A novel monoclonal antibody screening method using the Luminex-100 microsphere system.
We describe the development of a robust and sensitive assay system (detection limit <500 pg/ml biotin-IL-6, K(d)=75 ng/ml), using Luminex-100 microspheres, that could effectively screen for neutralizing antibody whenever a soluble form of the receptor for a target molecule is available. As an example, we coupled a recombinant human interleukin-6 soluble receptor to a Luminex carboxylated microsphere and used a biotin-labeled recombinant human interleukin-6 as a probe to assess binding competition. Three anti-human IL-6 monoclonal antibodies that bind distinct IL-6 epitopes were used as test articles to evince the stringency of the screen. Our assay was able to detect antibody concentration as low as 10 ng/ml without interference from hybridoma growth medium or cell supernatant. The time-saving benefits of this assay format make it ideal for high-throughput screening (HTS) applications for neutralizing monoclonal antibodies.
Seideman
Jonathan
J
Department of Immunobiology, Centocor, 200 Great Valley Parkway, Malvern, PA 19355, USA. jseidema@cntus.jnj.com
Peritt
David
D
D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001710:Biotin; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D008163:Luminescent Measurements; D008863:Microspheres; D009500:Neutralization Tests; D019947:Receptors, Interleukin-6 / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D001710:Biotin