A Multiplex Immunoassay Method for Simultaneous Quantification of Iron, Vitamin A and Inflammation Status Markers

Abstract

Deficiencies of vitamin A and iron affect a significant portion of the world's population, and efforts to characterize patterns of these deficiencies are hampered by a lack of measurement tools appropriate for large-scale population-based surveys. Vitamin A and iron are not easily measured directly, so reliable proxy markers for deficiency status have been identified and adopted. Measurement of inflammatory markers is necessary to interpret vitamin A and iron status markers, because circulating levels are altered by inflammation. We developed a multiplex immunoassay method for simultaneous measurement of five markers relevant to assessing inflammation, vitamin A and iron status: α-1-acid glycoprotein, C-reactive protein, retinol binding protein 4, ferritin and soluble transferrin receptor. Serum and plasma specimens were used to optimize the assay protocol. To evaluate assay performance, plasma from 72 volunteers was assayed using the multiplex technique and compared to conventional immunoassay methods for each of the five markers. Results of the new and conventional assay methods were highly correlated (Pearson Correlations of 0.606 to 0.991, p<.0001). Inter-assay imprecision for the multiplex panel varied from 1% to 8%, and all samples fell within the limits of quantification for all assays at a single dilution. Absolute values given by the multiplex and conventional assays differed, indicating a need for further work to devise a new standard curve. This multiplexed micronutrient immunoassay technique has excellent potential as a cost effective tool for use in large-scale deficiency assessment efforts.

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

“…The accurate assessment of MN status to indicate prevalence and severity of deficiencies is presently challenged in part by the high costs and in part by logistical barriers associated with biological sample collection, and subsequent measurement of biomarkers of multiple micronutrients [ 4 ]. This study expands upon our previous work that described a multiplexed tool for the simultaneous assessment of vitamin A (VA), iron deficiency (ID), and acute phase proteins (APP) by improving assay components and adding further biochemical indicators of iodine deficiency and recent or current malarial infection with Plasmodium falciparum [ 5 ].…”

“…Quansys Biosciences (Logan, UT, USA) conducted the prototype assay development using their proprietary Q-Plex™ technology described in detail elsewhere [ 5 , 38 ]. The original development work was expanded upon with the addition of quantitative assays HRP2 and Tg to the existing 5-plex array, accompanied by a redesign of the sTfR assay component as a key antibody is no longer commercially available [ 5 ]. Several candidate antibodies specific to HRP2, Tg, and sTfR were screened for initial performance on the Q-Plex™ technology to establish analytical-specificity and -sensitivity for each antibody [ 39 ].…”

“…Preliminary evaluation of the new assays for Tg and sTfR used commercially available ELISAs as reference assays (Tg [EIA-4126 from DRG International, Springfield, NJ, USA] and sTfR [DTFR1 from R&D Systems Inc., Minneapolis, MN, USA]). Following the optimization of the new assays for HRP2, Tg, and sTfR, the assays were integrated into a 7-Plex assay format with the 4 assays (RBP, CRP, AGP, and ferritin) developed previously [ 5 ].…”

“…We have developed a multiplexed immunoassay to quantify MN biomarkers using the Q-Plex™ platform (Quansys Biosciences, Logan, Utah, USA). This format significantly reduces the overall number of assays that need to be performed to provide the same or extra data but with less cost compared to commercial monoplexed immunoassays, labor, and time required to test large numbers of samples as compared to multiple analyses of individual biomarkers [ 5 , 36 ]. The multiplexed assay uses a protocol that is very similar to conventional ELISAs, a commonly used technique in most reference laboratories.…”

“…In this study, we describe the development of a revised and expanded version of the original multiplexed MN assessment tool [ 5 ] to the Human Micronutrient Assay (7-Plex) that includes 1) the quantitative measurement of Tg and HRP2 biomarkers; 2) improvements to the performance of the original assay [ 5 ], including revision of the sTfR assay using different antibodies, a full revision of the calibration of each assay, new calibrator material, and adding a reference spot to assist with image capture prior to data analysis; and 3) the refinement of the new assay design using reference standards and a USA donor training panel screened with predicate tests for the biomarkers assayed by the 7-plex. The panel allowed for the iterative design changes to the 7-plex assay.…”