ISSN:
0952-3499
Keywords:
cytokine
;
receptor
;
kinetics
;
biosensor
;
interaction
;
screening
;
ELISA
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring as ELSIA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5Rα-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor α subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of theFc chimera attached by oriented immobilization via protein A. Hence, shIL5Rα-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5Rα-Fc receptor complex. The binding was high affinity (Kdapp=6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5Rα. The results demonstrate a novel use of real-time biosensor technology as a macromolecular interaction analysis tool to help develop other assay using immobilized ligands, including high throughout screening assays to identify antagonist leads. The biosensor technology also offers a means to evaluate the mechanism of action of lead compounds by competition binding, as well as by direct binding if the leads are large enough or can be immobilized to the sensor surface.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jmr.300080109
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