Jaschke Paul R
Department of Molecular Sciences, Macquarie University, Sydney, 2109, New South Wales, Australia.
Biochem Mol Biol Educ. 2020 Jan;48(1):67-73. doi: 10.1002/bmb.21304. Epub 2019 Sep 18.
The ability to separate, identify, and quantify proteins from complex mixtures are key foundational methods across biochemistry teaching and research. In particular, enzyme-linked immunosorbent assay (ELISA) is an important technique that is used to measure antigen concentrations in both industry and academia. There are four categories of ELISA, direct, indirect, competitive, and sandwich, each with their own applications. Sandwich ELISAs are used to determine antigen concentrations from complex mixtures of protein, such as a cell lysates, and are regularly used as medical diagnostics to diagnose illness and diseases ranging from hepatitis to celiac disease. One major problem with teaching the sandwich ELISA technique to students is the prohibitive cost due to the need to coat a 96-well plate with a capture antibody. One solution to this problem would be to significantly reduce the role of each student in the lab, but this does not adequately prepare students to perform the procedure in a research or industry lab. Instead, this laboratory exercise teaches students the procedural knowledge needed to perform a direct sandwich ELISA, but uses a simulated experience performed within a wet-lab environment. The presented scenario is the analysis of phosphorylated proteins within a synthetic signaling pathway, but because the lab uses simulated samples, it can be tailored to different topics and educational aims. The procedure is 10- to 26-fold less expensive per student to deploy than an authentic sandwich ELISA. Students in the course report that the ELISA lab significantly strengthened the connection between theory and practice. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):67-73, 2020.
从复杂混合物中分离、鉴定和定量蛋白质的能力是生物化学教学和研究的关键基础方法。特别是，酶联免疫吸附测定（ELISA）是一种重要技术，在工业和学术界都用于测量抗原浓度。ELISA有四类，即直接法、间接法、竞争法和夹心法，每种都有其自身的应用。夹心ELISA用于从蛋白质复杂混合物（如细胞裂解物）中测定抗原浓度，并且经常用作医学诊断方法来诊断从肝炎到乳糜泻等各种疾病。向学生教授夹心ELISA技术的一个主要问题是成本过高，因为需要用捕获抗体包被96孔板。解决这个问题的一种方法是大幅减少每个学生在实验室中的操作，但这并不能充分让学生为在研究或工业实验室中执行该程序做好准备。相反，本实验练习教授学生进行直接夹心ELISA所需的程序知识，但使用在湿实验室环境中进行的模拟体验。所呈现的场景是对合成信号通路中磷酸化蛋白质的分析，但由于实验室使用模拟样品，它可以针对不同主题和教育目标进行调整。与真实的夹心ELISA相比，每个学生开展该程序的成本要低10至26倍。该课程的学生报告说，ELISA实验显著加强了理论与实践之间的联系。© 2019国际生物化学与分子生物学联盟，48(1):67 - 73, 2020。
