Purpose: Immunomodulation checkpoint blockade, such as programmed cell death protein 1 (PD-1) inhibitor, is emerging as a promising therapy for various types of cancer. In order to quantify PD-1 inhibitor, anti-PD-1 antibody drug in human serum, a sensitive enzyme linked immunosorbent assay (ELISA) method was developed and validated.
Methods: The human PD-1 extracellular domain peptide is coated onto a 96-well immunoassay plate. Calibration standards, quality control and serum test samples are added into the wells. PD-1 inhibitor present in each sample is bound by the immobilized capture antigen and further detected by mouse anti-human IgG4 antibody labeled with horseradish peroxidase (HRP). The TMB substrate of HRP is added to the wells and the color conversion in the presence of hydrogen peroxide is quantified by measuring the absorbance at 450 nm. The color intensity is proportional to the amount of the PD-1 inhibitor in the calibration standards, quality control samples and test samples.
The calibration standards were prepared in pooled human serum at concentrations of 256, 128, 90, 50, 30, 15, 8, 4, 2, and 1 ng/mL, and the 256, 2 and 1 ng/mL standards were used as anchor points. Validation samples (VSs) were prepared at 5 concentrations in pooled human serum. The nominal concentrations in the VSs were 128 ng/mL (VS5-ULOQ), 100 ng/mL (VS4-HQC), 25 ng/mL (VS3-MQC), 12 ng/mL (VS2-LQC), and 4 ng/mL (VS1-LLOQ).
Results: The standard calibration curve was evaluated and the results met the target acceptance criteria. The %CV of the mean back-calculated concentration of the calibration standards was ≤20% (≤25% at the LLOQ and ULOQ). The %Bias of the mean back-calculated concentration of the calibration standards was 100% ± 20% of its nominal concentration (100% ± 25% for the LLOQ and ULOQ). A representative calibration curve is presented in Figure 1.
Accuracy and precision of VS and QC samples were evaluated and the result summary is presented in Table 1.The %CV of the mean back-calculated concentration of each VS/QC was ≤20% at each concentration level (≤25% at the LLOQ and ULOQ). The %Bias of the mean measured concentration of each VS/QC was 100% ± 20% of the nominal concentration (100% ± 25% for the LLOQ and ULOQ).
The selectivity of the method was evaluated by analyzing 10 lots of normal human serum spiked with analyte at a nominal concentration of 4.0 ng/mL. The result of the evaluation demonstrated an acceptable selectivity for the PD-1 antibody drug in human serum (Table 2), no significant matrix effect was observed.
Conclusion: A sensitive ELISA method for quantification of anti-PD-1 antibody was developed and validated. Since the capture antigen is PD-1 extracellular peptide, the method is suited for quantification of any anti-PD-1 human antibody drug candidate with simple verification.
Hong Chen– Malvern, Pennsylvania