Isolation and characterization of soluble insulin-anti-insulin immune complexes formed in vitro and in vivo in sera from patients with diabetes mellitus
Kilpatrick, J.M.; Virella, G.
Clinical and Experimental Immunology 40(3): 445-452
1980
ISSN/ISBN: 0009-9104 PMID: 6998615 Document Number: 154369
The existence of soluble insulin-anti-insulin immune complexes in the serum of patients with diabetes mellitus was investigated. Formation of such immune complexes in vitro was studied by adding radioiodianted insulin to the sera of patients with anti-insulin antibodies [Ab]; immune complexes were formed readily, but apparently differed from patient to patient. Immune complexes formed in vitro were percipitated with 5% polyethylene glycol. They eluted in the high MW fractions when the precipitated material was fractionated by gel filtration and they remained bound at neutral pH when the high MW fractions were submitted to affinity chromatography on protein A-Sepharose. When the bound immune complexes were recovered by acid elution and immediately filtered through a Sephadex G-50 column equilibrated with the same acid buffer, free antigen (radiolabeled insulin) and Ab were recovered. This Ab, after neutralization, showed binding capacity when remixed with radioiodinated insulin. When this protocol was applied to a serum that gave positive results in several screening methods for soluble immune complexes, insulin was detected by radioimmunoassay in the high MW fractions separated from the 5% polyethylene glycol precipitate and in the fractions retained by protein A; no free insulin was detected after gel filtration in Sephadex G-50, perhaps due to excessive dilution. The high MW fraction had binding capacity for radioiodinated insulin. No insulin-binding protein could be recovered with a similar procedure from a serum negative by all screening tests for soluble immune complexes. Soluble immune complexes can be formed easily in sera containing anti-insulin Ab and can be recovered from sera of diabetic patients that show positive results in screening techniques for soluble immune complexes.