Department of Nanotoxicology and Molecular Epidemiology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 4, 142 00, Czech Republic; Department of Cell Biology, Faculty of Science, Charles University, Prague 2, 128 00, Czech Republic.
Department of Nanotoxicology and Molecular Epidemiology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 4, 142 00, Czech Republic; Department of Cell Biology, Faculty of Science, Charles University, Prague 2, 128 00, Czech Republic.
J Immunol Methods. 2021 Jun;493:113013. doi: 10.1016/j.jim.2021.113013. Epub 2021 Mar 6.
Nonspecific binding of conjugated antibodies represents a critical step which could significantly influence the results of immunostaining or flow cytometry. In this respect, various staining procedures and distinct cell types can alter the results obtained with different fluorochromes. In this study, we analysed nonspecific binding of R-phycoerythrin (R-PE)-conjugated antibodies to mouse mitogen-stimulated B and T lymphocytes. The cells were fixed, permeabilized and stained using isotype control antibodies conjugated with different fluorochromes and assessed by flow cytometry. R-PE-conjugated antibodies bound to LPS-stimulated B cells, in contrast to Con A-stimulated T cells, independently of their specificity. The percentage of R-PE positive B cells varied, according to the used antibodies or the fixation/permeabilization kit. Nevertheless, up to 30% of R-PE B cells after staining with R-PE-conjugated isotype control antibodies was detected. Furthermore, LPS-stimulated B cells bound nonspecifically, in a dose-dependent manner, unconjugated R-PE molecules. Con A-stimulated T cells slightly bound R-PE only in high concentrations. Similarly, the antibodies conjugated with other fluorochromes showed less than 1% of nonspecific binding independently of the manufacturer of antibodies or fixation/permeabilization kits. The data demonstrated that LPS-stimulated B cells, in contrast to Con A-stimulated T cells, bind R-PE nonspecifically following formaldehyde or paraformaldehyde fixation. Therefore, the results based on the use of R-PE-conjugated antibodies should be taken with a precaution.
非特异性结合的共轭抗体是一个关键步骤，它可能会显著影响免疫染色或流式细胞术的结果。在这方面，各种染色程序和不同的细胞类型可以改变不同荧光染料获得的结果。在这项研究中，我们分析了 R-藻红蛋白（R-PE）共轭抗体与小鼠有丝分裂原刺激的 B 和 T 淋巴细胞的非特异性结合。使用不同荧光染料偶联的同型对照抗体对固定、通透和染色的细胞进行分析，并通过流式细胞术进行评估。R-PE 共轭抗体与 LPS 刺激的 B 细胞结合，与 Con A 刺激的 T 细胞不同，与它们的特异性无关。根据所用抗体或固定/通透试剂盒的不同，R-PE 阳性 B 细胞的百分比有所不同。然而，在用 R-PE 共轭同型对照抗体染色后，R-PE 阳性 B 细胞的比例高达 30%。此外，LPS 刺激的 B 细胞以剂量依赖的方式非特异性地结合未偶联的 R-PE 分子。Con A 刺激的 T 细胞仅在高浓度下轻微结合 R-PE。同样，与其他荧光染料偶联的抗体也显示出低于 1%的非特异性结合，与抗体或固定/通透试剂盒的制造商无关。这些数据表明，与 Con A 刺激的 T 细胞相比，LPS 刺激的 B 细胞在甲醛或多聚甲醛固定后会非特异性地结合 R-PE。因此，使用 R-PE 共轭抗体获得的结果应谨慎对待。
