Immune complex formation analysed by high-performance size exclusion chromatography (HPLC-SEC) using either 125I-labelled antigen or enzyme-linked immunosorbent assay (ELISA) for detection.
A physical/immunochemical method has been developed for the analysis of soluble immune complexes. Human serum with high amount of antibody against bovine serum albumin (BSA) was incubated at 37 degrees with a wide range of 125I-labelled BSA and separated according to size by high-performance size exclusion chromatography (HPLC-SEC) on a TSK G 6000 PW column. Fractions were collected onto microplates and analysed either by gamma counting or by an enzyme-linked immunosorbent assay (ELISA) technique detecting anti-BSA antibody. The size of the immune complexes were the same when analysed by the two methods. The heaviest immune complexes were seen in moderate antibody excess where the majority of the complexes were eluted in a broad peak around Mr of 7 X 10(6). In large antibody excess the immune complexes eluted at around 3 X 10(6). Around equilibrium two peaks were seen corresponding to 3 X 10(6) and 5 X 10(5). In antigen excess the peak at 3 X 10(6) is diminished and free antigen appears. No changes in the distribution of the complexes were observed when the serum was made 20 mM in EDTA before incubation with BSA.
D000328:Adult; D000936:Antigen-Antibody Complex / Q000032:analysis*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004492:Edetic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008297:Male; D012710:Serum Albumin, Bovine / Q000276:immunology; D012711:Serum Albumin, Radio-Iodinated / Q000276:immunology
D000936:Antigen-Antibody Complex; D012711:Serum Albumin, Radio-Iodinated; D012710:Serum Albumin, Bovine; D004492:Edetic Acid