Author:
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Ubillos, Itziar; Aguilar, Ruth; Sanz, Héctor; Jiménez, Alfons; Vidal, Marta; Valmaseda, Aida; Dong, Yan; Gaur, Deepak; Chitnis, Chetan E.; Dutta, Sheetij; Angov, Evelina; Aponte, John J.; Campo, Joseph J.; Valim, Clarissa; Harezlak, Jaroslaw; Dobaño, Carlota, 1969-
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Abstract:
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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. |