Figure 5: Analyses of human milk by RP-HPLC and ELISA-Opiorphin. A. Analysis of individual milk samples using the ELISA-Opiorphin method. Standard curve (open squares) relating opiorphin concentration in ng/ml (log scale, X axis) to the B/B0 (the percentage of binding in the presence (B) of standard peptide / binding in the absence (B0) of the standard peptide: linear scale, Y axis) and sample curve (open circles) relating serially diluted (2-fold) individual purified milk sample (2-06) to the B/B0, % response. Note that there is a parallel between the dose-response standard curve and serial dilutions of the sample. Each point represents the mean ± SD of duplicates. B. HPLC chromatography in tandem with ELISA-opiorphin detection of individual milk extracts. The milk sample (2-06) was extracted by the C18 solid-phase extraction procedure and the dried extract was treated with DTPA, 16 mM final concentration, prior to loading onto the C18-ACE-HPLC column. After lyophilization each 1 min fraction was analyzed by ELISA-Opiorphin and the results expressed as concentration of immunoreactive (IR) opiorphin in ng/ml (Y axis) by HPLC-fraction in min (X axis).