Estimation of Aggregates of IgG and Their Effect on Quantitation of IgG

Abstract

Four quantitating techniques - radial immunodiffusion (RID),electroimmunoassay (EIA), thin-layer Sephadex gel filtration (GF), and GF with an added, specific immunoprecipitation step, immuno-gel filtration (IGF) - are compared for their accuracy in quantitating IgG of various molecular sizes - that is, pure 7S IgG and aggregated IgG, from large polymers to dimeric 10S molecular size. The aggregate fractions differed and were stable with regard to their migration in thin-layer gel filtration, their electromobility in agar and agarose, and their diffusibility in agarose. All four methods quantitated the largest aggregates four to five-fold lower and the next largest at no better than half the Lowry ratings. Only GF and IGF quantitated the two fractions of smaller aggregates within 10% of the expected value. The presence of about 50% of the two larger aggregates in prepared mixes with 7S IgG reduced quantitation results by about half to two thirds with all four methods; the smaller aggregates at 50% concentration were estimated within 16% of the correct values. Twenty percent aggragated IgG about the mean amount found in commercial gamma-globulin preparations, influenced measurments with RID, GF and IGF by less than 10% and the results with EIA by less than 16%.