Kinetic Analysis of Interaction Between Tumor Necrosis Factor and Its Soluble Receptors

The kinetic analysis of interaction between tumor necrosis factor (TNF) and its two soluble extracellular receptors was carried out by using surface plasmon resonance (SPR) technique in the present study. Two immobilization strategies were used. For the first one, TNF was covalently immobilized on the CM5 sensor chip by amine coupling method, and the interaction between TNF and its soluble receptors were analyzed. For the second one, Protein A was first covalently immobilized on the CM5 sensor chip by amine coupling method, and TNF sR I/Fc Chimera or TNF sR II/Fc Chimera was subsequently captured on the sensor chip through the specific interaction of their Fc moiety with Protein A. The kinetics of immobilized soluble receptors interacted with TNF was studied by surface plasmon resonance. The experiment results showed that the kinetic parameters obtained from these two strategies agreed well. The affinity constant KD of TNF with its soluble receptors was in the magnitude of 10−9 M, indicating a higher affinity than that of the antibody-antigen interaction (KD: 10−5–10−7 M)