Yang Xu, Vidunas Alexander J, Beniash Elia
Department of Oral Biology, School of Dental Medicine, University of PittsburghPittsburgh, PA, USA.
Department of Bioengineering, Center for Craniofacial Regeneration, Swanson School of Engineering, McGowan Institute for Regenerative Medicine, University of PittsburghPittsburgh, PA, USA.
Front Physiol. 2017 Apr 25;8:239. doi: 10.3389/fphys.2017.00239. eCollection 2017.
Non-specific fluorescence from demineralized enamel matrix can significantly compromise the immunofluorescence studies and lead to false positives. Our goal was to assess degrees of non-specific binding under different conditions and try to optimize procedures for immunofluorescence studies of forming enamel. Firstly, we compared two methods for background fluorescence elimination, i.e., sodium borohydride and Sudan Black B treatments. The results demonstrated that Sudan Black B is far superior to sodium borohydride in reducing the background fluorescence in dental tissues. We also studied the extent of non-specific binding of normal sera and purified polyclonal immunoglobulins (IgG) from five mammalian species, guinea pig, rat, rabbit, goat, and sheep, over a broad range of dilutions. For all sera tested fluorescence signals increased exponentially from 1:1000 to 1:100. Interestingly, the non-specific binding of sera from rodent species was below that of positive control in the whole range of dilutions. In contrast, incubation with sera from 3 non-rodent species produced much higher signals which surpassed the positive control signal at 1:250~1:500 dilution range. Most of the IgGs didn't show significant non-specific binding within 0.25-5 μg/ml range, except rabbit IgG which demonstrated extremely high affinity to the enamel matrix even at concentrations as low as 1 μg/ml. Further, studies confirmed that Fab fragments of purified normal rabbit IgG, not conserved Fc fragments, were involved in the interactions. Our observations suggest this high affinity is associated with the antigen binding sites of rabbit IgG. We anticipate that our results will help enamel researchers to optimize and standardize their immunochemical procedures.
脱矿釉质基质的非特异性荧光会严重影响免疫荧光研究并导致假阳性。我们的目标是评估不同条件下的非特异性结合程度，并尝试优化正在形成的釉质免疫荧光研究的程序。首先，我们比较了两种消除背景荧光的方法，即硼氢化钠处理和苏丹黑B处理。结果表明，在降低牙齿组织背景荧光方面，苏丹黑B远优于硼氢化钠。我们还研究了来自豚鼠、大鼠、兔子、山羊和绵羊这五种哺乳动物的正常血清和纯化多克隆免疫球蛋白（IgG）在广泛稀释范围内的非特异性结合程度。对于所有测试血清，荧光信号从1:1000到1:100呈指数增加。有趣的是，在整个稀释范围内，啮齿动物血清的非特异性结合低于阳性对照。相比之下，用三种非啮齿动物的血清孵育产生的信号要高得多，在1:250至1:500稀释范围内超过了阳性对照信号。除兔IgG即使在低至1μg/ml的浓度下仍对釉质基质表现出极高亲和力外，大多数IgG在0.25 - 5μg/ml范围内未显示出明显的非特异性结合。此外，研究证实参与相互作用的是纯化的正常兔IgG的Fab片段，而非保守的Fc片段。我们的观察结果表明这种高亲和力与兔IgG的抗原结合位点有关。我们预计我们的结果将有助于釉质研究人员优化和标准化他们的免疫化学程序。
