DIFFERENTIAL ELUTION OF CLQ, CLR AND CLS FROM HUMAN CL BOUND TO IMMUNE AGGREGATES - USE IN THE RAPID PURIFICATION OF CL SUB-COMPONENTS

IgG-ovalbumin aggregates were used to bind and activate Cl from human serum. The resulting Cl̄ bound to the insoluble support provided a convenient tool for studying the release of Cl̄ sub-components under various conditions of pH and ionic strength. In the presence of calcium, Cl̄r and Cl̄s were removed in parallel under all the conditions employed, with a minimum release at pH 7.0 and at low ionic strength. The elution behaviour of Clq was distinct from that of the Cl̄r-Cl̄s pair. The experimental conditions used demonstrated the presence of two separate entities in Cl̄: firstly, Clq and secondly, Cl̄r plus Cl̄s. Cl̄ bound to IgG-ovalbumin was employed for a rapid purification of Clq, Cl̄r and Cl̄s sub-components. Cl̄r plus Cl̄s were first selectively released with EDTA † † DFP, di-isopropyl phosphorofluoridate; EDTA, ethylene diamine tetracetic acid; SDS, sodium dodecyl sulphate; STI. soybean trypsin inhibitor; TAME, p-toluene sulfonyl-l-arginine methyl ester; RSC, relative salt concentration (the RSC of a buffer is equivalent to the molarity of a solution of NaCl giving the same conductivity as the buffer at 0 C).. Cl̄r was separated from Cl̄s by DEAE-cellulose chromatography, and Cl̄s was further purified on anti-Cl̄r IgG-Sepharose 6B in order to remove contaminant Cl̄r. Clq still bound to IgG-ovalbumin aggregates was removed at pH 10.0 in the presence of 0.7 M NaCl and further purified by CM-cellulose chromatography. Clq, Clr and Cl̄s were obtained in good yield and in pure form, as judged by immunodiffusion analysis and SDS-polyacrylamide gel electrophoresis. © 1979.