Due to the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for reliable high-throughput serological assays in order to evaluate the immunological responses against SARS-COV-2 virus and to enable population screening, as well as vaccines and drugs efficacy testing. Several serological assays for SARS-CoV-2 are now becoming available in the market. However, it has also become extremely important to have well-established assays with desirable high sensitivity and specificity. To date, the micro-neutralization (MN) assay, is currently considered the gold-standard being capable of evaluating and detecting, functional neutralizing antibodies (nAbs). Several protocols exist for micro-neutralization assays which vary in several steps of the protocol: cell seeding conditions, number of cells seeded, virus amount used in the infection step, virus-serum-cells incubation period etc. These potential differences account for a high degree of variability and inconsistency of the results and using a harmonized protocol for the micro-neutralization assay could potentially solve this. Given this situation, the main aim of our study was to carry out SARS-CoV-2 wild type virus MN assay in order to investigate which optimal tissue culture infective dose 50 (TCID50) infective dose in use is the most adequate choice for implementation in terms of reproducibility, standardization possibilities and comparability of results. Therefore, we assessed the MN by using two different viral infective doses: a standard dose of 100 TCID50/well and a lower dose of 25 TCID50/well. The results obtained, yielded by MN on using the lower infective dose (25 TCID50), were in line with those obtained with the standard infective dose; in some cases, however, we detected a titre that was one or two dilution steps higher, which maintained all negative samples negative. This suggesting that the lower dose can potentially have a positive impact on the detection and estimation of neutralizing antibodies present in a given sample, showing higher sensitivity but similar specificity and therefore, it would require a more accurate assessment and cross-laboratories standardisation especially when MN is employed as serological assay of choice for pre-clinical and clinical studies.

中文翻译：

---

中和试验中病毒剂量的理论与实践：对SARS-CoV-2“双重思考”效应的见解。

由于严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）在全球范围内的传播，迫切需要可靠的高通量血清学检测方法，以评估针对SARS-COV-2病毒的免疫反应并使其能够人群筛查以及疫苗和药物功效测试。市场上现已可以使用几种针对SARS-CoV-2的血清学检测方法。然而，具有完善的测定方法以及所需的高灵敏度和特异性也变得极为重要。迄今为止，微中和（MN）分析目前被认为是能够评估和检测功能性中和抗体（nAbs）的金标准。微中和测定法存在几种方案，它们在方案的几个步骤中有所不同：细胞接种条件，接种的细胞数，在感染步骤，病毒血清细胞潜伏期等中使用的病毒数量。这些潜在的差异说明了高度的可变性和结果的不一致，并且使用统一的方案进行微量中和测定可能会解决此问题。在这种情况下，我们的主要研究目的是进行SARS-CoV-2野生型病毒MN检测，以调查使用的最佳组织培养感染剂量50（TCID50）感染剂量最适合用于实施可重复性，标准化可能性和结果可比性方面。因此，我们通过使用两种不同的病毒感染剂量来评估MN：标准剂量为100 TCID50 /孔，而较低剂量为25 TCID50 /孔。MN使用较低的感染剂量（25 TCID50）获得的结果，与标准感染剂量获得的结果一致；但是，在某些情况下，我们检测到的滴度比稀释度高一到两个稀释步骤，从而使所有阴性样品均保持阴性。这表明较低的剂量可能会对给定样品中存在的中和抗体的检测和估计产生积极影响，显示出更高的敏感性但相似的特异性，因此，这将需要更准确的评估和跨实验室的标准化，尤其是当MN用于临床前和临床研究的血清学检测方法。