To ensure accurate quantitative assessment, the positive samples of the assay must dilute linearly and in parallel with the standard curve. To determine this linearity of dilution, human serum samples containing a high‐titer of ATI or a high concentration of IFX were used. The samples were diluted serially Kinase Inhibitor Library research buy 2-fold and tested using the ATI-HMSA and the IFX-HMSA, respectively. The observed values of ATI or IFX were plotted with the expected levels of ATI or IFX in the serum. As shown in Fig. 4, both the R2 values and the slopes of each linear regression curve for both assays show linearity. We studied the effects of potential substance interference in both

assays by spiking in common endogenous components of human serum and

drugs methotrexate (MTX) and Azathioprine into the three QC samples (high, mid, and low) to determine their percent recovery. VX-770 chemical structure As shown in Table 5, no significant interference was observed in the physiological levels of serum substances and typical serum concentration of drugs in the ATI-HMSA and IFX-HMSA as assessed by the recovery of the mid QC samples in the presence of the potential interfering substances because of the recovery values were within ± 10% of the mid QC control sample except for the lipemic serum sample at a concentration of 200 mg/mL in the IFX-HMSA and the TNF-α concentration at 250 ng/mL in the ATI-HMSA. TNF-α also had some interference in the IFX-HMSA when the concentrations were over 100 ng/mL because the recovery was greater than ± 10% of the mid QC control sample value. Substantial concentrations of IFX may be present in the serum from patients, even if the blood is drawn at the trough time point. As discussed previously, the presence of IFX in the patient serum significantly

Gefitinib affected the quantitative measurement of ATI using the bridging ELISA assay. To address this issue with the HMSA-based assays, we evaluated the potential impact of IFX level in patient serum on ATI-HMSA results by adding increased amounts of IFX (6.6, 20, and 60 μg/mL) to each of the eight ATI calibration standards to assess the effects on the standard curve. As seen in Fig. 5, the ATI-HMSA could detect ATI levels as low as 0.036 μg/mL in the serum sample containing up to 60 μg/mL of IFX, which is much higher than the maximum therapeutic level reached after infusion of the patient with IFX. To establish the cut point for the ATI-HMSA and the IFX-HMSA, we screened 100 serum samples collected from IFX drug-naïve healthy subjects for the measurement of ATI and IFX levels. No shifting of the IFX-488 to the bound complex areas was found in most of the samples of the ATI-HMSA (Fig. 6A). The proportion of shifted area over the total area was near the LOB and the mean value of the extrapolated ATI from standard curve (multiplied by the dilution factor) was 0.73 ± 0.23 μg/mL as shown in Fig. 6B. The cut point for ATI was determined by taking the mean value + 2 × SD, which yielded 1.19 μg/mL.