Antibody Immobilization On The Surface Of Micro/Nano Materials And Its High-sensitivity Digital ELISA Applications

Antibody functionalized micro/nano materials are used in various fields due to their high affinity and specificity.However,both uncontrolled antibody molecular state and non-specific adsorption reduce antigen binding avidity.We prepared a series of silica nanoparticles with different charged polymers,followed by surface immobilization of antibody.In this thesis we investigated the effects of surface and solution factors on antibody immobilization and antigen binding avidity,including antibody type,charge density,ionic strength and p H value,etc.In addition,we screened conditions for the detection of IL-17A(interleukin-17A)by high-sensitivity digital ELISA.In this project,first,to prepare nanoparticles with different electrical properties and charge densities,ten monomers(positive,negative and zwitterionic)were selected to grow polymer brushes by atom transfer radical polymerization.The positive charge densities are in the range from 26.9±1.2 m V to 43.7±1.6 m V,while the negative charge densities are in the range from-20.9±2.4 m V to-40.4±1.8 m V.Then,antibody was immobilized on the nanoparticles via physical adsorption.The results indicated that on positively charged nanoparticles the Fab domain of antibody oriented toward solution.As the positive charge densities increased,the amount of immobilized antibody decreased,and antigen binding avidity increased then followed by a decrease.On negatively charged nanoparticles Fc domain oriented away from solution.With increasing negative charge density,antigen binding avidity decreased first then followed by a increase.Consequently,we investigated the effects of three major factors(pH,ionic strength and hydrodynamic diameter of nanoparticles)on K_d value.Finally,we examined conditions for the detection of IL-17A by high-sensitivity digital ELISA.According to calculated values of R²,C_calculated/C_real and coefficient of variation(CV value),the optimized incubation conditions were determined to be:two-step incubation,detection antibody concentration of 0.3?g/m L,enzyme concentration of 40 p M and incubation time of 2 h.The CV values of IL-17A detection are in the range of 1.98-9.78%through 5repetition,with good reproducibility and accuracy.The limit of detection is 0.1 pg/m L,which is 180 times more sensitive than commercial IL-17A ELISA kits.