Signal strength of test and control line as a result of location of sample application.

<p>Diluted Ov-16-positive plasma samples were applied to either the conjugate pad upstream of the dried conjugate (BC), directly onto the conjugate (OC) or on the nitrocellulose upstream of the test and control lines (NC). Line scans show average pixel intensity across the width of the nitrocellulose strip.</p

Example soft cassette tests run with standard wick (Ahlstrom 243) or compressed cellulose wick.

<p>All tests were run with positive-spiked blood samples. Vivid samples shown below were run with 5 µl of blood and Cytosep samples shown were run with 30 µl of blood.</p

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

Fraction of tests that failed due to blood on membrane after dry.

<p>Tests that had blood on the membranes after the test was dry were counted as failed tests. This failure rate is shown as a function of the volume, blood filter, and wick material. A minimum of 10 tests were run for each condition shown.</p

Performance of the Ov-16 lateral flow test and ELISA on a panel of sera samples against <i>Onchocerca volvulus</i> (Ov) status and Ov-16 enzyme immunoassay (ELISA).

<p>Performance of the Ov-16 lateral flow test and ELISA on a panel of sera samples against <i>Onchocerca volvulus</i> (Ov) status and Ov-16 enzyme immunoassay (ELISA).</p

Thickness over time of three compressed hydrophilic materials.

<p>Sample thicknesses were measured after exposure to 37°C and 80% relative humidity over time.</p

Schematic and pictures of Ov-16 soft cassette test.

<p>A) top view schematic of the basic Ov-16 lateral flow strip components, with compressed cellulose at the wick end noted (although Ahlstrom 243 absorbent material was also used when specified as standard wick material). B) Side view schematic of the test as assembled in the soft cassette housing, before fluid actuation. C) Side view schematic of a running test shown, with expansion of cellulose resulting in lift of overlay material and blood sample filter. D) Top and side views of the soft cassette housing, with test components labeled on top view.</p

Sensitivity and specificity of the lateral flow test at different read times.

<p>Four different people read the test results over these time points. At days 30 and 70 the tests were read by two readers indicated by superscripts 1 and 2. For this sample size (n = 100), a change of 0.02 in sensitivity or specificity represents a discordance in 1 test result. For test development results throughout drying, the correlation coefficients for 1 hour and 24 hours were calculated relative to the 20 minutes test results(*). The remaining time points give the correlation coefficients calculated relative to the 24-hour dry reads.</p

Inventory of specimens used to evaluate the Ov-16 lateral flow test.

<p>Inventory of specimens used to evaluate the Ov-16 lateral flow test.</p

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