The optimal concentration of HRP-conjugated streptavidin was determined in the same way. The calibrator consisted of the culture supernatant TSA HDAC mouse from DG44 CHO cells expressing recombinant CL-11. A two-fold serial dilution of the culture supernatant was used to generate an eight-point calibrator curve with a range from 0.26 to 34.8 ng/ml. A five-parameter fit model was applied to the calibrating samples and used to estimate the concentration of unknown samples. The calibrator was stored as single-use aliquots at − 80 °C. The QCs consisted of a pool of serum or plasma from five healthy volunteers diluted 1/11, 1/80 and 1/500 in dilution buffer to

represent high, medium and low concentrations of CL-11, respectively. The QCs were stored as single ready-to-use aliquots at − 80 °C. To study parallelism, the calibrator serial dilution curve was compared to the serial dilution curves of two batches of purified recombinant CL-11 and serial dilutions curves of plasma and serum from two blood donors (analyzed in duplicates). OD data were GDC-0980 evaluated using regression analysis on logistically transformed values, an algorithm that comprised several steps. Due to the maximum limit of the OD determination,

a number of consecutive measurements of OD = 4.0 was observed in each dilution series. Only the last value of OD = 4.0 was maintained in each dilution series, while the prior maximum determinations were omitted.

Subsequently, all OD values were divided by 4.1 to transform the OD data to values above 0, but below 1, as required for the subsequent logistic transformation, y′ = ln[y/(1 − y)]. A background level of OD = 0.05 was observed, and values below the corresponding logistically transformed values were omitted from further analysis. A linear regression was fitted to the remaining data points and multiple comparisons among slopes using Tukey’s HSD test were used to compare the parallelism of the different serial dilutions. The statistical analyses were performed using the Analyse-it software (Analyse-it Software, Ltd, Leeds, UK). Ten two-fold serial dilutions of serum and plasma samples from five blood donors were analyzed in triplicates. Coefficients of variation Y-27632 2HCl (CV) were calculated for the triplicate measurements of each dilution. A “measured/mean” ratio was expressed for each sample using the triplicate measurements and calculating the mean of the triplicates. To study linearity, the CL-11 concentration calculated for each dilution and multiplied by the dilution factor was compared to a mean of the CL-11 concentration that was back-calculated from four dilutions of each sample (1/16–1/128 for serum samples and 1/20–1/160 for plasma samples). The working range was determined as the CL-11 concentrations for which CV was < 10% and the measured/mean ratios deviated < 20%.