ISGlobal, Hospital Clínic-Universitat de Barcelona, Barcelona, Catalonia, Spain.
CIBER Epidemiology and Public Health (CIBERESP), Barcelona, Spain.
PLoS One. 2018 Jul 2;13(7):e0199278. doi: 10.1371/journal.pone.0199278. eCollection 2018.
Reducing variability of quantitative suspension array assays is key for multi-center and large sero-epidemiological studies. To maximize precision and robustness of an in-house IgG multiplex assay, we analyzed the effect of several conditions on variability to find the best combination. The following assay conditions were studied through a fractional factorial design: antigen-bead coupling (stock vs. several), sample predilution (stock vs. daily), temperature of incubation of sample with antigen-bead (22°C vs. 37°C), plate washing (manual vs. automatic) and operator expertise (expert vs. apprentice). IgG levels against seven P. falciparum antigens with heterogeneous immunogenicities were measured in test samples, in a positive control and in blanks. We assessed the variability and MFI quantification range associated to each combination of conditions, and their interactions, and evaluated the minimum number of samples and blank replicates to achieve good replicability. Results showed that antigen immunogenicity and sample seroreactivity defined the optimal dilution to assess the effect of assay conditions on variability. We found that a unique antigen-bead coupling, samples prediluted daily, incubation at 22°C, and automatic washing, had lower variability. However, variability increased when performing several couplings and incubating at 22°C vs. 37°C. In addition, no effect of temperature was seen with a unique coupling. The expertise of the operator had no effect on assay variability but reduced the MFI quantification range. Finally, differences between sample replicates were minimal, and two blanks were sufficient to capture assay variability, as suggested by the constant Intraclass Correlation Coefficient of three and two blanks. To conclude, a single coupling was the variable that most consistently reduced assay variability, being clearly advisable. In addition, we suggest having more sample dilutions instead of replicates to increase the likelihood of sample MFIs falling in the linear part of the antigen-specific curve, thus increasing precision.
降低定量悬浮阵列分析的变异性是多中心和大规模血清流行病学研究的关键。为了最大限度地提高内部 IgG 多重分析的精密度和稳健性，我们分析了几种条件对变异性的影响，以找到最佳组合。通过部分因子设计研究了以下分析条件：抗原-珠偶联（储备液与几种）、样品预稀释（储备液与每日）、样品与抗原-珠孵育的温度（22°C 与 37°C）、板清洗（手动与自动）和操作人员的专业知识（专家与学徒）。在测试样品、阳性对照和空白中测量了针对 7 种 Pfalciparum 抗原的 IgG 水平，这些抗原具有不同的免疫原性。我们评估了每种条件组合及其相互作用的变异性和 MFI 定量范围，并评估了实现良好可重复性所需的最小样品和空白重复数量。结果表明，抗原免疫原性和样品血清反应性定义了评估分析条件对变异性影响的最佳稀释度。我们发现，独特的抗原-珠偶联、每日预稀释的样品、22°C 孵育和自动清洗具有较低的变异性。然而，当进行多次偶联和在 22°C 与 37°C 孵育时，变异性增加。此外，在独特偶联时，温度没有影响。操作人员的专业知识对分析变异性没有影响，但会降低 MFI 定量范围。最后，样品重复之间的差异最小，两个空白足以捕捉分析变异性，正如三个和两个空白的恒内类相关系数所建议的那样。总之，单次偶联是最能一致降低分析变异性的变量，显然是明智的。此外，我们建议增加更多的样品稀释度而不是重复，以增加样品 MFIs 落入抗原特异性曲线线性部分的可能性，从而提高精密度。
