(M1330-06-36) Establishment of an Immunoassay to Quantify Total TL1α Target in Monkey Serum Samples

Purpose: Quantification of target is frequently required for bioanalysis during therapeutic antibody development. However, it is challenging to quantify a target accurately when target levels are lower and/or when levels of therapeutic antibody are higher. To develop an assay for total (free plus bound) target TL1α measurement with sufficient sensitivity (<40 pg/mL) and drug tolerance (up to 50 μg/mL anti- TL1α antibody), we evaluated several assay formats in terms of sensitivity and tolerance to drug interference for quantification of total amount of TL1a in the presence of an excess amount of anti-TL1α antibody in monkey sera.

Methods: Four Sandwich assay formats employ an anti-TL1a non-competitive (non-human) monoclonal antibody as capture reagent, and anti-human IgG-HRP (Format A of Figure 1) or biotinylated anti-human IgG (Formats B, C, D of Figure 1) as the detection reagent after a saturation step by the excessive drug (a human anti-TL1α antibody). Additionally, a direct immunoassay is built with sample pre-treatment with purification and acid dissociation steps, followed by detection with a biotinylated anti-target non-competitive (non-human) monoclonal antibody (Format E of Figure 1). Two detection technologies are used in these assays - Except electrochemiluminescence detection system for the 4th assay format (Format D of Figure 1), all other assay formats use optical density detection technology. Assay performance was evaluated and compared among these formats in terms of assay sensitivity and drug tolerance. One assay format is selected for validation.

Results: Compared to the direct assay, all four Sandwich assays exhibited greater sensitivity (36-105 pg/mL versus 1216 pg/mL). Specifically, the sensitivity ranks in the order: format C ≈ format D > format A ≈ format B > format E (Table 1). Among these assays, the ability to tolerate drug interference ranks in the order: format C ≈ format D ≈ format B > format A > format E (Table 2). The final selected assay (format C) has the capability to accurately quantify TL1α as low as 36 pg/mL in the presence of the anti-TL1α antibody at a level of up to 50 μg/mL. Validation results of the assay all passed acceptance criteria.

Conclusion: Among 5 assays tested for total TL1α target analysis in this study, formats C and D have been found to display greater assay sensitivity as well as the drug tolerance when compared to other assays. The selected assay format C demonstrated sufficient assay sensitivity, precision, accuracy, and drug tolerance for the intended application.


Figure 1. Assay design and formats


Table 1. Determination of assay sensitivity

Table 2. Drug tolerance assessment of total target assay