Quantitative enzyme-linked immunosorbent assay determination of an abundant hemoglobin-derived anti-infective peptide in human placenta.
A fragment of the human beta-chain of hemoglobin (HEM), hHEMbeta111-146, 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 hHEMbeta111-146 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 450nm. It showed no cross-reactivity with the corresponding gamma- and alpha-HEM regions and low cross-reactivity with the beta-HEM region and full-length HEM. The sample preparation procedure enabled a prepurification of hHEMbeta111-146, completely eliminating cross-reactive proteins and HEM peptides. The linear range of detection in step 3 was 20-200ng/well (200-2000microg/L) with a limit of quantification of 23ng/well (230microg/L) and a limit of detection of 7ng/well (70microg/L). The assay was characterized by good linearity (r(2)>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 0.74mg/g placenta tissue of the hHEMbeta111-146 in different placenta samples with an average concentration of 0.57mg/g. This abundant concentration supports an important physiological role of hHEMbeta111-146 in the placenta infective barrier.
Ständker
Ludger
L
Clinic of Immunology and Rheumatology, Experimental and Clinical Peptide Chemistry, Medical University Hannover, 30625 Hannover, Germany. L.standker@gmx.de
Zachgo
Veronica
V
Hillemanns
Peter
P
Rösinger
Marcel
M
Forssmann
Wolf-Georg
WG
Hass
Ralf
R
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000032:analysis* / Q000302:isolation & purification; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy
D023181:Antimicrobial Cationic Peptides; D006454:Hemoglobins