A method for preparing IgG F(ab')2 fragments using small amounts of serum.
An alternative method devised to isolate functionally active antibody F(ab')2 fragments required fewer manipulations and used less serum than do methods generally used. The method involved pepsin digestion of the whole globulin fraction precipitated from as little as 3 ml of serum. Chromatographic separation of the digest on Sephadex G-150 yielded two distinct peaks: Peak I consisted of lipoprotein and showed no immunoglobulin determinants; Peak II, as established by immunodiffusion analysis, contained immunoglobulin F(ab')2 fragments. Radioimmunoassays performed on Peak II protein to determine the presence of allotypic markers revealed less than 10% contamination by non-immunoglobulin protein; coprecipitation tests, used to characterize Peak II further, also showed less than 10% contamination by non-immunoglobulin protein. Recovery of total serum IgG F(ab')2 was 90% or greater using this technique, compared with a potential 20-25% recovery using standard isolation procedures.
D000818:Animals; D002850:Chromatography, Gel; D005779:Immunodiffusion; D007126:Immunoglobulin Allotypes; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification*; D007074:Immunoglobulin G / Q000032:analysis; D011817:Rabbits
D007126:Immunoglobulin Allotypes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G