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. 2017 Jul 17;16(1):287.
doi: 10.1186/s12936-017-1933-6.

An analytical approach to reduce between-plate variation in multiplex assays that measure antibodies to Plasmodium falciparum antigens

Rui Fang  1 , Andrew Wey  1 , Naveen K Bobbili  2 , Rose F G Leke  3 , Diane Wallace Taylor  2 , John J Chen  4
Affiliations

An analytical approach to reduce between-plate variation in multiplex assays that measure antibodies to Plasmodium falciparum antigens

Rui Fang et al. Malar J. .

Abstract

Background: Antibodies play an important role in immunity to malaria. Recent studies show that antibodies to multiple antigens, as well as, the overall breadth of the response are associated with protection from malaria. Yet, the variability and reliability of antibody measurements against a combination of malarial antigens using multiplex assays have not been well characterized.

Methods: A normalization procedure for reducing between-plate variation using replicates of pooled positive and negative controls was investigated. Sixty test samples (30 from malaria-positive and 30 malaria-negative individuals), together with five pooled positive-controls and two pooled negative-controls, were screened for antibody levels to 9 malarial antigens, including merozoite antigens (AMA1, EBA175, MSP1, MSP2, MSP3, MSP11, Pf41), sporozoite CSP, and pregnancy-associated VAR2CSA. The antibody levels were measured in triplicate on each of 3 plates, and the experiments were replicated on two different days by the same technician. The performance of the proposed normalization procedure was evaluated with the pooled controls for the test samples on both the linear and natural-log scales.

Results: Compared with data on the linear scale, the natural-log transformed data were less skewed and reduced the mean-variance relationship. The proposed normalization procedure using pooled controls on the natural-log scale significantly reduced between-plate variation.

Conclusions: For malaria-related research that measure antibodies to multiple antigens with multiplex assays, the natural-log transformation is recommended for data analysis and use of the normalization procedure with multiple pooled controls can improve the precision of antibody measurements.

Keywords: Antibodies; Between-plate variation; Malaria; Multiplex assay; Normalization procedure; Placental malaria.

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Figures

Fig. 1
Fig. 1
Nine pairs of MFI difference values (M) versus average MFI values (A) plots using both linear and natural-log scales
Fig. 2
Fig. 2
An illustration of reduction of the plate effect using data on the natural-log scale for each antigen using all seven pooled controls. Each dot represents the estimated plate effect of one pooled control on each of the six plates, i.e., after subtracting the overall mean of the corresponding pooled control across all six plates. Each horizontal dotted line represents the final estimated plate effect, which is the average across the seven pooled controls
Fig. 3
Fig. 3
Concordance of unadjusted versus adjusted MFI values between two plates in 60 test samples for all antigens on the natural-log scale. The sample MFI values closer to the line Y = X line indicate reduction of plate-to-plate variation after the adjustment on the natural-log scale. p values were based on paired t tests, comparing between unadjusted and adjusted values in between-plate variations
See this image and copyright information in PMC

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