Work

Development of a sandwich ELISA for recombinant feline GM-CSF and determination of its serum half-life

Year: 2026

Type: article

Abstract: Introduction: clearance of native GM-CSF requires frequent administration, which limits its clinical translation. To overcome this limitation, a recombinant GM-CSF-Fc protein was constructed using Fc fusion technology. Pharmacokinetic evaluation of GM-CSF-Fc, however, is constrained by the lack of analytical approaches capable of simultaneously quantifying differently engineered forms of recombinant GM-CSF. Methods: In this study, recombinant feline GM-CSF was expressed in a CHO eukaryotic expression system and used to immunize rabbits to generate high-affinity polyclonal antibodies. Based on these antibodies, a sandwich ELISA was established. Results: = 0.993), and its validated quantitative range was 50-2000 pg/mL, covering the expected concentration fluctuations of recombinant GM-CSF under physiological and immune-activated conditions in cats. The assay demonstrated good precision and accuracy, and no cross-reactivity with related feline cytokines (IL-3, IL-5, M-CSF, and G-CSF) was observed. Notably, the system efficiently quantified differently engineered forms of recombinant feline GM-CSF (His-tagged and Fc-fused) with comparable recovery, overcoming the limitations of conventional assays optimized for a single protein format. Pharmacokinetic analysis following subcutaneous administration in cats showed that Fc fusion substantially prolonged the apparent terminal elimination half-life of immunoreactive GM-CSF from 5.05 ± 0.91 h to 40.89 ± 3.41 h, supporting the improved pharmacokinetic profile of the Fc-fused construct. Discussion: In conclusion, this study provides a versatile and sensitive analytical platform for the preclinical evaluation of Fc-fused feline GM-CSF formulations and serves as an important methodological reference for the quantitative assessment of Fc-fused veterinary biologicals.

Source: Frontiers in Veterinary Science

DOI: 10.3389/fvets.2026.1958789