Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon α-2A

Crystals of the complex between the Fab fragment of a human anti-interferon α therapeutic antibody and human interferon α-2A have been obtained and diffracted to 3.0 Å resolution

Meeting the challenges of implementing rapid genomic testing in acute pediatric care

Authors: Stark, Z. Lunke, S. Brett, G. Tan, N. Stapleton, R. Kumble, S. Yeung, A. Phelan, D. Chong, B. Fernandez, M.F. Marum, J.E. Hunter, M. Jarmolowicz, A. Yael Prawer, Y. Riseley, J.R. Regan, M. Elliott, J. Melissa Martyn, M. Best, S. Tan, T. Clara L Gaff, C.L. and White, S.M

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

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

Pairing mAbs 3B10 and 1C1 results in enhanced EphA2 detection sensitivity in conditioned media.

<p>(A) Binding of detection antibody mAb 3B2 plotted against EphA2 concentrations. (B) Logarithmic scale display with binding signals in ∼0.3–30 RU (or ∼0.03–3 ng/cm²) range. The bi-epitope 3B10-1C1 surface detected the lowest EphA2 concentration (15.6 pM at a binding signal of 6 RU or 0.6 ng/cm²), an ∼100- and 200-fold improvement in detection limits when compared with the corresponding 3B10 (1.3 nM) and 1C1 (3.1 nM), respectively, single-epitope surfaces.</p

EphA2 binding to individual mAbs 3B10 (A), 1C1 (B) and corresponding mixture (C) immobilized at high density levels.

<p>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).</p

Generation and characterization of high density bi-epitope SPR sensor surfaces.

<p>(A) Immobilization sensorgrams of mAbs 3B10, 1C1 and 3B10-1C1 mixture. The immobilization profiles are comparable and yielded a high density surface (∼5,000–5,500 RU or ∼500–550 ng/cm²). (B) Confirmation of the co-existence of functional antibodies on the bi-epitope surfaces. Excess of mAbs 3B10 or 1C1 (1 µM) inhibited EphA2 binding to the single-epitope 3B10 or 1C1 surfaces, respectively, but not to the bi-epitope 3B10-1C1 surface.</p

Binding and epitope characterization of various anti-EphA2 mAbs.

<p>(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.</p