Correlation of zeta-globin ELISA with PCR for (--SEA) deletion and clinical diagnosis for 1 alpha-thal-1 trait.
Correlation of zeta-globin ELISA with PCR for (--SEA) deletion and clinical diagnosis for 1 alpha-thal-1 trait.
复制标题
zeta-珠蛋白 ELISA 与 PCR 的 (--SEA) 缺失和 1 α-thal-1 性状临床诊断的相关性。
DOI：
10.1111/j.1749-6632.1998.tb10512.x
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发表时间：
1998
影响因子：
5.2
通讯作者：
Daoust,PR
中科院分区：
文献类型：
--
作者：
Simkins,RA;Than,KA;Schapiro,B;Choi,ES;Daoust,PR
材料和方法样本是300个连续的血液标本送到医院的临床实验室进行血红蛋白（Hb）电泳，其中62%的人口是东南亚（SEA）。另外检查了9份样本，其人口统计学数据未知。对全血样本进行初步诊断和PCR。对点样于Schleicher & Schuell 903滤纸上的全血（75 μl）进行ELISA。在原始样本中，有18份无法进行PCR分析。在环境温度下，通过在含有0.15 M NaCl（TS）的150 μl 50 mM Tris-HCl缓冲液（pH 7.5）中从干燥血点中提取1/8”穿孔物（约3 ml）进行ELISA。在环境温度下放置30分钟后，将浸提液在相同缓冲液中以1：5稀释，并使其与Costar微量滴定板结合30分钟。用含有0.05%吐温20的TS洗涤三次后，将在含有1%BSA的TS中稀释的生物素化抗体加入到每个孔中，并使其反应30分钟。再洗涤三次后，向每个孔中加入适当稀释的辣根缀合的链霉亲和素，反应15分钟，洗涤三次，然后用含有50 mM ABTS试剂的底物向每个孔中加入（2，2′-连氮基-双（3-乙基苯并噻唑啉-6-磺酸）二铵盐加0.03%H2O2的柠檬酸盐缓冲液（pH 4.2），显影15 - 30分钟，用0.1%SDS终止反应。在酶标仪中在405 nm处读取平板。
MATERIALS AND METHODSSamples were 300 consecutive blood specimens sent to the hospital's clinical laboratory for hemoglobin (Hb) electrophoresis from a population of which 62% were Southeast Asian (SEA). Nine additional specimens were examined for which demographic data were not known. The initial diagnosis and PCR were performed on whole blood samples. The ELISA was performed on whole blood spotted (75 μl) onto Schleicher & Schuell 903 filter paper. Of the original samples, 18 were not available for PCR analysis. The ELISA was performed at ambient temperature, by extracting a 1/8” punch (∼ 3 ml) from the dried blood spots in 150 μl of 50 mM Tris-HCl buffer, pH 7.5 containing 0.15 M NaCl (TS). After 30 minutes at ambient temperature, the extract was diluted 1: 5 in the same buffer and allowed to bind to a Costar mictotiter plate for 30 minutes. After three washes with TS containing 0.05% Tween 20, biotinylated antibody diluted in TS containing 1% BSA was added to each well and allowed to react for 30 minutes. After three more washes an appropriate dilution of horseradish-conjugated streptavidin was added to each well, reacted for 15 minutes, and washed three times before substrate containing 50 mM ABTS reagent (2, 2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) diammonium salt plus 0.03% H 2 O 2 in citrate buffer pH 4.2 was added to each well, allowed to develop 15 to 30 minutes, and the reaction stopped with 0.1% SDS. The plates were read at 405 nm in a plate reader.