Dvelopment of microanalysis for biological substances detected by bioluminescent assay

生物发光检测生物物质微量分析方法的进展

基本信息

批准号：
09557219
负责人：
MAEDA Masako
金额：
$ 7.04万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Recently, bioluminescence is extensively investigated by several researchers for its high sensitivity with the aim of applying several assays. And the various luciferases and photoprotein genes were cloned and expressed in E.coli or saccharomyces serevisiae. Especially, recombinant firefly luciferase is now commercially available and has been employed for enzyme immunoassay(EIA). However, the firefly luciferase tends to lose much of its activity when treated with the chemical cross linkage reagents used in the conjugation. To dissolve this problem, we have established two highly sensitive bioluminescent assays for thermostable acetate kinase (AK). After EIA using AK as label enzyme, AK activity was determined by measuring the amount of produced AT P using firefly luciferase.Furthermore, we have introduced well known streptavidin-biotin system to EIA using biotinyted AK. Using these systems, we applied to assay for biologically substances such as hCG, mIL-6 and other clinically importan … More t substances. Especially, in the TSH assay, sensitivity was about 25 times higher than that of RIA. It will be useful for detection of the low concentration of TSH values such as hyperthyroidism. It have been applied to BLEIA in combination with the streptavidin-biotin system with biotinylated AK and appeared sensitive, simple, and convenient. Using the streptavidin-biotin system, mIL-6 in mouse tissue and plasma, and PACAP 38 in rat tissues: it could be accurately measured without the concentration of samples or any other pretreatment using the BLEIA. More recently, Kajiyama et al conducted mutational analysis of firefly luciferase and showed that they emit light of different colors due to single amino acid change. The study demonstrated multiple simultaneous antigen detection using the two kinds of biotinylated luciferase-streptavidin-bioanylated antibody complexes that were capable of emitting difference colors of light. As clinical application using this system, PGI and PGII in serum can be assayed, satisfactory. Further investigation is now ongoing to determine if multiple analyte in the same sample can be assayed by BLEIA. Less

最近，一些研究人员主要研究生物发光的高灵敏度，并在大肠杆菌或酿酒酵母中克隆和表达各种荧光素酶和发光蛋白基因，特别是重组萤火虫荧光素酶现已商业化。已用于酶免疫测定（EIA），然而，当用化学交联处理时，萤火虫荧光素酶往往会失去大部分活性。为了解决这个问题，我们建立了两种热稳定性醋酸激酶（AK）的高灵敏度生物发光测定法，使用 AK 作为标记酶进行 EIA 后，通过使用萤火虫荧光素酶测量产生的 ATP 量来测定 AK 活性。此外，我们还使用生物素化的 AK 将众所周知的链霉亲和素-生物素系统引入 EIA，使用这些系统，我们应用于 hCG 等生物物质的测定。 mIL-6等临床上重要的物质，特别是在TSH测定中，灵敏度比RIA高约25倍，对于检测甲状腺功能亢进等低浓度TSH值很有用。已将其与具有生物素化 AK 的链霉亲和素-生物素系统结合应用于 BLEIA，并且使用链霉亲和素-生物素系统、小鼠组织和血浆中的 mIL-6 以及 PACAP 显得灵敏、简单且方便。大鼠组织中的 38：使用 BLEIA 无需样品浓缩或任何其他预处理即可准确测量。最近，Kajiyama 等人对萤火虫荧光素酶进行了突变分析，结果表明它们由于单一氨基酸的变化而发出不同颜色的光。该研究证明了使用两种能够发出不同颜色光的生物素化荧光素酶-链霉亲和素-生物酰化抗体复合物进行多种抗原检测作为使用该系统的临床应用，PGI和。可以对血清中的 PGII 进行检测，目前正在进行进一步研究以确定是否可以通过 BLEIA 检测同一样品中的多种分析物。