Therapeutic drugs that are based on biological materials, like fusion proteins, enzymes, cytokines, hormones, and antibodies, may have the potential to trigger an immune response in the host.
Immunogenicity of a therapeutic can be described as the undesirable stimulation of an immune response by the host to the therapeutic. Immune response measurement is often evaluated by determining the antibodies produced by the host against the therapeutic molecule. A therapeutic’s immunogenicity can be a significant barrier in the development of a product.
This article describes a proof-of-concept assay to show how the Simoa platform can be used as a tool for measuring the levels of anti-drug antibodies (ADA) against adalimumab. In order to compare performance, equivalent assays were ran using a regular plate-based ELISA and the Simoa platform.
Reagents and Methods
A fully human monoclonal (IgG1/kappa) antibody-drug, was used in both assays as the capture reagent. Here, the detection reagent was the same kind of drug marked with biotin. The fully human monoclonal anti-adalimumab IgG1, being the target, was spiked into samples at different concentrations.
Using a high-binding ELISA plate, the adalimumab antibody drug was coated overnight at 1 µg/mL. The plate was then washed and blocked with 5% BSA in PBST. Next, the anti-adalimumab antibody was titrated between 1 and 1000 ng/mL in a 2% BSA + PBST + 10% human serum buffer for the calibration curve.
Once the anti-adalimumab antibody was captured and the ELISA plate was washed, the detection was carried out with biotinylated adalimumab at 1 µg/mL in the same buffer. After detection, the plate was again washed and the SbG enzyme was added into the Quanterix SbG diluent at 500 pM. After the enzyme was labeled, the ELISA plate was washed again and subsequently incubated with RGP substrate. Finally, resorufin fluorescence was determined as an assay readout.
Figure 1. Schematic image of ADA bridging assay. Monoclonal antibody-drug (gold) as capture antibody and detection antibody labeled with biotin. A fully human anti-idiotypic antibody, Ig format (blue). Image source Bio-rad.
Using EDC+SNHS chemistry, adalimumab was conjugated to magnetic microparticles. The anti-adalimumab antibody was then titrated between 1 and 100 ng/mL in a 2% BSA + PBST + 10% human serum buffer for the calibration curve. The same detection antibody—1 µg/mL biotinylated adalimumab—used for the ELISA assay was also used for the Simoa assay.
Labeling was carried out with 50 pM SBG. Then, the assay was used on the Simoa HD-1 platform as a three-step assay with 60- 15-, and 15 minute-incubation times for each of the steps.
Both the ELISA assay and the Simoa assay displayed a dose-dependent response to the anti-adalimumab antibody in a calibration curve, suggesting that either of the assays can be used for measuring the concentration of autoantibodies against the adalimumab antibody drug in patient samples. The Simoa assay showed approximately eight times higher signal-to-background ratios across the calibration curve (<100 ng/mL).
Figure 2. The signal-to-background ratio of the Simoa assay versus plate ELISA for calibrators under 115 ng/mL.
In addition, both the ELISA assay and the Simoa assay displayed relatively linear dose responses of around 100 ng/mL. The plate ELISA assay has an upper limit of quantification (ULOQ) of 215 ng/mL, while that of the Simoa assay is 200 ng/mL. The lower limit of quantification (LLOQ) for the Simoa assay is 0.14 ng/mL, while that of the plate ELISA assay is 0.98 ng/mL.
Figure 3. Calibration curve for Simoa assay (0.137–100 ng/mL)
For the Simoa assay, the limit of detection (LOD), measured as three standard deviations above the zero calibrator, was 0.05 ng/mL, while it was LOD of 0.5 ng/mL for the plate ELISA assay.
Figure 4. Calibration curve for plate ELISA assay (1.37–1000 ng/mL)
Figure 5. Simoa versus ELISA calibration curve (relative units)
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