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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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Binding and epitope characterization of various anti-EphA2 mAbs.(A) Binding kinetics of mAbs 1C1, 3F2, 3B10 and 3B2. Measurements were conducted using a ProteOn XPR36. Each antibody was immobilized at low density (∼200–600 RU or ∼20–60 ng/cm2) using amine coupling and EphA2 injected over the resulting surfaces. All 4 antibodies exhibit fast dissociation rates in the 10−2−10−3 s−1 range. (B) Epitope binning. Cross-competition binding studies between any pair of mAbs 1C1, 3F2, 3B10 and 3B2 was performed using a ProteOn XPR36 instrument. Injections are indicated by arrows. A response from the second injection indicated that each mAb in a given pair binds to a different epitope. (C) 3 distinct epitopes were identified, including 1 shared between mAbs 3B10 and 3F2.
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pone-0112070-g001: Binding and epitope characterization of various anti-EphA2 mAbs.(A) Binding kinetics of mAbs 1C1, 3F2, 3B10 and 3B2. Measurements were conducted using a ProteOn XPR36. Each antibody was immobilized at low density (∼200–600 RU or ∼20–60 ng/cm2) using amine coupling and EphA2 injected over the resulting surfaces. All 4 antibodies exhibit fast dissociation rates in the 10−2−10−3 s−1 range. (B) Epitope binning. Cross-competition binding studies between any pair of mAbs 1C1, 3F2, 3B10 and 3B2 was performed using a ProteOn XPR36 instrument. Injections are indicated by arrows. A response from the second injection indicated that each mAb in a given pair binds to a different epitope. (C) 3 distinct epitopes were identified, including 1 shared between mAbs 3B10 and 3F2.

Mentions: Kinetics and affinity measurements, as well as epitope binning were performed on the 4 anti-EphA2 mAbs 1C1, 3F2, 3B10 and 3B2. All mAbs exhibited fast dissociation rates ranging from 1.3×10−2 to 1.0×10−3 s−1 (Figure 1A). These fast dissociation rates would prevent their usage as capture reagents in sensitive immunoassays. Additionally, mAbs 3B10 and 3F2 were found to recognize the same or largely overlapping epitope(s) (Figure 1B) and as such were not paired to generate a bi-epitope surface. mAbs 1C1 and 3B2 each recognized a distinct epitope from 3B10 and 3F2, as shown in Figure 1B. In summary, 3 distinct epitopes were identified (Figure 1C).


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

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

Binding and epitope characterization of various anti-EphA2 mAbs.(A) Binding kinetics of mAbs 1C1, 3F2, 3B10 and 3B2. Measurements were conducted using a ProteOn XPR36. Each antibody was immobilized at low density (∼200–600 RU or ∼20–60 ng/cm2) using amine coupling and EphA2 injected over the resulting surfaces. All 4 antibodies exhibit fast dissociation rates in the 10−2−10−3 s−1 range. (B) Epitope binning. Cross-competition binding studies between any pair of mAbs 1C1, 3F2, 3B10 and 3B2 was performed using a ProteOn XPR36 instrument. Injections are indicated by arrows. A response from the second injection indicated that each mAb in a given pair binds to a different epitope. (C) 3 distinct epitopes were identified, including 1 shared between mAbs 3B10 and 3F2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4221230&req=5

pone-0112070-g001: Binding and epitope characterization of various anti-EphA2 mAbs.(A) Binding kinetics of mAbs 1C1, 3F2, 3B10 and 3B2. Measurements were conducted using a ProteOn XPR36. Each antibody was immobilized at low density (∼200–600 RU or ∼20–60 ng/cm2) using amine coupling and EphA2 injected over the resulting surfaces. All 4 antibodies exhibit fast dissociation rates in the 10−2−10−3 s−1 range. (B) Epitope binning. Cross-competition binding studies between any pair of mAbs 1C1, 3F2, 3B10 and 3B2 was performed using a ProteOn XPR36 instrument. Injections are indicated by arrows. A response from the second injection indicated that each mAb in a given pair binds to a different epitope. (C) 3 distinct epitopes were identified, including 1 shared between mAbs 3B10 and 3F2.
Mentions: Kinetics and affinity measurements, as well as epitope binning were performed on the 4 anti-EphA2 mAbs 1C1, 3F2, 3B10 and 3B2. All mAbs exhibited fast dissociation rates ranging from 1.3×10−2 to 1.0×10−3 s−1 (Figure 1A). These fast dissociation rates would prevent their usage as capture reagents in sensitive immunoassays. Additionally, mAbs 3B10 and 3F2 were found to recognize the same or largely overlapping epitope(s) (Figure 1B) and as such were not paired to generate a bi-epitope surface. mAbs 1C1 and 3B2 each recognized a distinct epitope from 3B10 and 3F2, as shown in Figure 1B. In summary, 3 distinct epitopes were identified (Figure 1C).

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