胰岛素自身抗体微量平板放射免疫法的建立与应用

目的建立胰岛素自身抗体（IAA）的微量平板放射免疫（RIA）法，并探讨其临床应用价值。方法^125I-胰岛素与血清在微量平板4℃保温72h后，免疫复合物转移至已包被蛋白A的Millipore过滤平板中并洗涤，于多功能液体闪烁发光分析仪上测计数。以IAA指数≥0．06作为阳性标准，通过参加第四次国际糖尿病自身抗体检测标准化评估（DASP 2005），评价方法的灵敏度与特异性。并检测71例1型糖尿病（T1DM）患者、551例初诊2型糖尿病（T2DM）患者以及317名健康对照者IAA水平。采用SPSS 11．5软件进行t检验、非参数检验χ^2检验和直线相关分析；一致率用Kappa值评估。结果（1）优化的检测条件为5μl血清与2×10^4计数·min^-1的^125I-胰岛素于4℃缓慢振摇保温72h。（2）该方法批内CV4．8％～8．9％，批间CV6．4％～10．5％，DASP 2005反馈结果显示该法诊断T1DM灵敏度50％（25／50），特异性97％（97／100）。与国产IAA RIA试剂盒进行比较，结果判定总体一致率为72．9％（Kappa值0．402），相关系数（r）为0．678（P〈0．001）；26例结果不相符的标本中25例为该法检测阳性而国产RIA试剂盒检测阴性。（3）该方法检测T1DM患者的阳性率为19．7％（16／71），显著高于健康对照组的0．9％（3／317，χ^2=54．36，P〈0．001），其中0～9岁T1DM组9例中IAA阳性5例。检测初诊T2DM患者IAA阳性率1．5％，与健康对照组差异无统计学意义（χ^2=0．95，P〉0．05）。结论IAA微量平板RIA法灵敏度与特异性好，可应用于临床，IAA在婴幼儿T1DM患者中阳性率较高。

A novel microtiter plate radioimmunoassay of insulin autoantibody

Objective Insulin autoantibody (IAA) is known to exist in sera of type 1 diabetes mellitus (T1DM) patients and pre-T1DM individuals. The aim of this study was to establish a novel microtiter plate radioimmunoassay (RIA) for IAA and evaluate its clinical value. Methods Diluted 125Ⅰ-insulin was mixed with 5 ul serum samples in a 96-well microtiter plate and then incubated for 72 h on an orbital plate shaker (4℃). The immunocomplexes were transferred to another protein a coated Millipore plate, and then the plate was washed with Tri-Buffered Saline Tween-20 (TBT) buffer. Counts per minute (CPM) was measured with liquid scintillation and luminescence counter. The positive cut-off point of IAA index was defined as ≥0.06 based on the 99-percentile of the distribution in 317 healthy individuals. The specificity and sensitivity of the assay were calculated from the samples provided by the fourth Diabetes Autoantibodies Standardization Program (DASP 2005). The IAA levels were determined in 71 T1 DM and 551 newly diagnosed type 2 diabetes (T2DM) patients, and 317 healthy controls. The t test, non-parametric test, x2 test and linear correlation analysis were performed on the data using SPSS 11.5 software. The concordance rate was estimated with Kappa value. Results (1) The optimized testing condition was described as 2×104 CPM of 125Ⅰ-insulin, 5 ul serum sample and slowly horizontal shaking for 72 h. (2) The intra-assay CV was 4.8%-8.9% and inter-assay CV was 6.4%-10.5%. Based on DASP 2005 samples, the specificity and sensitivity of the assay were 97% (97/100) and 50% (25/50), respectively. Ninety-six serum samples with different IAA levels were selected and tested to compare between our new method and a domestic IAA RIA kit. The results showed that the IAA indices from the two methods were positively correlated (r= 0.678, P<0.001). The concordance rate was 72.9 %(Kappa value=0.402). There were 25 samples with discordant results, which were positive for IAA titer using the corresponding microtiter plate RIA but negative using the novel RIA kit. (3) In TIDM group the positive rate of IAA was 19.7% (16/71), higher than the healthy controls (0.9%, x2=54.36, P<0.001). The subgroup of T1DM children (with 0-9 years) showed the highest IAA positive rate (55.6% ,x2=4.85, P<0.05). In T2DM group the frequency of IAA was 1.5% (8/551), which had no significant difference comparing with that of healthy controls (x2= 0.95, P >0.05). Conclusions Our proposed microtiter plate RIA method for IAA is highly sensitive and specific, likely to be feasible for clinical application. The frequency of IAA is high in children with T1DM.