Basic Study on Development and Application of Ultrasensitive Enzyme Immunoassay for Interleukins

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We developed a highly sensitive two-site enzyme immunoassay technique and applied it to the measurement of antigens at attomole levels. However, the sensitivity by this technique is not sufficiently high for the measurement of interleukins (IL). For example, the detection limits of hIL-1alpha, hIL-1beta and hIL-2 were 5, 30 and 200 amol, respectively. Therefore, attempts were made to develop more sensitive enzyme immunoassay techniques for interleukins.1. In order to trap antigens onto antibody-coated polystyrene balls with high efficiency, polyclonal antibody IgG is usually purified by affinity chromatography on a column of antigen-Sepharose 4B. Unfortunately, the antibody IgG preparations thus affinity-purified contain more or less antigens released from antigen-Sepharose 4B, which cause high background to limit the sensitivity of two-site enzyme immunoassay. To eliminate these antigens, antigen-biotinylated nonspecific rabbit IgG conjugate was coupled to CNBr-activated Sepharose 4B. … More Antibody IgG affinity-purified by elution from a column of antigen-biotinylated nonspecific rabbit IgG-Sepharose 4B was passed through a column of streptavidin-Sepharose 4B and was subjected to gel filtration. When erythropoietin was used as model antigen, the background of two-site enzyme immunoassay using affinity-purified anti-erythropoietin IgG-coated polystyrene balls was significantly lowered, improving the sensitivity 3 fold as compared with that before affinity-purification.2. Sandwich transfer enzyme immunoassay. The complex formed of antigen with biotinyl dinitrophenyl antibody IgG and antibody Fab^1-beta-D-galactosidase conjugate was trapped onto affinity-purified anti-dinitrophenyl IgG-coated polystyrene balls. After eliminating excess of the conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with streptavidin. beta-D-Galactosidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorimetry. When ferritin was used as model antigen, nonspecifically bound beta-D-galactosidase activity considerably decreased with less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 3 milliattomoles.3. On the basis of these results, it is being planned to develop highly sensitive two-site enzyme immunoassay for interleukins and measure them in the culture supernatants of immunocompetent cells. Less