Abstract
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 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 in vitro 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.
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Chenais F., Virella G., Patrick C. C., Fudenberg H. H. Isolation of soluble immune complexes by affinity chromatography using staphylococcal protein A--Sepharose as substrate. J Immunol Methods. 1977;18(1-2):183–192. doi: 10.1016/0022-1759(77)90169-7. [DOI] [PubMed] [Google Scholar]
- Deaton C. D., Maxwell K. W., Smith R. S., Creveling R. L. Use of laser nephelometry in the measurement of serum proteins. Clin Chem. 1976 Sep;22(9):1465–1471. [PubMed] [Google Scholar]
- Digeon M., Laver M., Riza J., Bach J. F. Detection of circulating immune complexes in human sera by simplified assays with polyethylene glycol. J Immunol Methods. 1977;16(2):165–183. doi: 10.1016/0022-1759(77)90051-5. [DOI] [PubMed] [Google Scholar]
- Gerbitz K. D., Kemmler W. Method for rapid quantitation and characterization of insulin antibodies. Clin Chem. 1978 Jun;24(6):890–894. [PubMed] [Google Scholar]
- Herbert V., Lau K. S., Gottlieb C. W., Bleicher S. J. Coated charcoal immunoassay of insulin. J Clin Endocrinol Metab. 1965 Oct;25(10):1375–1384. doi: 10.1210/jcem-25-10-1375. [DOI] [PubMed] [Google Scholar]
- Irvine W. J., Al-Khateeb S. F., Di Mario U., Feek C. M., Gray R. S., Edmond B., Duncan L. J. Soluble immune complexes in the sera of newly diagnosed insulin-dependent diabetics and in treated diabetics. Clin Exp Immunol. 1977 Oct;30(1):16–21. [PMC free article] [PubMed] [Google Scholar]
- Jayarao K. S., Faulk W. P., Karam J. H., Grodsky G. M., Forsham P. H. Measurement of immune complexes in insulin-treated diabetics. J Immunol Methods. 1973 Dec;3(4):337–345. doi: 10.1016/0022-1759(73)90035-5. [DOI] [PubMed] [Google Scholar]
- Virella G., Hipp W. A., John J. F., Jr, Kahaleh B., Ford M., Fudenberg H. H. Nephelometric detection of soluble immune complexes: methodology and clinical applications. Int Arch Allergy Appl Immunol. 1979;58(4):402–410. doi: 10.1159/000232219. [DOI] [PubMed] [Google Scholar]