Kinetic characterization of the interaction of biotinylated human interleukin 5 with an Fc chimera of its receptor alpha subunit and development of an ELISA screening assay using real-time interaction biosensor analysis.
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 an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5R alpha-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor alpha 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 the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5R alpha-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/shIL5R alpha-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 a microtiter 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 shIL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Bennett
D
D
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, PA 19406, USA.
Morton
T
T
Breen
A
A
Hertzberg
R
R
Cusimano
D
D
Appelbaum
E
E
McDonnell
P
P
Young
P
P
Matico
R
R
Chaiken
I
I
D000818:Animals; D015374:Biosensing Techniques; D001710:Biotin; D003001:Cloning, Molecular; D004330:Drosophila; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015870:Gene Expression; D006801:Humans; D015848:Interleukin-5 / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011961:Receptors, Fc / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D015848:Interleukin-5; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001710:Biotin