Labeling systems, color concentrates

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

In another Eu-Tb two-color immunoassay for two low concentration components, highly luminescent labels, BPTA-Tb3+ and BHHCT-Eu3+ were used for human serum AFP and CEA (carcinoembryonic antigen) by Yuan et al. (2001) (fig. 12). As shown in fig. 10, a well was coated with AFP and CEA antibodies, and after the serum sample was applied, anti-AFP antibody labeled with BHHCT-Eu3+ and anti-CEA antibody labeled with biotin were reacted. After washing the well and measuring the emission of the Eu chelate at 615 nm, SA labeled with BPTA-Tb3+ was added and the emission of the Tb chelate at 545 nm was measured. In this system, the detection limits of AFP and CEA were very low, 44 pg/ml and 76 pg/ml, respectively. 

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