The specific aims of this grant application are (1) to fractionate CF serum ultrafiltrates by HPLC (2) to isolate this serum factors(s) as close to homogeneity as possible and to characterize this material and (3) to use the enriched subfractions from CF serum as antigens to produce antibodies to CF factor(s). Our previous experimental data demonstrated a calcium dependence for both the ciliary dyskinesia response in tracheal explants and K+ efflux from submandibular fragments. We are devoting the majority of our time to isolation and characterization studies of low molecular weight materials from serum of CF patients. These studies include use of high performance liquid chromatography, gel permeation chromatography, SDS-PAGE, ultrafiltration and isoelectric focusing. Fractions generated by ultrafiltration are being subfractionated by HPLC producing reproducible profiles that demonstrate specific difference between CF serum on the one hand and control and asthmatic serum on the other. We are now testing these fractions for both K+ efflux activity and ciliary dyskinesia and will characterize the material found in the active fractions. Biologically active material, purified to homogeneity will be used as antigen to raise an antibody that is necessary to establish if this material is directly related to the molecular abnormality that is the basis of this genetic disease.
| Neri, M; Phillips, S F; Fich, A et al. (1991) Canine ileocolonic sphincter: flow, transit, and motility before and after sphincterotomy. Am J Physiol 260:G284-9 |
