Institute of Biomedical Engineering & Shandong Provincial Key Laboratory of Microbial Engineering, Shandong Institute of Light Industry, Jinan 250353, China.
J Pharm Biomed Anal. 2013 Sep;83:129-34. doi: 10.1016/j.jpba.2013.04.036. Epub 2013 May 15.
The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was <10%. The comparison between this immunoassay and a polystyrene bead-based flow cytometric immunoassay showed a correlation coefficient of 0.998 for serum CEA or 0.996 for serum CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein.
基于聚苯乙烯珠的流式细胞术免疫测定法已被广泛报道。然而，功能化聚苯乙烯珠的制备仍然不方便。本研究描述了一种简单易用的基于细菌的流式细胞术免疫测定法，用于蛋白质定量，其中金黄色葡萄球菌（SAC）被用作抗体-抗原载体代替聚苯乙烯珠。SAC 珠通过羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）标记、多聚甲醛固定和抗体结合来制备。癌胚抗原（CEA）和细胞角蛋白 19 片段（CYFRA 21-1）蛋白被用作测试系统中的模型。使用制备的 SAC 珠、生物素化蛋白和链霉亲和素-藻红蛋白（SA-PE），通过定量样品中的 CEA 和 CYFRA 21-1 来验证基于细菌的流式细胞术免疫测定法。获得的数据表明，蛋白质浓度的对数与藻红蛋白平均荧光强度（MFI）的对数之间存在一致的结果。该免疫测定法的检测限（LOD）至少为 0.25ng/ml。精密度和准确度评估表明，相对标准偏差（R.S.D.）或相对误差（R.E.）均<10%。该免疫测定法与基于聚苯乙烯珠的流式细胞术免疫测定法的比较显示，血清 CEA 的相关系数为 0.998，或血清 CYFRA 21-1 的相关系数为 0.996。总之，基于细菌的流式细胞术免疫测定法可用于血清蛋白的定量。
