Spectroscopic Studies of Interaction between Hapten and Monoclonal Antibody (McAb) against Iso-tenuazonic Acid
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Abstract:
The interaction between iso-tenuazonic acid (ITeAH) hapten and monoclonal antibody (McAb) against ITeAH was studied by ultraviolet-visible (UV-vis) spectroscopy, fluorescence spectroscopy, and circular dichroism (CD). Fluorescence results were verified using enzyme-linked immunosorbent assay (ELISA). The results suggested that fluorescence intensity of McAb was quenched by ITeAH, while the quenching mechanism was based on static quenching and nonradioactive energy-transfer theory. The binding constants (Ka) at 298, 310, and 318 K were calculated as 1.9 × 106, 1.6 × 106, and 1.4 × 106 L/moL, respectively, consistent with the values determined by ELISA. The binding point (n) number was about 1, while the average binding distance (r) was 3.35 nm. Furthermore, thermodynamic parameters evaluated during the binding process showed that the binding force between ITeAH and McAb constituted of mainly electrostatic attraction, which affected the extent of binding between ITeAH and McAb, when studied under different pH conditions by using ELISA. The synchronous fluorescence spectra indicated that the addition of ITeAH did not change the microenvironment of tyrosine and tryptophan residues in the McAb, and that the binding site for ITeAH was more inclined to tyrosine residues. CD analysis showed significant changes in the secondary structure of McAb after interaction with ITeAH.
