Canvax Biotech, Parque Científico y Tecnológico Rabanales 21, c/Astrónoma Cecilia Payne s/n, Edificio Orión, 14014 Córdoba, Spain.
Department of Cell Biology, Physiology and Immunology, Campus de Excelencia Internacional Agroalimentario (ceiA3), University of Córdoba, 14014 Córdoba, Spain.
Biosensors (Basel). 2021 Dec 20;11(12):524. doi: 10.3390/bios11120524.
Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.
酶联免疫吸附测定（ELISA）常用于检测与多种疾病相关的生物分子，有助于这些疾病的诊断和监测，以及降低其死亡率的可能性。已经有几种方法被用于通过将抗体固定在微孔板上来提高 ELISA 的灵敏度。在这里，我们开发了一种通过四嗪（Tz）-反式环辛烯（TCO）反应将抗体固定在表面上以提高 ELISA 灵敏度的策略，从而增加抗体的表面密度。为此，我们制备了带有四嗪基团的表面，而捕获抗体则与 TCO 偶联。四嗪表面通过两种不同的方式制备：（1）从氨基化板上，（2）从 Tz-BSA 涂覆的板上。使用两种夹心 ELISA 模型评估表面，其中一种使用低亲和力抗体抗 c-myc 作为捕获抗体来检测 c-myc-GST-IL8h 重组蛋白，另一种用于检测人癌胚蛋白（CEA）。与标准未修饰表面相比，两种经过四嗪处理的表面的灵敏度都有所提高。在两种四嗪表面上，c-myc-GST-IL8h 的检测灵敏度提高了约 10 倍，而在 Tz-BSA 涂覆的表面上，CEA ELISA 的检测灵敏度提高了 12 倍。总之，我们表明，使用这种固定化系统提高 ELISA 灵敏度是可行的，其中捕获抗体与表面发生共价结合。
