Figure 3: Analyses of healthy and CBAVD human semen by RP-HPLC and ELISA-Opiorphin. A. Box plot representations of sperm opiorphin levels in healthy young men under different extraction conditions. Single individual values below the 10th or above the 90th percentile are represented as open circles. The box intersecting line represents the median and the box upper and lower limits represent the 75% and 25% percentile, respectively. **P<0.01 by MWT; (n) number of subjects. B. HPLC chromatography–tandem ELISA-opiorphin detection of two semen extracts. Both samples, 3a-13 (healthy donor) and 3b-02 (CBAVD patient) were extracted by an acid-methanol procedure prior to loading onto a C18-Luna-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).