用铕螯合物标记的链霉亲和素-聚乙烯胺缀合物：在免疫测定、免疫组织化学和微阵列中的应用。

Streptavidin-polyvinylamine conjugates labeled with a europium chelate: applications in immunoassay, immunohistochemistry, and microarrays.

作者信息

Scorilas A, Bjartell A, Lilja H, Moller C, Diamandis E P

机构信息

Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.

出版信息

Clin Chem. 2000 Sep;46(9):1450-5.

PMID:10973889

Abstract

BACKGROUND

The favorable properties of lanthanide chelates compared with conventional fluorescent probes have attracted considerable interest. A Eu(3+) chelator, 4,7-bis(chlorosulfophenyl)-1, 10-phenanthroline-2,9-dicarboxylic acid (BCPDA), has been synthesized previously.

METHODS

We here describe immunoassay, immunohistochemistry, and microarray applications of a new streptavidin-based universal polyvinylamine (PVA) detection reagent that is multiply labeled with the europium chelate of BCPDA. Solid-phase time-resolved immunofluorometric assays for biotinylated mouse IgG and prostate-specific antigen (PSA) were developed using the new conjugate as a detection reagent. The new conjugate was also used for the immunohistochemical localization of PSA expression in paraffin-embedded prostatic tissues. A model microarray with spotted biotinylated antibody as target was also performed.

RESULTS

Approximately 50-100 BCPDA moieties were covalently bound to PVA, which was then linked to streptavidin via biotin interaction. The macromolecular complex successfully recognized and bound biotinylated detection reagents, e.g., antibodies. The new reagent enabled measurement of solid phase-immobilized biotinylated mouse IgG with a detection limit of approximately 1 pg/assay and demonstrated excellent linearity. In an ELISA-type sandwich PSA assay that included two PSA monoclonal antibodies using the new conjugate as detection reagent, we detected 0.001 microg/L PSA ( approximately 100 fg or approximately 3 amol/assay). Serum samples analyzed for PSA by this method and a commercial assay gave highly correlated results. The new reagent enabled excellent immunohistochemical localization of PSA expression in prostate tissues. Using the new reagent in a model microarray experiment with biotinylated mouse IgG as target, we demonstrated excellent spatial resolution of 5- to 10-nL microspots.

CONCLUSIONS

The new detection reagent may find important applications in biotechnology.

摘要

背景

与传统荧光探针相比，镧系螯合物的优良特性引起了广泛关注。一种铕（3+）螯合剂，4,7-双（氯磺苯基）-1,10-菲咯啉-2,9-二羧酸（BCPDA），此前已被合成。

方法

我们在此描述一种基于链霉亲和素的新型通用聚乙烯胺（PVA）检测试剂的免疫测定、免疫组织化学和微阵列应用，该试剂用BCPDA的铕螯合物进行了多重标记。使用这种新的偶联物作为检测试剂，开发了用于生物素化小鼠IgG和前列腺特异性抗原（PSA）的固相时间分辨免疫荧光测定法。这种新的偶联物还用于石蜡包埋前列腺组织中PSA表达的免疫组织化学定位。还进行了以点样生物素化抗体为靶标的模型微阵列实验。

结果

约50 - 100个BCPDA部分共价结合到PVA上，然后通过生物素相互作用与链霉亲和素相连。这种大分子复合物成功识别并结合了生物素化的检测试剂，如抗体。这种新试剂能够检测固相固定的生物素化小鼠IgG，检测限约为1 pg/测定，线性良好。在使用这种新偶联物作为检测试剂的ELISA型夹心PSA测定中，我们检测到0.001 μg/L PSA（约100 fg或约3 amol/测定）。用该方法和商业测定法分析的血清PSA样本结果高度相关。这种新试剂能够在前列腺组织中对PSA表达进行出色的免疫组织化学定位。在以生物素化小鼠IgG为靶标的模型微阵列实验中使用这种新试剂，我们展示了5 - 10 nL微点的出色空间分辨率。