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Analysis of factors affecting the variability of a quantitative suspension bead array assay measuring IgG to multiple Plasmodium antigens

Author

Listed:
  • Itziar Ubillos
  • Ruth Aguilar
  • Hector Sanz
  • Alfons Jiménez
  • Marta Vidal
  • Aida Valmaseda
  • Yan Dong
  • Deepak Gaur
  • Chetan E Chitnis
  • Sheetij Dutta
  • Evelina Angov
  • John J Aponte
  • Joseph J Campo
  • Clarissa Valim
  • Jaroslaw Harezlak
  • Carlota Dobaño

Abstract

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.

Suggested Citation

  • Itziar Ubillos & Ruth Aguilar & Hector Sanz & Alfons Jiménez & Marta Vidal & Aida Valmaseda & Yan Dong & Deepak Gaur & Chetan E Chitnis & Sheetij Dutta & Evelina Angov & John J Aponte & Joseph J Campo, 2018. "Analysis of factors affecting the variability of a quantitative suspension bead array assay measuring IgG to multiple Plasmodium antigens," PLOS ONE, Public Library of Science, vol. 13(7), pages 1-21, July.
  • Handle: RePEc:plo:pone00:0199278
    DOI: 10.1371/journal.pone.0199278
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    References listed on IDEAS

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    1. Angelique Biancotto & Abigail Wank & Shira Perl & Wendell Cook & Matthew J Olnes & Pradeep K Dagur & J Christopher Fuchs & Marc Langweiler & Ena Wang & J Philip McCoy, 2013. "Baseline Levels and Temporal Stability of 27 Multiplexed Serum Cytokine Concentrations in Healthy Subjects," PLOS ONE, Public Library of Science, vol. 8(12), pages 1-1, December.
    2. Evelina Angov & Collette J Hillier & Randall L Kincaid & Jeffrey A Lyon, 2008. "Heterologous Protein Expression Is Enhanced by Harmonizing the Codon Usage Frequencies of the Target Gene with those of the Expression Host," PLOS ONE, Public Library of Science, vol. 3(5), pages 1-10, May.
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