Kilpatrick J M, Virella G
Clin Exp Immunol. 1980 Jun;40(3):445-52.
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.
对糖尿病患者血清中可溶性胰岛素 - 抗胰岛素免疫复合物的存在情况进行了研究。通过向含有抗胰岛素抗体的患者血清中添加放射性碘化胰岛素来研究此类免疫复合物的形成；免疫复合物很容易形成，但显然患者之间存在差异。形成的免疫复合物用5%聚乙二醇沉淀。当通过凝胶过滤对沉淀物质进行分级分离时，它们在高分子量级分中洗脱，并且当将高分子量级分在蛋白A - 琼脂糖上进行亲和层析时，它们在中性pH下仍保持结合。当通过酸洗脱回收结合的免疫复合物并立即通过用相同酸缓冲液平衡的Sephadex G - 50柱过滤时，可回收游离抗原（放射性碘化胰岛素）和抗体。这种抗体在中和后，与放射性碘化胰岛素重新混合时显示出结合能力。当将该方案应用于在几种可溶性免疫复合物筛查方法中呈阳性结果的血清时，通过放射免疫测定在从5%聚乙二醇沉淀中分离出的高分子量级分以及蛋白A保留的级分中检测到胰岛素；然而，在Sephadex G - 50凝胶过滤后未检测到游离胰岛素，可能是由于过度稀释。高分子量级分确实对放射性碘化胰岛素具有结合能力。通过类似程序，从所有可溶性免疫复合物筛查试验均为阴性的血清中无法回收胰岛素结合蛋白。这些结果证明，在含有抗胰岛素抗体的血清中可轻易形成可溶性免疫复合物，并且可以从在可溶性免疫复合物筛查技术中呈阳性结果的糖尿病患者血清中回收。
