Probe antibody immobilization on matrix surface plays a key role in solid phase immunoassays.The traditional amide bond formation by EDC/NHS chemistry often leads to random orientation as it binds the antibody via amino acid residues which may locate at,or close to antigen-binding site,thus results in relative low antigen-binding capacity.Another binding strategy via oxidized glycochains linkage involves periodate oxidation of the carbohydrate moieties locating at the Fc region of the antibody into aldehyde group,following their coupling to amino or hydrazino groups on the matrix by a Schiff base or hydrazone formation,should lead to antibody-matrix conjugates that possess higher antigen binding activity.In this study,both immobilization schemes were illustrated to quantitatively investigate the efficacy of antibody immobilization.Both mouse anti goat monoclonal antibody-matrix conjugates using the above two methods were incubated individually with Alexa488 goat anti mouse reporter antibody and Alexa488 goat anti rabbit reporter antibody.The detected fluorescence signal from Alexa488 goat anti mouse reporter antibody characterized the amount of total binding antibody and the signal fromAlexa488 goat anti rabbit reporter antibody characterized the amount of oriented binding antibody.The result showed that the orientation effect using glycochains linkage-based binding was three times better than that using traditional amino-based binding,while the total amount of binding antibody had a decrease.