Mujawar Liyakat Hamid, Moers Antoine, Norde Willem, van Amerongen Aart
Biomolecular Sensing and Diagnostics, Food and Biobased Research, Wageningen University and Research Centre, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.
Anal Bioanal Chem. 2013 Sep;405(23):7469-76. doi: 10.1007/s00216-013-7192-7. Epub 2013 Aug 4.
We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.
我们基于标记特异性抗体分子的固定、双标记扩增子的结合以及作为二级信号的表面带有中性抗生物素蛋白和碱性磷酸酶融合蛋白分子的黑碳纳米颗粒共轭物，开发了一种快速乳腺炎检测试验。将抗体喷墨打印到三种不同的硝酸纤维素膜载玻片上，即Unisart（赛多利斯）、FAST（通用医疗沃特曼）和Oncyte-Avid（格雷斯生物实验室），并比较这些载玻片上的最终检测信号。通过平板扫描和使用TotalLab图像分析软件评估像素灰度体积来确定斑点的黑度。黑点肉眼即可轻松读取。我们成功证明了在不到3小时内即可检测到乳腺炎致病病原体的特异性扩增子。使用类似的方案，我们还表明可以在同一垫上检测来自四种不同乳腺炎致病病原体（六种菌株）的特异性扩增子。还比较了两种不同的打印缓冲液，即磷酸盐缓冲盐水（pH 7.4）和碳酸盐缓冲液（pH 9.6）对一抗功能的影响。
