TNF alpha is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNF alpha antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNF alpha and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNF alpha was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The K-D values for rhTNF alpha interactions with their antagonists were obtained by noncompetitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFa under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNF alpha monomers. For the first time, the interactions of rhTNF alpha with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high K-D values (<10(-9) M and >10(-8) M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNF alpha-antagonists with rhTNF alpha monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNF alpha and its antagonists.