Lengfeld Justin, Zhang Hongtao, Stoesz Steven, Murali Ramachandran, Pass Franklin, Greene Mark I, Goel Peeyush N, Grover Payal
Martell Diagnostic Laboratories, Inc., Roseville, MN, 55113, USA.
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.
Breast Cancer (Dove Med Press). 2021 Oct 14;13:575-593. doi: 10.2147/BCTT.S331844. eCollection 2021.
Breast cancer is a highly prevalent malignancy that shows improved outcomes with earlier diagnosis. Current screening and monitoring methods have improved survival rates, but the limitations of these approaches have led to the investigation of biomarker evaluation to improve early diagnosis and treatment monitoring. The enzyme-linked immunosorbent assay (ELISA) is a specific and robust technique ideally suited for the quantification of protein biomarkers from blood or its constituents. The continued clinical relevancy of this assay format will require overcoming specific technical challenges, including the ultra-sensitive detection of trace biomarkers and the circumventing of potential assay interference due to the expanding use of monoclonal antibody (mAb) therapeutics. Approaches to increasing the sensitivity of ELISA have been numerous and include employing more sensitive substrates, combining ELISA with the polymerase chain reaction (PCR), and incorporating nanoparticles as shuttles for detection antibodies and enzymes. These modifications have resulted in substantial boosts in the ability to detect extremely low levels of protein biomarkers, with some systems reliably detecting antigen at sub-femtomolar concentrations. Extensive utilization of mAb therapies in oncology has presented an additional contemporary challenge for ELISA, particularly when both therapeutic and assay antibodies target the same protein antigen. Resolution of issues such as epitope overlap and steric hindrance requires a rational approach to the design of diagnostic antibodies that takes advantage of modern antibody generation pipelines, epitope binning techniques and computational methods to strategically target biomarker epitopes. This review discusses technical strategies in ELISA implemented to date and their feasibility to address current constraints on sensitivity and problems with interference in the clinical setting. The impact of these recent advancements will depend upon their transformation from research laboratory protocols into facile, reliable detection systems that can ideally be replicated in point-of-care devices to maximize utilization and transform both the diagnostic and therapeutic monitoring landscape.
乳腺癌是一种高度常见的恶性肿瘤，早期诊断可改善其预后。目前的筛查和监测方法提高了生存率，但这些方法的局限性促使人们对生物标志物评估进行研究，以改善早期诊断和治疗监测。酶联免疫吸附测定（ELISA）是一种特异性强且稳健的技术，非常适合定量检测血液或其成分中的蛋白质生物标志物。要使这种检测形式在临床上持续具有相关性，需要克服特定的技术挑战，包括超灵敏检测痕量生物标志物以及应对由于单克隆抗体（mAb）治疗药物使用增加而可能产生的检测干扰。提高ELISA灵敏度的方法有很多，包括使用更灵敏的底物、将ELISA与聚合酶链反应（PCR）相结合，以及将纳米颗粒用作检测抗体和酶的载体。这些改进极大地提高了检测极低水平蛋白质生物标志物的能力，一些系统能够可靠地检测到亚飞摩尔浓度的抗原。mAb疗法在肿瘤学中的广泛应用给ELISA带来了另一个当代挑战，特别是当治疗性抗体和检测抗体靶向同一蛋白质抗原时。解决表位重叠和空间位阻等问题需要一种合理的诊断抗体制备方法，该方法要利用现代抗体生成流程、表位分组技术和计算方法来策略性地靶向生物标志物表位。本文综述了迄今为止在ELISA中实施的技术策略及其解决当前临床环境中灵敏度限制和干扰问题的可行性。这些最新进展的影响将取决于它们能否从研究实验室方案转变为简便、可靠的检测系统，理想情况下可在即时检测设备中复制，以最大限度地提高利用率，并改变诊断和治疗监测格局。
