Comparison and development of two different solid phase chemiluminescence ELISA for the determination of albumin in urine Original Research Article

Two chemiluminescence enzyme-linked immunosorbent assay (ELISA) methods based on avidin–biotin system and Fluorescein-iso-thiocyanate (FITC)-anti-FITC system as two different solid phase for the determination of albumin in urine were optimized, characterized and compared. Avidin or anti-FITC antibody was coated in the microplates to provide a universal solid phase which improved the variability and sensitivity. Alkaline phosphatase (ALP) labeled albumin and albumin from standard or patient samples compete for biotinylated-antibody or FITC labeled antibody binding sites. Enzyme activity in the bound fraction was detected with the chemiluminescence substrate 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane) (AMPPD). The influence of several physico-chemical parameters, such as incubation time, detergent concentration and solid phase conditions were also studied. For the two solid phase, both the linear range and the limit of detection of albumin were 0.15–15 and 0.089 μg/mL, compared with the commercially ELISA kit, a good correlation were obtained. Moreover, three different solid phases include avidin–biotin system, FITC–anti-FITC system and the anti-albumin Ab directly coated the microtitre plates were compared mainly from the cost and precision, the results showed: (1) when the avidin–biotin system as solid phase, the amounts of the antibody used was only the 1/10 than those of the anti-FITC antibody as solid phase and antibody directly coated the microtiter plates; (2) the C.V. of these two solid-phase CL ELISA methods were lower than that of antibody directly coated solid phase.