Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA; Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA; Duke Center for AIDS Research, Duke University Medical Center, Durham, NC 27710, USA.
J Immunol Methods. 2023 Apr;515:113452. doi: 10.1016/j.jim.2023.113452. Epub 2023 Feb 28.
The use of conventional serum for supplementation of media in cell-based and single-cell functional assays has been a major challenge for assay performance, standardization, optimization, and reproducibility. It has been identified as the leading cause of variability and suboptimal performance in large, international Elispot proficiency panels (Janetzki et al., 2008; Rountree et al., 2016). Extensive pretesting and optimization activities are one approach to overcome these challenges, but they are time-consuming and resource-intensive because suitable lots of serum are difficult to identify and secure in sufficient quantities to provide stability in long-term studies. Advancements in manufacturing methods have resulted in a new class of serum with the potential to solve these challenges. An IFNɣ Elispot study was designed by the External Quality Assurance Program Oversight Laboratory (EQAPOL) at Duke Human Vaccine Institute's (DHVI) Immunology and Virology Quality Assessment Center (IVQAC) to test this new class of serum against their in-house, validated control serum, which is regarded as a global standard in performance for high functionality, recovery, and viability. Commonly used serum-free media were also included in the study. The results of this study compellingly demonstrate that this new class of serum produces high responses and low background reactivity comparable to the included serum standard, with excellent recovery and viability of cells. A protocol for ongoing testing has been developed to continuously validate new batches of this serum with the goal to make available to the field a pretested and validated serum that can be used with confidence in functional cell-based assays.
在基于细胞和单细胞功能测定的测定中，使用常规血清来补充培养基一直是测定性能、标准化、优化和重现性的主要挑战。它已被确定为导致大型国际 Elispot 能力验证小组中变异性和性能不佳的主要原因（Janetzki 等人，2008 年；Rountree 等人，2016 年）。广泛的预测试和优化活动是克服这些挑战的一种方法，但它们既耗时又资源密集，因为难以确定合适的血清批次，并难以确保足够数量的血清来提供长期研究的稳定性。制造方法的进步产生了一类具有潜在能力的新血清，可以解决这些挑战。杜克人类疫苗研究所 (DHVI) 免疫学和病毒学质量评估中心 (IVQAC) 的外部质量保证计划监督实验室 (EQAPOL) 设计了一项 IFNɣ Elispot 研究，以测试这种新的血清类与他们内部验证的对照血清进行对比，该对照血清被认为是高功能、高回收率和高活力的全球性能标准。该研究还包括了常用的无血清培养基。该研究的结果令人信服地表明，这种新的血清类产生的高反应和低背景反应与包括的血清标准相当，具有出色的细胞回收率和活力。已经制定了一个正在进行测试的方案，以不断验证这种血清的新批次，目标是为该领域提供一种经过预测试和验证的血清，可在基于细胞的功能测定中放心使用。
