B型钠尿肽固相时间分辨免疫荧光分析法的建立及其临床应用

Development of solid-phase time-resolved fluoroimmunoassay of B-type natriuretic peptide and its clinical application

目的采用一种新的铕螯合物BHHCT,制备高灵敏度荧光标记物,建立B型钠尿肽(BNP)固相时间分辨荧光免疫分析法(TrFIA).方法使用2株匹配的单克隆抗体,其中1株可以识别BNP分子C-端区,另1株生物素化单抗能识别分子内环结构,分别作为捕获抗体和检测抗体,并利用制备的SA-BHHCT-Eu3+作为示踪物,建立双抗体夹心法,检测正常对照组和心功能Ⅰ～Ⅳ级心力衰竭患者组血液BNP含量.结果本法测定BNP的灵敏度为12 pg/ml,批内和批间CV分别为3.9%和5.2%,平均回收率为97.3%,线性范围为20～4 000 pg/ml.对照组(n=110)、心功能Ⅰ级(n=31)、Ⅱ级(n=30)、Ⅲ～Ⅳ级(n=25)各组血浆BNP浓度分别为(69±37)、(84±46)、(346±134)和(932±326)pg/ml;心衰各组BNP水平显著高于对照组(P＜0.05或P＜0.001).以BNP 100pg/ml为cut off值,诊断心力衰竭(HF)的特异性为82.4%,敏感性为91.6%.结论应用铕螯合物BHHCT建立的固相TrFIA法测定BNP,是一个很好的诊断心衰和监测其严重程度的实验诊断指标.

Objeelive To develop a solid-phase time-resolved fluoroimmunoassay （TRFIA） with a new europium fluorescent chelate BHHCT for highly sensitive detection of B-type natriuretic peptide（BNP）. Methods Two coupled monoclonal antibodies （MoAbs） to BNP32 were used,a two-side “sandwich” immunoassay which applied SA-BHHCT-Eu^3 ± as the marking substance to detect plasma BNP levels of healthy people（ n = 110 ） and heart failure （ HF ） patients （ n = 86 ）. Results The lowest detection level （ mean 3s ） was 12 pg/ml. The CVs of inter-and intra-assay were 3.9% and 5.2% , respectively. The mean recovery rate was 97.3%. The linear range of the method for BNP detection was 20 to 4000 pg/ml. The mean levels of plasma BNP concentration in healthy controls, HF patients （ with NYHA classification） class Ⅰ （ n = 31 ）, class Ⅱ （ n = 30） and class Ⅲ - Ⅳ （ n = 25 ） were 69 ± 37 pg/ml, 84 ± 46 pg/ml, 336 -± 144 pg/m1,912 ± 379 pg/ml ,respectively （ P 〈0.05 or P 〈0. 001 ）. BNP levels in the HF groups were significantly higher than that in healthy controls. Its sensitivity and specificity for the diagnosis of HF were 91% and 82.4% with a cut-off value of 100 pg/ml. Conclusions Our solid-phase TRIFA is a good method which can be used to detect the plasma BNP level of HF patients. Increased BNP indicates increment of the severity of HF according to the NYHA classification.