Characterization of Antibody Interaction Using Surface Plasmon Resonance for the Application in Nanoparticle-Based Immunoassays
Author
Deimantaviciute, Egle
Term
4. term
Education
Publication year
2017
Submitted on
2017-06-12
Pages
102
Abstract
Dette studie undersøgte, hvordan en laboratoriefremstillet version af NS1-proteinet fra denguevirus type 2 binder til to monoklonale antistoffer, testet enten som frie antistoffer eller bundet til nanopartikler. Vi brugte overfladeplasmonresonans (SPR), en metode der måler binding i realtid uden mærker, samt en sandwich-opstilling til at vurdere, om to antistoffer kan binde proteinet samtidigt. Antistofferne blev kemisk fastgjort til sensoroverfladen ved aminkobling. For begge antistoffer målte vi associerings- og dissociationshastigheder samt den samlede bindingsstyrke (affinitet). Flere målinger var inkonsistente, hvilket tyder på, at forsøgsbetingelserne ikke var optimale. For ét antistof, Mer 39, gav sandwich-analysen en associeringskonstant på 1.13e5 1/(M*s), en dissociationskonstant på 1.23e-4 1/s og en affinitet (KD) på 1.09e-9 M. Sandwich-forsøgene undersøgte også epitop-overlap, dvs. om de to antistoffer genkender samme område på NS1. Epitoperne overlappede ikke, men begge antistoffer kunne kun binde, når komplekset blev dannet i en bestemt rækkefølge. Overordnet kræves der mere arbejde for at afgøre, hvor velegnet SPR er til en fuld karakterisering af, hvordan nanopartikel-konjugerede antistoffer interagerer med NS1.
This study investigated how a laboratory-made version of the dengue virus type 2 NS1 protein binds to two monoclonal antibodies, tested either in free form or attached to nanoparticles. We used surface plasmon resonance (SPR), a real-time, label-free method, and a sandwich setup to assess whether two antibodies can bind the protein at the same time. The antibodies were chemically fixed to the sensor surface by amine coupling. For both antibodies we measured association and dissociation rates and overall binding strength (affinity). Several measurements were inconsistent, indicating that the experimental conditions were not yet optimal. For one antibody, Mer 39, sandwich analysis gave an association rate constant of 1.13e5 1/(M*s), a dissociation rate constant of 1.23e-4 1/s, and an affinity (KD) of 1.09e-9 M. The sandwich experiments also probed epitope overlap, that is, whether the two antibodies recognize the same region on NS1. The epitopes did not overlap, but both antibodies could bind only when the complex formed in a specific order. Overall, further work is needed to determine how suitable SPR is for fully characterizing how nanoparticle-conjugated antibodies interact with NS1.
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