Quantitative enzyme-linked immunosorbent assay determination of an abundant hemoglobin-derived anti-infective peptide in human placenta

A fragment of the human β-chain of hemoglobin (HEM), hHEMβ111-146, was shown to have broad antimicrobial properties. The 3.9-kDa peptide was postulated to occur in high concentrations in placenta tissue. We established a reliable method to quantify hHEMβ111-146 in placenta tissue. Our methodology consists of a tissue extraction step (step 1), a chromatographic enrichment step (step 2), and a final quantification step (step 3) by enzyme-linked immunosorbent assay (ELISA). The specificity of the ELISA reaction was confirmed by parallel analysis of the samples via Western blot (step 4). The ELISA measured the absorbance of a tetramethylbenzidine substrate at 450 nm. It showed no cross-reactivity with the corresponding γ- and α-HEM regions and low cross-reactivity with the β-HEM region and full-length HEM. The sample preparation procedure enabled a prepurification of hHEMβ111-146, completely eliminating cross-reactive proteins and HEM peptides. The linear range of detection in step 3 was 20-200 ng/well (200-2000 μg/L) with a limit of quantification of 23 ng/well (230 μg/L) and a limit of detection of 7 ng/well (70 μg/L). The assay was characterized by good linearity (r2 > 0.99), intraday precision (coefficient of variation [CV] = 2.2-8.3%), interday precision (CV = 1.8-9.1%), and accuracy (76-109%). The mean recovery of the ELISA was determined to be 97%, and the overall recovery during steps 1-3 was found to be 40.3 ± 2.5%. We measured concentrations from 0.28 to 0.74 mg/g placenta tissue of the hHEMβ111-146 in different placenta samples with an average concentration of 0.57 mg/g. This abundant concentration supports an important physiological role of hHEMβ111-146 in the placenta infective barrier.