Isolation of normal human IgG3. Identical molecular weight for normal and monoclonal gamma-3 chains

Abstract

Staphylococcal protein A has been shown to bind all IgG subclasses except IgG3 (Kronvall & Williams, 1969). This differential binding provides a method for the purification of normal IgG3, since chromatography of normal IgG on insolubilized protein A (e.g. protein A-Sepharose) should result in the elution of unbound IgG3 with neutral buffer. whereas IgGl. IgG2 and IgG4 molecules would be retained and would require a low pH for elution. This procedure was first used by Hjelm (1975) but his estimate of 56.000 for the mol. wt of normal gamma-3 chains was significantly lower than the previously reported mol. w’t of 59.50&60,950 for gamma-3 chains released by monoclonal IgG3 proteins (Saluk & Clem, 1971 ; Virella & Parkhouse, 1972). This discrepancy led us to attempt to repeat the purification of normal IgG3 and to determine the mol. wt of normal gamma-3 chains. Normal IgG was separated from the freshly collected serum of a normal donor by precipitation with ammonium sulfate at 33”)” saturation followed by ion-exchange chromatography in DE-52 (Whatman) using 0.01 A4 sodium phosphate buffer, pH 6.5. for the elution of IgG. This is a standard procedure in our laboratory, which has been found to yield IgG free of contamination with other proteins or immunoglobulins. The purified IgG was chromatographed in a protein A-Sepharose (Pharmacia) column measuring 0.9 x Scm. with a bed volume of 4.3 ml. Unbound protein was washed with 0.1 M sodium phosphate buffer, pH 7.0. to which 0.2 M epsilon-aminocaproic acid was added to protect IgG3 from proteolytic degradation (Virella & Yeh. 1977). Bound protein was eluted with 1 A4 acetic acid containing the same concentration of epsilon-aminocaproic acid. Fractions containing unbound and bound protein were pooled separately and concentrated by negative pressure ultrafiltration, with simultaneous dialysis against phosphate-buffered saline. The concentrated and neutralized pools were then characterized for their IgG subclass content by double immunodiffusion (Ouchterlony, 1962) in 17, (w/v) Noble agar (Difco) in 0.3 M potassium phosphate buffer. pH 8.0, using sheep antisera to IgG subclasses produced in the Department of Experimental Pathology, University of Birmingham, U.K. and absorbed in our laboratory (except for anti-IgGl, which was provided in absorbed form). The molecular weights of the heavy chains released by the bound and unbound fractions of normal human IgG