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January 15, 2011 (Vol. 31, No. 2)

Antibodies Against Membrane Protein Targets

Lipoparticle Technology Facilitates Discovery in This Challenging Protein Class

  • Modified Lipoparticles

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    Figure 2. (A) Biotinylated Lipoparticles containing CXCR4 were captured onto streptavidin-coated ELISA plates and probed with the conformation-specific anti-CXCR4 antibody 12G5. (B) A phage library panned using Lipoparticles resulted in enrichment of phage reactivity against Lipoparticle targets. (C) Biotinylated Lipoparticles containing CXCR4 were captured onto a streptavidin biosensor chip and increasing concentrations of the conformational antibody 12G5 were used as the flowed analyte. (D) Fluorescent Lipoparticles were bound to beads and visualized by fluorescence microscopy. Beads were then immunostained using 12G5 (inset) to demonstrate conformational integrity of the incorporated receptor.

    The addition of chemical moieties to proteins represents a strategy utilized in many phases of mAb discovery to capture and visualize target proteins. For example, the biotinylation of proteins enables their attachment to avidin surfaces with high affinity while the addition of fluorophores allows for protein localization, quantification, and sorting. These biochemical modifications have been applied to Lipoparticles to enable membrane proteins to be immobilized on solid supports and visualized without disrupting the conformation of incorporated receptor targets during mAb characterization.

    For example, biotinylated Lipoparticles attached to neutravidin microplates have been used to screen antibodies and hybridoma supernatants by ELISA for highly reactive monoclonal antibodies (Figure 2A). Biotinylated Lipoparticles immobilized onto avidin wells and beads have also been used to enrich phage libraries for reactive antibodies (Figure 2B). Finally, biotinylated Lipoparticles have been immobilized onto streptavidin-coated biosensor chips to enable detailed kinetic measurements of mAb binding to membrane protein targets (Figure 2C).

    Lipoparticles that have been biochemically modified to contain fluorophores enable advanced visualization, quantification, and sorting of target proteins by microscopy, microplate fluorescent readers, and flow cytometry (Figure 2D). For example, fluorescent Lipoparticles have been used to facilitate antibody discovery strategies such as yeast display where yeast cells express a library of antibodies and those with desired specificity are sorted by their interactions with fluorescent Lipoparticles.

    Lipoparticles represent a novel approach to discovering antibodies against previously intractable and clinically relevant membrane protein targets. When used as an immunogen, the high concentration of structurally intact membrane proteins helps elicit a focused and robust immune response. Modifications to Lipoparticles, including biotinylation and fluorescence, can facilitate a variety of screening strategies for isolating and characterizing monoclonal antibodies. These tools have the potential for helping develop key diagnostic and therapeutic reagents against this challenging class of proteins.

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