Comparison of a New Multiplex Immunoassay for Measurement of Ferritin, Soluble Transferrin Receptor, Retinol-Binding Protein, C-Reactive Protein and α1-Acid-glycoprotein Concentrations against a Widely-Used s-ELISA Method

Abstract

Recently, a multiplex ELISA (Quansys Biosciences) was developed that measures ferritin, soluble transferrin receptor (sTfR), retinol-binding protein (RBP), C-reactive protein (CRP), α1-acid glycoprotein (AGP), thyroglobulin, and histidine-rich protein 2. Our primary aim was to conduct a method-comparison study to compare five biomarkers (ferritin, sTfR, RBP, CRP, and AGP) measured with the Quansys assay and a widely-used s-ELISA (VitMin Lab, Willstaett, Germany) with use of serum samples from 180 women and children from Burkina Faso, Cambodia, and Malaysia. Bias and concordance were used to describe the agreement in values measured by the two methods. We observed poor overall agreement between the methods, both with regard to biomarker concentrations and deficiency prevalence estimates. Several measurements were outside of the limit of detection with use of the Quansys ELISA (total n = 42 for ferritin, n = 2 for sTfR, n = 0 for AGP, n = 5 for CRP, n = 22 for RBP), limiting our ability to interpret assay findings. Although the Quansys ELISA has great potential to simplify laboratory analysis of key nutritional and inflammation biomarkers, there are some weaknesses in the procedures. Overall, we found poor comparability of results between methods. Besides addressing procedural issues, additional validation of the Quansys against a gold standard method is warranted for future research.

The Quansys multiplex immunoassay for serum ferritin, C-reactive protein, and α-1-acid glycoprotein showed good comparability with reference-type assays but not for soluble transferrin receptor and retinol-binding protein

“…As such, the median relative differences between the Q-Plex and reference assay in our study (Fer -2.4%, sTfR 107%, CRP 0.03%, AGP -1.3%, and RBP 51%) do not correspond well with the 2 previously reported relative differences between the Q-Plex and the commonly-used sandwich ELISA [8,11]: Fer 88% and 108%, sTfR 70% and 148%, CRP -33% and -1%, AGP -53% and -37%, and RBP -16% and 12%. Several reasons could explain these discrepancies: different antibodies with different specificities and affinities used in these comparisons; different sample sets may result in different assay relationships either due to different concentration ranges and/or due to sample composition; the statistical approach used to assess the assay agreement varies across studies and non-constant variance and/or non-constant differences may not have been addressed in previous studies; in previous studies, the authors used the slope to describe the proportional difference between the assays without giving consideration to the intercept, which in some cases was quite large.…”

“…Conversion equations may be helpful in the future to allow comparison of Q-Plex assay data with US population data generated with the reference-type assays in the National Health and Nutrition Examination Survey. Previous studies that compared the Q-Plex and sandwich-ELISA assay with samples from African and Asian countries, appeared to cover similar concentration ranges to our study based on visual inspection of the scatter plots or Bland-Altman plots [8,11]. Still, the equations derived in this study should be confirmed with other sample sets, particularly from countries where analyte concentrations are different from those in the United States.…”

“…Furthermore, we carefully conducted the statistical analysis to assess the agreement between the test and reference assay, ensuring that we appropriately address assumption violations such as non-constant variance and non-constant difference. Because previous method comparison studies [8,11] used different approaches, it is difficult to compare the findings.…”

“…The 7-plex microarray was validated against a laboratory-developed test (LDT) sandwich-ELISA from the VitMin Lab [10], which is widely used in micronutrient surveys, using 206 heparinized plasma samples from Nigerian pregnant women [8]. In another comparison of these 2 assays (7-plex and LDT) using 180 serum samples from women and children from Burkina Faso, Cambodia, and Malaysia, poor comparability between the methods was reported [11].…”

Standardization of laboratory practices and reporting of biomarker data in clinical nutrition research

“…Yet, for several of their surveys, samples were analyzed at the VitMin lab (Juergen Erhardt; http://www.nutrisurvey.de/blood_samples/ ), which uses a sandwich ELISA method to measure ferritin, retinol-binding protein, soluble transferrin receptor, CRP, and AGP ( 9 ). The VitMin lab has been a valued resource in the global micronutrient research community for many years; an initial validation study of its ELISAs was promising ( 9 ), although recent comparisons of the VitMin method to a new commercial assay showed poor concordance ( 10 ). For the surveys for which samples were analyzed at the VitMin lab, detailed measures of assay technique, measures of precision, and limits of quantification could have been feasibly obtained and assessed as part of the BRINDA project.…”