Ultrasensitive detection of pepsinogen I and pepsinogen II by a time-resolved fluoroimmunoassay and its preliminary clinical applications
Ultrasensitive detection of pepsinogen I and pepsinogen II by a time-resolved fluoroimmunoassay and its preliminary clinical applications
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DOI：
10.1016/j.aca.2006.04.053
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发表时间：
2006-06-30
影响因子：
6.2
通讯作者：
Jin, J
中科院分区：
文献类型：
--
作者：
Huang, B;Xiao, HL;Jin, J
建立了一种快速、高灵敏度的胃蛋白酶原I （PG 1）和胃蛋白酶原H （PG H）的时间分辨荧光免疫法（TRFIA）检测方法，用于检测胃肠道疾病患者血清中PG I和PG 11的含量。在非竞争性实验中，一个单克隆抗体（McAb）包被在孔上，直接针对PG I或PG II上的特定抗原位点。该单克隆抗体被称为标记单克隆抗体，由N-（对异硫氰酸酯）-二乙烯三胺- nnnn -四乙酸的铕螯合物制备，并针对PG I或PG H分子上的不同抗原位点。水洗结合/自由分离后，测定结合的Eu3+-McAb的荧光计数。采用autoDELFIA（1235）系统，采用PG I和PG II TRFIA检测患者或健康志愿者血清中的PG水平。PG I-TRFIA的测定范围为3.5 ~ 328.0 μ g -1， PG II-TRFIA的测定范围为2.0 ~ 55.0 μ g -1。PG I-TRFIA的组内和组间cv分别为1.9%和4.7%，PG II-TRFIA的组内和组间cv分别为2.1%和3.8%。PG I-TRFIA和PG II-TRFIA的回收率分别为102.7%和104.6%。PG I和PG II的检出限分别为0.05 μ g L-1和0.02 μ g L-1。稀释实验表明，PG I-TRFIA的期望值为93.2 ~ 102.3%，PG H-TRFIA的期望值为97.3 ~ 110.6%。PG I和PG II之间的交叉反应速率可以忽略不计。放射性同位素测定法（RIA）与TRFIA测定的相关系数分别为0.926和0.959。铕标记的单克隆抗体在-20℃下至少稳定一年，相同试剂的TRFIA结果在一年内也可重复。1600名健康志愿者血清PG I平均值为162.4 +/- 52.1 μ g -1， PG II平均值为11.7 +/- 6.8 μ g -1， PG I/PG II比值平均值为13.8 +/- 7.4。健康志愿者血清PG I水平正常范围为58.2 ~ 266.6 μ g -1， PG II水平低于25.3 μ g -1。一种高度敏感、可靠、方便的PG- trfia定量PG的方法的可用性，将允许研究该分析在各种临床情况下的可能诊断价值，包括胃癌、十二指肠溃疡、胃溃疡和胃炎。该方法的灵敏度和重现性较好，适合临床应用。(c) 2006 Elsevier B.V.版权所有
A fast and highly sensitive assay for pepsinogen I (PG 1) and pepsinogen H (PG H) by using time-resolved fluoroimmunoassay (TRFIA) detection technique has been developed for the determination of serum PG I and PG 11 against gastrointestinal diseases. On the noncompetitive assay, one monoclonal antibody (McAb) coated on wells was directed against a specific antigenic site on the PG I or PG II. The McAb, called as labelling McAb, was prepared with the europium-chelate of N-(p-isothiocyanatobenzyl)-diethylenetriamine-NNNN-tetraacetic acid and directed against a different antigenic site on the PG I or PG H molecule. After bound/free separation by washing, the fluorescence counts of bound Eu3+-McAb were measured. The levels of PG in sera from patients or healthy volunteers were determined by PG I and PG II TRFIA using the autoDELFIA(1235) system. The measurement ranges of PG I-TRFIA were 3.5-328.0 mu g L-1 and those of PG II-TRFIA were 2.0-55.0 mu g L-1. The within-run and between-run CVs of the PG I-TRFIA were 1.9% and 4.7%, respectively, and those of PG II-TRFIA were 2.1% and 3.8%, respectively. The recovery rates of PG I-TRFIA and PG II-TRFIA were 102.7% and 104.6%, respectively. The detection limitations of PG I and PG II were 0.05 mu g L-1 and 0.02 mu g L-1, respectively. The dilution experiments showed the percentage of expected value by PG I-TRFIA was 93.2-102.3% and of PG H-TRFIA was 97.3-110.6%. The cross-reacting rate between PG I and PG II was negligible. The linear correlation of radioinununoassay (RIA) and TRFIA measurements resulted in a correlation coefficient as 0.926 of PG I and as 0.959 of PG II. The europium-labelling McAbs were stable for at least one year at -20 degrees C, and the results of the TRFIA with same reagents were reproducible over one year as well. The means of 1600 healthy volunteers were 162.4 +/- 52.1 mu g L-1 for serum PG I, 11.7 +/- 6.8 mu g L-1 for serum PG II, and 13.8 +/- 7.4 for the PG I/PG II ratio. The normal ranges of Serum PG I levels for healthy volunteers were 58.2-266.6 mu g L-1, and those of serum PG II levels were less than 25.3 mu g L-1. The availability of a highly sensitive, reliable, and convenient PG-TRFIA method for quantifying PG will allow investigations into the possible diagnostic value of this analysis in various clinical conditions, including gastric carcinoma, duodenal ulcer, gastric ulcer and gastritis. The sensitivity and reproducibility of the assay were satisfactory for clinical applications. (c) 2006 Elsevier B.V. All rights reserved.