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As a crucial member of the Interleukin-1 (IL-1) family, IL-33 plays an indispensable role in modulating inflammatory responses. Here, we developed an effective anti-human IL-33 monoclonal antibody (mAb) named 5H8. Importantly, we have identified an epitope (FVLHN) of IL-33 protein as a recognition sequence for 5H8, which plays an important role in mediating the biological activity of IL-33. We observed that 5H8 significantly suppressed IL-33-induced IL-6 expression in bone marrow cells and mast cells in a dose-dependent manner in vitro. Furthermore, 5H8 effectively relievedHDM-induced asthma and PR8-induced acute lung injury in vivo. These findings indicate that targeting the FVLHN epitope is critical for inhibiting IL-33 function. In addition, wedetected that the Tm value of 5H8 was 66.47℃ and the KD value was 173.0 pM, which reflected that 5H8 had good thermal stability and high affinity. Taken together, our data suggest that our newly developed 5H8 antibody has potential as a therapeutic antibody for treating inflammatory diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)