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Background: An affibody molecule obtained from a bioengineered staphylococcal protein was previously shown to act as an affinity binder for a wide range of targets and develop Tumour Necrosis Factor α (TNF-α)-binding clones. Methods: In this study, we demonstrated that affibody molecules against TNF-α could bind to recombinant TNF-α on the membrane for biochemical detection. In addition, we examined whether the affibody molecules could block binding between recombinant TNF-α and its receptor on MH7A synovial cells. Results: When a TNF-α-binding affibody was added, the production level of inflammatory mediators IL-6 and MMP-3 in MH7A were found to decrease up to 44%. Additionally, proliferation of synovial cells was also inhibited by the addition of TNF-α to cultivation media. Conclusion: These results suggest that affibody molecules against TNF-α could be candidate molecules for the detection of TNF-α during biochemical analysis and pharmacotherapy for rheumatoid arthritis.