Isolation and characterization of soluble insulin-anti-insulin immune complexes formed in vitro and in vivo in sera from patients with diabetes mellitus.

The existence of soluble insulin–anti-insulin immune complexes in the serum of patients with diabetes mellitus was investigated. Formation of such immune complexes was studied by adding radioiodinated insulin to the sera of patients with anti-insulin antibodies; immune complexes were formed readily, but apparently differed from patient to patient. Immune complexes formed were precipitated with 5% polyethylene glycol. They eluted in the high-molecular weight fractions when the precipitated material was fractionated by gel filtration, and they remained bound at neutral pH when the high-molecular weight 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 (radiolabelled insulin) and antibody were recovered. This antibody, 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-molecular weight fractions separated from the 5% polyethylene glycol precipitate and in the fractions retained by protein A; however, no free insulin was detected after gel filtration in Sephadex G-50, perhaps due to excessive dilution. The high-molecular weight fraction did have 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. These results prove that soluble immune complexes can be formed easily in sera containing anti-insulin antibodies and can be recovered from sera of diabetic patients that show positive results in screening techniques for soluble immune complexes.