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Bi-epitope SPR surfaces: a solution to develop robust immunoassays.

Peng L, Damschroder MM, Wu H, Dall'Acqua WF - PLoS ONE (2014)

Bottom Line: The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and 'single-epitope' surfaces were then compared.For all antibody pairs tested, bi-epitope surfaces exhibited an ∼ 10-100-fold improvement in apparent binding affinities when compared with single-epitope ones.This led to an ∼ 100-200-fold enhancement in EphA2 limit of detection in crude cell supernatants.

View Article: PubMed Central - PubMed

Affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, United States of America.

ABSTRACT
Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called 'bi-epitope' sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and 'single-epitope' surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼ 10-100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼ 10(-8) M) and fast dissociation rates (∼ 10(-2) s(-1)), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼ 10(-10) M and 10(-4) s(-1), respectively. This led to an ∼ 100-200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development.

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EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.When using the single-epitope high density surfaces, dissociation rates were fast and similar to that of the corresponding low density surfaces. Surfaces immobilized with the antibody pair allowed for an ∼100-fold increase in the apparent dissociation rate (∼10−4 s−1).
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pone-0112070-g003: EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.When using the single-epitope high density surfaces, dissociation rates were fast and similar to that of the corresponding low density surfaces. Surfaces immobilized with the antibody pair allowed for an ∼100-fold increase in the apparent dissociation rate (∼10−4 s−1).

Mentions: For any of the single-epitope high density surface, captured EphA2 quickly decayed with a similar dissociation rate to that of the corresponding low density surface (see Figure 3A–B with mAbs 3B10 and 1C1 as an example). In contrast, bi-epitope surfaces showed an ∼10–100-fold enhancement in their apparent dissociation rates (∼10−4–10−5 s−1, Table 1) when compared with that of the corresponding high density single-epitope surfaces. In particular, mixing mAbs 3B10 and 1C1, each possessing a very fast dissociation rate of ∼10−2 s−1, yielded a biosensor surface with an apparent dissociation rate of 1.4×10−4 s−1 (Figure 3C and Table 1), an ∼100-fold improvement.


Bi-epitope SPR surfaces: a solution to develop robust immunoassays.

Peng L, Damschroder MM, Wu H, Dall'Acqua WF - PLoS ONE (2014)

EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.When using the single-epitope high density surfaces, dissociation rates were fast and similar to that of the corresponding low density surfaces. Surfaces immobilized with the antibody pair allowed for an ∼100-fold increase in the apparent dissociation rate (∼10−4 s−1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221230&req=5

pone-0112070-g003: EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.When using the single-epitope high density surfaces, dissociation rates were fast and similar to that of the corresponding low density surfaces. Surfaces immobilized with the antibody pair allowed for an ∼100-fold increase in the apparent dissociation rate (∼10−4 s−1).
Mentions: For any of the single-epitope high density surface, captured EphA2 quickly decayed with a similar dissociation rate to that of the corresponding low density surface (see Figure 3A–B with mAbs 3B10 and 1C1 as an example). In contrast, bi-epitope surfaces showed an ∼10–100-fold enhancement in their apparent dissociation rates (∼10−4–10−5 s−1, Table 1) when compared with that of the corresponding high density single-epitope surfaces. In particular, mixing mAbs 3B10 and 1C1, each possessing a very fast dissociation rate of ∼10−2 s−1, yielded a biosensor surface with an apparent dissociation rate of 1.4×10−4 s−1 (Figure 3C and Table 1), an ∼100-fold improvement.

Bottom Line: The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and 'single-epitope' surfaces were then compared.For all antibody pairs tested, bi-epitope surfaces exhibited an ∼ 10-100-fold improvement in apparent binding affinities when compared with single-epitope ones.This led to an ∼ 100-200-fold enhancement in EphA2 limit of detection in crude cell supernatants.

View Article: PubMed Central - PubMed

Affiliation: Department of Antibody Discovery and Protein Engineering, MedImmune, One MedImmune Way, Gaithersburg, MD, 20878, United States of America.

ABSTRACT
Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called 'bi-epitope' sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and 'single-epitope' surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼ 10-100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼ 10(-8) M) and fast dissociation rates (∼ 10(-2) s(-1)), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼ 10(-10) M and 10(-4) s(-1), respectively. This led to an ∼ 100-200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development.

Show MeSH
Related in: MedlinePlus