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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.
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Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.

机译:用荧光素修饰牛分枝杆菌胞外蛋白MPB70,以通过荧光偏振快速检测特异性血清抗体。

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The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.
机译:Perrin(F. Perrin,J. Phys.Radiium 7:390-401,1926)描述的荧光偏振原理被用于开发一种新的测定方法,该方法使用荧光素标记的牛分枝杆菌分泌蛋白MPB70快速检测抗-来自三种牛分枝杆菌感染物种(麋鹿,伊拉玛鱼和野牛)的选定血清中的-MPB70抗体。用异硫氰酸荧光素标记纯化的MPB70,导致每个MPB70分子掺入0.96 +/- 0.08(平均+/-标准偏差; n = 3)荧光素基团。标记蛋白在被493 nm附近的波长激发时,在518 nm处发出最大的荧光,并发出强烈的荧光,并且它与抗MPB70单克隆抗体4C3 / 17的免疫反应性不会因荧光素的修饰而改变。通过将0.05%的十二烷基硫酸锂混入测定缓冲液中,优化了荧光偏振测定方案以分析血清样品,从而防止了某些非特异性相互作用的发生。根据表现出抗MPB70抗体的存在而选择的牛分枝杆菌感染动物的血清(通过酶联免疫吸附测定(ELISA)检测)与荧光素标记的MPB70反应,导致向上极化增加到330毫极化单位,与未感染血清的值(167至178 mP)相比,该值接近于不存在特异性抗体时的值(164.7 +/- 3.3 mP; n = 6)。这些结果证明了使用荧光素标记的MPB70通过荧光偏振检测抗MPB70抗体的可行性,并表明此处描述的检测方法可以替代ELISA或其他抗体检测系统。讨论了这种原始方法的优点及其在传染病诊断中的一般适用性。

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