2.5. Validation of RIA
The validation of two RIA systems (RIA-1 and RIA-3) used to measure the PAG concentrations in the plasma samples had been previously described (El Amiri et al., 2007). The main characteristics of RIA-2 and RIA-4 used for PAG measurements in the plasma samples as well as those of RIA-1 to RIA-4 used for measurements in milk are summarized in Table 2. The MDL were calculated as the mean concentration minus twice the standard deviations (mean −2 SD) of 10 duplicates of the zero (B0) standard (Skelley et al., 1973). In plasma samples, MDL varied from 0.16 ng/ml (RIA-2) to 1.12 ng/ml (RIA-4). In milk samples, they varied from 0.16 ng/ml (RIA-3) to 0.9 ng/ml (RIA-4).
Reproducibility was determined by calculating the intra- and inter-assay coefficients of variation (CV) as follows: [%CV = (SD/mean)*100]. For intra-assay CV, the same serum was assayed 10 times within the same assay. The inter-assay reproducibility was assessed by analyzing each serum in 10 consecutive assays (Rodbard, 1974).
Cut-off points corresponding to the maximum value of sensitivity, specificity, and accuracy of pregnancy diagnosis for each RIA system varied from 0.25 ng/ml (RIA-2) to 2.0 ng/ml (RIA-4) in plasma samples. In milk samples they were quite similar and varied from 0.6 ng/ml (RIA-1 and RIA-2) to 0.9 ng/ml (RIA-4).