Semi-automatic analysis of proteins and protein complexes by automated enzyme immunoassay after separation by high-performance gel-permeation chromatography. Size distribution of C3-IgG complexes.
Enzyme immunoassay (ELISA) was applied in the establishment of an immunological method for the characterization of column effluents. Serum samples, containing aggregated immunoglobulin (IgG), were separated by high-performance gel-permeation chromatography on a TSK column and fractions were collected directly in 96-well ELISA microplates. IgG was determined on anti-IgG-coated plates, followed by development with biotin-labelled anti-IgG and enzyme-labelled avidin. Complement factor C3 was determined on anti-C3-coated plates, developed with biotin-labelled anti-C3 and enzyme-labelled avidin. Complexes between IgG and complement factor C3 were determined on anti-IgG-coated plates, developed with biotin labelled anti-C3 antibody and enzyme-labelled avidin. Complex formation between C3 and IgG after incubation of serum with aggregated IgG was demonstrated. The methodology described is generally applicable to the analysis of biological components and is uniquely useful for the analysis of complexes between different components.
D000818:Animals; D001322:Autoanalysis / Q000379:methods; D001710:Biotin; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003176:Complement C3 / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006868:Hydrolysis; D007074:Immunoglobulin G / Q000032:analysis*; D010434:Pepsin A; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D005719:gamma-Globulins
D003176:Complement C3; D007074:Immunoglobulin G; D011506:Proteins; D005719:gamma-Globulins; D001710:Biotin; D010434:Pepsin A