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Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

ABSTRACT: Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

SUBMITTER: Bee C

PROVIDER: S-EPMC3340344 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

Bee Christine C Abdiche Yasmina N YN Stone Donna M DM Collier Sierra S Lindquist Kevin C KC Pinkerton Alanna C AC Pons Jaume J Rajpal Arvind A

PloS one 20120430 4

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnit ...[more]

PMID: 22558410

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Project description:Serum-to-2i interconversion of mouse Embryonic Stem Cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary but not sufficient to establish these interactions, as confirmed by Capture Hi-C on Eed-/- serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation. To study dynamics in chromatin architecture and to characterize long-range interaction, we performed Hi-C using DpnII as the restriction enzyme, potentially reaching a genome-wide coverage at a less than 1Kb resolution. We subsequently performed enrichment of interaction by a target capture similar to the exome sequencing approach. We enriched for DNaseI hyper-sensitive sites (DHSâs) in chromatin from mESCs. Probes were designed against the union of all DHSâs of Serum and 2i mESCs. Capture Hi-C reveals Extremely Long-Range Interactions (ELRI) in Serum but not in 2i ESCs. We observed H3K27me3 as a prominent characteristic, but not exclusive feature of ELRI loci in Serum mESCs. To further elucidate the involvement of constituents of PRC1 and PRC2 in ELRI, we performed ChIP-seq experiment on Suz12 and Ring1B during serum-to-2i transition. In addition, RNA-seq was performed to compare the expression levels of genes.

Dataset Information

Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

Publications

Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions.

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