Kohno T, Ishikawa E
J Biochem. 1986 Nov;100(5):1247-51. doi: 10.1093/oxfordjournals.jbchem.a121830.
A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23 M HCl and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7 pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10,000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.
本文描述了一种用于检测豚鼠血清中抗胰岛素抗体的高灵敏度酶免疫测定法。将豚鼠抗胰岛素血清用非特异性豚鼠血清进行不同程度的稀释，然后与胰岛素孵育。孵育后，通过用葡聚糖 - 活性炭处理，将游离胰岛素与胰岛素 - 抗胰岛素抗体复合物分离。复合物中的抗胰岛素抗体通过与0.23 M HCl孵育而与胰岛素解离并失活。使用抗胰岛素IgG包被的聚苯乙烯球和亲和纯化的抗胰岛素Fab'-辣根过氧化物酶缀合物，通过夹心酶免疫测定法测量解离的胰岛素量。豚鼠血清中抗胰岛素抗体的检测限为6.7 pg/测定或150 ng/升血清。目前的酶免疫测定法比先前描述的酶免疫测定法灵敏10000倍，在先前的方法中，胰岛素包被的聚苯乙烯球与稀释的豚鼠抗胰岛素血清孵育，随后与兔（抗豚鼠IgG）Fab'-辣根过氧化物酶缀合物孵育。
