Development of a sandwich ELISA for recombinant feline GM-CSF and determination of its serum half-life
- 1College of Veterinary Medicine, Huazhong Agricultural University , ,
- 2Hubei Jiangxia Laboratory , ,
Abstract
Introduction: Feline granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key cytokine involved in the regulation of myeloid immune responses and represents an important therapeutic target for leukopenia. However, the rapid in vivo 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: The assay exhibited excellent linearity within 7.8–2000 pg/mL (R2 = 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.
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BibTeX
@article{yu2026development,
title = {{Development of a sandwich ELISA for recombinant feline GM-CSF and determination of its serum half-life}},
author = {Chun Yu and Miao Wang and Li Chen and Tianliang Wang and Ya Zhao and Hetao Song and Meilin Jin},
journal = {Frontiers in veterinary science},
year = {2026},
month = oct,
doi = {10.3389/fvets.2026.1958789},
url = {https://europepmc.org/article/PMC/PMC13725428},
note = {CC BY},
}
RIS
TY - JOUR
TI - Development of a sandwich ELISA for recombinant feline GM-CSF and determination of its serum half-life
AU - Chun Yu
AU - Miao Wang
AU - Li Chen
AU - Tianliang Wang
AU - Ya Zhao
AU - Hetao Song
AU - Meilin Jin
PY - 2026
DA - 2026/10/19
JO - Frontiers in veterinary science
DO - 10.3389/fvets.2026.1958789
UR - https://europepmc.org/article/PMC/PMC13725428
AB - Introduction: Feline granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key cytokine involved in the regulation of myeloid immune responses and represents an important therapeutic target for leukopenia. However, the rapid in vivo 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: The assay exhibited excellent linearity within 7.8–2000 pg/mL (R2 = 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.
SN - 2297-1769
ER -