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Tumor necrosis factor α (TNFα) is a cytokine that is mainly produced by macrophages and monocytes. It is a member of a large family of proteins, the TNF family. A lot of research in different species has been undertaken regarding the usefulness of this protein as a marker for a variety of diseases. The aim of this study was to develop a simple, reproducible protocol for the purification of recombinant feline TNFα from modified Escherichia coli, the partial characterization of the rfTNFα protein, the production of anti-rfTNFα antibodies, as well as the development and analytical validation of an ELISA for the measurement of feline TNFα in serum from cats. We also aimed to measure serum fTNFα concentrations in cats with chronic enteropathies.
An efficient and reproducible protocol for the purification of rfTNFα from Escherichia coli, modified to express fTNFα was established. The specific absorption and the molecular weight of the protein were evaluated. An ELISA for the measurement of fTNFα concentrations in serum from cats was developed and analytically validated. A reference interval was established by determination of the central 95th percentile in a group of 20 healthy cats. Lastly, serum concentrations of fTNFα in cats with chronic enteropathies were evaluated.
The specific absorbance of rfTNFα was found to be 1.75. The relative molecular mass was estimated at 16.93 and 17.57 kDa for 2 isoforms of rfTNFα. The N-terminal amino acid sequences for both isoforms of the protein were found to be R-T-P-S-D-K-P-V-A-H-V and SS- S-R-T-P-S-D-K-P-V, respectively. Both sequences showed 100% homology with the amino acid sequence of mature feline TNFα, as predicted by the nucleotide sequence of the protein. The isoelectric point of rfTNFα was estimated at approximately 5.3.
The ELISA for the measurement of serum concentrations of fTNFα in cats was determined to be sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use. The reference interval for fTNFα in serum from healty cats was established at <223.5 ng/l. Feline TNFα was only detectable in 33.3% of cats with chronic enteropathies.
In conclusion, a sensitive, linear, accurate, precise, and reproducible assay for the measurement of TNFα in cat serum was developed. Further studies are needed to explore if serum fTNFα concentrations could serve as a useful marker for the diagnosis, staging, and monitoring of feline patients with chronic enteropathies.