The SD of the y-intercepts and the mean slope were obtained from anti-glucocerebrosidase antibody calibration curves. The assay cut point was determined by testing treatment-naïve patient serum samples and calculating the mean plus 1.645 standard deviation of assay values, where 1.645 is the 95th percentile of the one-sided normal t-distribution (Mire-Sluis et al., 2004). A minimum of 67 samples from individual treatment-naïve patients with Gaucher disease were tested to set the antibody-positive cut points for the screening assays for both velaglucerase alfa and imiglucerase. The test design included at least three analysts testing replicate samples using a minimum of three different

microwell plate lots over a period of at least 14 days. Two MSD instruments were used randomly for a minimum of 1170 Forskolin determinations for each assay. The assay cut points for anti-velaglucerase

alfa or anti-imiglucerase antibodies were established on the basis of raw ECL counts and estimated to be 1.67 and 3.28 ng/mL, respectively (Table 2) by interpolation on a calibration curve. The assay sensitivity was estimated as the assay cut point multiplied by the minimum sample dilution factor. The assay sensitivities were therefore calculated to be 33.4 ng/mL for anti-velaglucerase alfa antibodies and 65.6 ng/mL for anti-imiglucerase antibodies. The LOD and LOQ values were calculated from the anti-glucocerebrosidase antibody calibration curves. Of note, the assay cut point values are below or near the instrument Ibrutinib limit of detection. The assay LOD values are greater than the instrument LOD. Precision, accuracy, and sensitivity of this assay were determined

as previously described (FDA, 2001, ICH, 2005 and EMEA, 2009) and are given in Table 3. The lowest LOD and lowest LOQ were determined according to the signal-to-noise method, where a signal-to-noise Erastin mouse ratio of 3 is considered acceptable for estimating the detection limit and a signal-to-noise ratio of 10 is considered acceptable for estimating the quantitation limit (EMEA, 2009). The mouse anti-glucocerebrosidase monoclonal antibody calibration curve was used to convert the raw CPM values. It is widely accepted that the positive cut point for antibody screening assays should be selected such that a false-positive rate of 5% is anticipated with 95% confidence (Mire-Sluis et al., 2004), as described in the previous section. However, little has been discussed regarding the establishment of an appropriate antibody-positive cut point for antibody confirmatory assays. The assay cut point of this confirmatory assay was established as the mean plus 3 standard deviations of assay values obtained from treatment-naïve patient serum samples. A total of 59 samples from individual, treatment-naïve patients with Gaucher disease were tested to set the antibody-positive cut point for the radioimmunoprecipitation confirmatory assays for both velaglucerase alfa and imiglucerase.