Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic cytokine that participates in both innate and adaptive immune responses. It is constitutively expressed in various cell types and rapidly released upon exposure to inflammatory stimuli, playing a pivotal role in regulating macrophage functions—including phagocytosis, cytokine production, and immune activation. Beyond immune regulation, MIF is also involved in modulating cell proliferation, apoptosis, and autophagy. Clinically, it has been implicated in the pathogenesis of multiple diseases, such as sepsis, autoimmune disorders, and various malignancies. Mechanistically, MIF binds to Cluster Of Differentiation 74 (CD74)—the invariant chain of major histocompatibility complex (MHC) class II complexes—with high affinity. As MIF’s primary high-affinity receptor, CD74 mediates the initiation of pro-inflammatory signaling pathways downstream of MIF binding.To detect the activity of recombinant MIF, a functional ELISA assay was performed to evaluate the interaction between recombinant human MIF and recombinant rat CD74.Briefly, biotin-linked MIF were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to CD74-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50μl stop solution to the wells and read at 450nm immediately. The binding activity of MIF and CD74 was shown in Figure 1, the EC50?for this effect is 0.16 μg/mL.