Newly‐Developed In‐House ELISA Methods for Measuring Serum Ferritin, Soluble Transferrin Receptor, C‐Reactive Protein and Alpha‐1‐Acid Glycoprotein
Authors: Esmaeili Razieh , Zhang Mindy , Mapango Carine , Pfeiffer Christine M 
Published: November 2023

Assessment of population iron status has been an importantelement in nutrition surveys to identify the population groups at risk for iron deficiency and to monitor the impact of iron intervention. The commercially available kit assays performed on clinical analyzers to measure biomarkers of iron and inflammation status require specimen volumes and resources that are usually not available in nutrition surveys conducted in low resource settings. The goal of this project was to develop in‐house ELISA assays for testing serum ferrit in (FER), soluble transferrin receptor (sTfR), C‐reactive protein (CRP), and alpha‐1‐acidglycoprotein (AGP) that require small specimen volume and are inexpensive and simple to perform; furthermore, to evaluate the potential application of these methods in nutrition surveys.We developed four sandwich ELISA assays by screening and pairing commercially available capture and detection(conjugated with horse radish peroxidase) antibodies for FER, sTfR, CRP, and AGP. We optimized the assay conditions for each analyte with regards to plate coating procedure, antibody concentration, sample dilution, incubation time, washing procedure, and timing for color development for detection. Each ELISA plate contained a 6‐point (FER) or 8‐point calibration curve (sTfR, CRP and AGP), 2 levels of quality control samples, 1 adjuster sample, 1 blank sample and 32 unknown samples (duplicate measurement each). Twenty‐five μL of serum was sufficient to test all four biomarkers. The reportable ranges were 8–360 ng/mL for FER, 0.1–15μg/mL for sTfR, 0.3–38 μg/mL for CRP, and 0.064–8.2 mg/mL for AGP. The intra‐ and inter‐assay variability was acceptable:7% (n=6) and 7% (n=25) for FER, 11% (n=6) and 8% (n=25) for sTfR, 5% (n=6) and 13%(n=40) for CRP, and 6% (n=6) and 12% (n=12) for AGP, respectively. We observed complete dilution linearity recovery (2‐fold dilution below or above routine dilution): 101±5% for FER, 101±14% for sTfR, 103±7% for CRP, and 100±9% for AGP. However, the sTfR assay exhibited a concentration dependent bias. An initial comparison between the in‐house ELISA assays and the Roche Mod PE clinical analyzer demonstrated good correlation (Pearson r=0.99 (n=82) for FER, r=0.95 (n=38)for sTfR, and r=0.99 (n=33) for CRP) and an acceptable mean difference of ‐6% for FER, ‐3% for sTfR, and ‐7% for CRP. For AGP, the comparison between the in‐house ELISA and the Quantikine AGP kit showed a weaker correlation (Pearson r=0.7 (n=18))and a high mean difference of 75%. However, our AGP ELISA assay (0.642 mg/mL)produced comparable results to the international reference material ERM‐DA470(0.656 mg/mL). In conclusion, the performance for these four in‐house ELIS Aassays is acceptable with regards to sensitivity, precision, dilution linearity recovery, and agreement with commercially available methods or in the case of AGP, with an international standard. These in‐house ELISA assays require only a small specimen volume and are designed for use as a routine procedure. They allow the laboratory control over assay long‐term stability. To evaluate their robustness for nutrition surveys, we plan to conduct more experiments in a routine assay setting.