Simultaneous assessment of iodine, iron, vitamin A, malarial antigenemia, and inflammation status biomarkers via a multiplex immunoassay method on a population of pregnant women from Niger

<div><p>Deficiencies of vitamin A, iron, and iodine are major public health concerns in many low- and middle-income countries, but information on their status in populations is often lacking due to high costs and logistical challenges associated with assessing micronutrient status. Accurate, user-friendly, and low-cost analytical tools are needed to allow large-scale population surveys on micronutrient status. We present the expansion of a 7-plex protein microarray tool for the simultaneous measurement of up to seven biomarkers with relevance to the assessment of the key micronutrients iron, iodine, and vitamin A, and inflammation and malaria biomarkers: α-1-acid glycoprotein, C-reactive protein, ferritin, retinol binding protein 4, soluble transferrin receptor, thyroglobulin, and histidine-rich protein II. Assay performance was assessed using international reference standards and then verified by comparing the multiplexed and conventional immunoassay results on a training panel of plasma samples collected from US adults. These data were used to assign nominal concentrations to the calibrators of the assay to further improve performance which was then assessed by interrogating plasma samples from a cohort of pregnant women from Niger. The correlation between assays for each biomarker measured from this cohort was typically good, with the exception of thyroglobulin, and the sensitivity ranged from 74% to 93%, and specificity from 81% to 98%. The 7-Plex micronutrient assay has the potential for use as an affordable tool for population surveillance of vitamin A, iron, and iodine deficiencies as well as falciparum malarial parasitemia infectivity and inflammation. The assay is easy-to-use, requires minimal sample volume, and is scalable, rapid, and accurate—needing only a low-cost reader and basic equipment present in most reference laboratory settings and so may be employed by low and middle income countries for micronutrient surveillance to inform on status in key populations. Micronutrient deficiencies including iron, iodine, and vitamin A affect a significant portion of the world’s population. Efforts to assess the prevalence of these deficiencies in vulnerable populations are challenging, partly due to measurement tools that are inadequate for assessing multiple micronutrients in large-scale population surveys. We have developed a 7-plex immunoassay for the simultaneous measurement of seven biomarkers relevant to assessing iodine, iron, and vitamin A status, inflammation and <i>Plasmodium falciparum</i> parasitemia by measuring levels of thyroglobulin, ferritin, soluble transferrin receptor, retinol binding protein 4, α-1-acid glycoprotein, C-reactive protein, and histidine-rich protein II. This 7-plex immunoassay technique has potential as a rapid and effective tool for use in large-scale surveys and assessments of nutrition intervention programs in low- and middle-income countries.</p></div

Scatter plots of the 7-Plex results versus NiMaNu conventional immunoassay results.

<p>Concentrations of each analyte as measured in the 7-Plex (x-axes) plotted against concentrations measured using conventional assays (y-axes) for the NiMaNu panel. Solid line is linear least squares regression (y = mx+b). For ferritin, 2 outliers were excluded from regression and for Tg, 9 outliers were excluded from regression (the outliers are included in the plots marked as x rather open circles); 3 extremely high NiMaNu Tg values excluded from plot. For HRP2, the plot reflects assay signal intensity rather than concentration as concentration was not available from the NiMaNu dataset. Horizontal dotted line indicates cutoff for positive results based on the conventional immunoassay and vertical line indicates 7-Plex assay results beyond assay saturation. AGP, α-1-acid glycoprotein; CRP, C-reactive protein; HRP2, histidine rich protein II; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin.</p

Assay lower limits of detection (LLD), precision, and assay linearity for 7-Plex and conventional ELISAs.

<p>Assay lower limits of detection (LLD), precision, and assay linearity for 7-Plex and conventional ELISAs.</p

The orientation of assays and a reference spot on the 7-Plex planar array displayed in each well of a 96-well plate.

<p>The image on the right is an example of test wells and the levels of chemiluminescence of each test spot on the 7-Plex array when challenged with different levels of analyte: 8 wells with 4 duplicate samples screened using a high calibrator (top left) where the sandwich assay spots give a bright signal and the competitive assay spots are muted, negative controls (bottom left) where the competitive assays have a strong signal whilst the sandwich assays are faint, and two samples (top and bottom right). The reference spot in each well remains at a similar intensity across all wells shown. AGP, α-1-acid glycoprotein; CRP, C-reactive protein; Fer, ferritin; HRP2, histidine rich protein II; Ref, reference spot; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin. ^a Indicates a competitive ELISA format, other assays are in a sandwich format.</p

7-Plex assay calibration ranges and recovery via comparisons with conventional ELISAs from USA donor training panel volunteers.

<p>7-Plex assay calibration ranges and recovery via comparisons with conventional ELISAs from USA donor training panel volunteers.</p

Bland Altman plots, 7-Plex vs NiMaNu immunoassay results.

<p>Bland-Altman plots showing percent difference between the 7-Plex and the NiMaNu conventional immunoassay results on the y-axes plotted against average concentration on the x-axes. Heavy horizontal line and light horizontal lines indicate mean ± 2 standard deviations of percent difference. AGP, α-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin.</p

Spearman correlations and recovery, 7-Plex assays vs NiMaNu data.

<p>Spearman correlations and recovery, 7-Plex assays vs NiMaNu data.</p