Particle concentration fluorescence as an alternative to radioactivity for cytokine receptor binding assays.
To detect inhibitors of the type-I interleukin-1 receptor in a safe and cost-effective nonisotopic environment, the use of particle concentration fluorescence (PCF) as a detection method for a high volume receptor binding assay (RBA) has been explored. A cell-free PCF-RBA has been developed using a soluble form of the type-I IL-1 receptor (sIL-1R), a nonneutralizing anti-sIL-1R monoclonal antibody and two fluorescein-labelled tracers: IL-1 alpha and IL-1ra fused to the Escherichia coli maltose binding protein (MBP-IL-1ra). Both ligands showed saturable specific binding (KA values in the 10(9) M-1 range), although signal intensity was about 3-fold higher with MBP-IL-1ra. The data presented here suggest that the combined use of recombinant DNA and PCF technologies can replace the traditional cell-based, radioactive RBA when screening for inhibitors of cytokine receptors.
Sarubbi
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Lepetit Research Center, Hoechst Marion Roussel, Gerenzano, Italy.
D001667:Binding, Competitive / Q000276:immunology; D019793:Fluorescein; D005452:Fluoresceins; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D017879:Isothiocyanates; D008863:Microspheres; D010316:Particle Size; D011137:Polystyrenes; D011869:Radioligand Assay; D017472:Receptors, Interleukin-1 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility
D005452:Fluoresceins; D017879:Isothiocyanates; D011137:Polystyrenes; D017472:Receptors, Interleukin-1; D019793:Fluorescein