Plikaytis B D, Holder P F, Pais L B, Maslanka S E, Gheesling L L, Carlone G M
Biostatistics and Information Management Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30333.
J Clin Microbiol. 1994 Oct;32(10):2441-7. doi: 10.1128/jcm.32.10.2441-2447.1994.
There is a lack of consensus among investigators who use a variety of immunoassay techniques (e.g., enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay) regarding the protocols for describing and forming standard reference or calibration curves and interpolating serum antibody concentrations. This confounds the issue of detecting the presence or absence of parallelism between standard reference serum and serially diluted serum sample curves. These curves must be parallel to support the assumption that the antibody-binding characteristics are similar enough to allow the determination of antibody levels in the diluted serum sample. There is no universal and widely adopted strategy for assessing parallelism in bioassays, and without an assurance of parallelism, investigators are not able to calculate reliable estimates for antibody concentrations in serum samples. Furthermore, single-point (dilution) serum assays do not provide information related to parallelism and nonparallelism, and this, too, may lead to considerable error when calculating antibody concentrations. When assay methodology, technique, and precision improve to the extent that standard reference serum and serially diluted serum sample curves are fit with little error, standard analysis of variance techniques are overly sensitive to negligible departures from parallelism. We present a series of guidelines that compose a protocol for assessing parallelism between bioassay dilution curves that are applicable to data derived from ELISAs. These criteria should be applicable, with minor modifications, to most immunoassay experimental situations and, most importantly, are not dependent on the mathematical model used to form the standard reference curve. These guidelines have evolved in our laboratories over the past 4 years during the performance of thousands of ELISAs for antibodies to the capsular polysaccharides of Neisseria meningitidis groups A and C and Haemophilus influenzae type b.
使用各种免疫测定技术（例如酶联免疫吸附测定[ELISA]和放射免疫测定）的研究人员在描述和形成标准参考曲线或校准曲线以及内插血清抗体浓度的方案方面缺乏共识。这使得检测标准参考血清和系列稀释血清样品曲线之间是否平行的问题变得复杂。这些曲线必须平行，以支持抗体结合特性足够相似，从而能够测定稀释血清样品中抗体水平的假设。在生物测定中，没有普遍且广泛采用的评估平行性的策略，并且如果不能确保平行性，研究人员就无法计算血清样品中抗体浓度的可靠估计值。此外，单点（稀释）血清测定不提供与平行性和非平行性相关的信息，这在计算抗体浓度时也可能导致相当大的误差。当测定方法、技术和精密度提高到标准参考血清和系列稀释血清样品曲线拟合误差很小时，标准方差分析技术对与平行性的微小偏差过于敏感。我们提出了一系列指南，这些指南构成了一个评估生物测定稀释曲线之间平行性的方案，适用于从ELISA获得的数据。这些标准经过 minor 修改后应适用于大多数免疫测定实验情况，最重要的是，不依赖于用于形成标准参考曲线的数学模型。在过去4年中，我们实验室在对A群和C群脑膜炎奈瑟菌以及b型流感嗜血杆菌荚膜多糖抗体进行数千次ELISA检测的过程中，逐步形成了这些指南。
