pI values of 7.85 to 8.10, have gamma electrophoretic mobility and a molecular weight of approximately 100,000-150,000.