Kohno T, Hashida S, Ishikawa E
J Biochem. 1985 Aug;98(2):379-84. doi: 10.1093/oxfordjournals.jbchem.a135292.
An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20 degrees C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37 degrees C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to beta-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem. 84, 93-102).
通过降低正常豚鼠IgG的非特异性结合并增强抗胰岛素IgG的特异性结合，提高了豚鼠血清中抗胰岛素IgG的酶免疫测定的灵敏度。用正常兔IgG包被硅橡胶片或聚苯乙烯球，然后用戊二醛偶联胰岛素。将包被有胰岛素-正常兔IgG的硅橡胶片或聚苯乙烯球与正常兔IgG孵育，然后在较低温度（20℃）下在正常兔IgG存在的情况下与稀释的豚鼠抗胰岛素血清孵育。最后，将固相与兔（抗豚鼠IgG）Fab'-辣根过氧化物酶缀合物孵育，以测量结合的豚鼠IgG的量。与根据先前报道（加藤，K.等人（1978年）《生物化学杂志》84卷，93 - 102页）通过在37℃下将包被有胰岛素-牛血清白蛋白的固相与稀释的豚鼠抗胰岛素血清孵育，然后与缀合有来自大肠杆菌的β-D-半乳糖苷酶的兔（抗豚鼠IgG）Fab'进行酶免疫测定相比，豚鼠血清中抗胰岛素IgG的检测限提高了10至100倍。
