The minimum acceptable criteria were < 20% for CV and < 25% for accuracy. Linearity of the ATI-HMSA and the IFX-HMSA was determined by performing a two-fold serial dilution of an ATI-

or an IFX-positive sample to graphically determine the relationship between the observed and the expected concentrations. Both the R2 value and the slope of each linear regression curve were calculated to evaluate the linearity of the assays. Serum samples from drug-naïve healthy donors (n = 100; Golden West Biologics. Temecula, CA) were analyzed to determine the screen cut point for the ATI-HMSA and IFX-HMSA. We set the cut point to have an upper negative limit of approximately 97.5%. It was calculated by using the mean value of individual samples interpolated from the standard curve plus Wnt inhibitor 2.0 times the standard deviation (SD), where 2.0 was the 97.5th percentile of the normal distribution. Receiver operating characteristic analysis was also used to estimate the clinical specificity and sensitivity for the ATI-HMSA. The principles of the ATI-HMSA and the IFX-HMSA are illustrated in Fig. 1A and B, respectively. The ATI-HMSA in Fig. 1A involved incubating an ATI-containing serum sample with IFX-488/IC at RT for 1 h to form IFX-488/ATI immune complexes. At the end of the incubation, the immune complexes

and the buy RAD001 remaining free IFX-488 were separated by SE-HPLC and the peak areas of the bound IFX-488 and the free IFX-488 were quantified by fluorescence detection. A pooled ATI-positive serum was

used as the calibration standard. When serial dilutions of the ATI calibration standard were incubated with IFX-488, dose-dependent immune complexes were formed with concomitant reduction of the free IFX-488, all of which could be resolved by SE-HPLC analysis, as shown in Fig. 2A. Fig. 2B shows the standard curve generated by plotting the data from Fig. 2A. The lowest concentration of ATI in the standard curve was 0.006 μg/mL. Fig. 1B illustrates the principle of the IFX-HMSA, which is similar to that of the ATI-HMSA. Incubation of the fluorescently labeled TNF-α (TNF-488) with the anti-TNF antibody IFX resulted in the formation of higher molecular weight immune complexes (TNF-488/IFX). Aprepitant The immune complexes and the remaining free TNF-488 were separated and quantified by SEC-HPLC. Purified IFX spiked in NHS at a concentration of 93.75 μg/mL was used as the IFX calibration standard. Using similar methodology to the ATI-HMSA, the immune complexes formed by combining the IFX calibration standards with TNF-488 were separated from the remaining free TNF-488 (Fig. 3A) and a standard curve was generated with the results (Fig. 3B). To validate the standard curve, the performance characteristics of the ATI calibration standards within the concentration range of 0.006–0.