(M1230-01-07) Development, Optimization and Qualification of an IFN-γ ELISPOT Assay for Human PBMCs

Purpose: The enzyme-linked immunoSpot (ELISPOT) assay represents a highly sensitive, specific, and high-throughput method for cellular immune response assessment for AAV-based gene therapies and vaccines. However, this assay is subjected to many preanalytical variables (e.g. whole blood transport and storage conditions, PBMCs isolation/recovery protocols). This study aims to assess effects of these variables and to evaluate an optimized condition for improved assay performance.

Methods: An ELISPOT assay is built on PBMCs with IFN-γ spot-forming unit (SFU) as the assay readout. Assay optimization experiments are conducted to: 1) compare specimen transport conditions (room temperature vs 2–8°C) and PBMC recovery conditions; 2) define an optimal cell seeding density and an optimal cell incubation time. After an optimized condition is established, assay qualification experiments are carried out to assess assay sensitivity, quantification range, precision, robustness, and linearity.

Results: As showed in Table 1, assay optimization experiments demonstrated that: 1) specimens should be transported at 2–8℃ and processed within 24 hours post-collection; 2) cell seeding densities should be maintained between 1×10⁴ and 10×10⁴ cells/well to ensure linear response dynamics; 3) incubation time should be strictly controlled at 18–22 hours. Using the optimal condition, method qualification results are shown in Table 2 and Figure 1, which demonstrates good assay sensitivity, an acceptable linearity, and an adequate precision profile and assay robustness.

Conclusion: This study defines an optimized condition for PBMC sample pre-treatment in ELISPOT assays. Following assay optimization, a sensitive, robust method is established for evaluating cellular immunity using IFN-γ SFU readout in PBMCs.

Table 1. IFN-γ SFU of PBMCs isolated under different whole blood shipping conditions.

Table 2. Assay precision of IFN-γ ELISPOT assay.

Figure 1. Linearity of IFN-γ ELISpot assay.