Research On The Oriented Immobilization Using Peptide Ligand

Antibody is immobilized on a solid-phase, which is usually used in immunoassay.Because of the random immobilization, the lower activity of antibody will beachieved. Some methods have been reported to make the antibody successfullyorientedly immobilized on the solid-phase. In this paper, we developed a new methodfor oriented immobilization of antibody using a peptide ligand which had a highaffinity with the Fc fragment of antibody.In this paper, dispersion polymerization was used to prepare monodispersedmicrospheres pGMA with a surface of epoxy groups. Modification of the microspheresurface was carried to achieve different function groups, including sulphuric acid,epichlorohydrin, ethanediamine and peptide HWRGWVC. Then a systematiccharacteration was carried, including particle size analyzer, scanning electronmicroscopy (SEM), fourier transform infrared spectra (FTIR) and reversed–phasechromatography (RPC). The results showed that we successfully preparedmonodispersed microspheres of1.6μm, while the SEM results showed themicrospheres were uniform and spherical, with no pores on the surface. Besides,FTIR and RPC results showed all the modification were effective. The adsorptionresults showed IgG could be adsorbed to the microspheres coupled with the peptidewith a capacity of3-4mg/g. The activity of antibody immobilized on themicrospheres was also determined and proved to be higher than the EDC method.Besides, emulsion polymerization was used to prepare monodispersednanoparticles pGMA of110nm. The adding of crosslinking agent EDMA couldimprove the stability of nanoparticles. Then amino groups were introduced and usedfor the carbodiimide reaction with the peptide HWRGWV. In the end, we studied thestability of nanoparticles before and after adding the EDMA and the result showednanoparticle size were more stable after adding EDMA. In the end, we studied theadsorption behavior of IgG on the nanoparticles with different function groups.Meanwhile, ITC was used to study the interation between the IgG and thesenanoparticles. The results showed the nanoparticles with a surface of peptide andamino could adsorb IgG. The former one had a stronger interaction through thehydrophobic interaction produced by the peptide, while the latter one had a weakerinteration through the electrostatic interaction and hydrogen bond produced by theamino groups on the nanoparticle surface.