Evaluation of Immunoassay Interference Reduction Technologies

Wendy D. Nelson, Ph.D., Gary Opperman, Tim Jentz, Drew Pauly, Shannon Kuklok

SurModics, Inc., Eden Prairie, MN

Contact InfoWendy D. Nelson, Ph.D.wnelson@surmodics.com

IntroductionOptimization of immunoassay applications is often troubled by issues such as non-specific binding, matrix interferences, destabilization of antibody/antigen interactions, and limited sensitivity. SurModics offers tools to address these problems and provides the framework to develop sensitive, reproducible, and robust immunoassays. For both blocking and stabilization, protein-protein interactions are an essential consideration during development, and often the best option is a synthetic formulation to eliminate cross-reactivity issues observed with protein-based formulations. SurModics offers synthetic formulations for many immunoassay formats such as ELISA, western blotting and in-solution applications, all of which are illustrated in the following poster. For microarray applications, assay interference can be reduced through the use of novel, passivating surfaces. Here the surface coating provides the optimal interface for stabilizing specific protein-protein interactions while significantly reducing non-specific binding. In addition, these surfaces can be modified to support cell-based assays. Whether it is in solution or on a surface, SurModics provides the tools necessary to develop and optimize immunoassays that are sensitive, reproducible, and robust.

Microarray Applications

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Protein Stability/Matrix Interference________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

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Figure #1: Dried antibody stability with StabilCoat® and StabilGuard® stabilizers

Methods: A capture antibody was stabilized with different commercially available stabilizers. This accelerated stability study challenged the captured antibody at 37ºC. The retained activity of the captured antibody was evaluated in a sandwich ELISA over one year by comparing the immunoassay signal produced by the 4ºC control versus 37ºC. Retained activity values are determined by dividing the average optical density @ 37ºC by the average optical density at 4ºC, then multiplying by 100.

Results: At the one-year stability time point, StabilCoat and StabilGuard stabilizers demonstrated greater than 90% retained activity. The sustained functional activity suggests SurModics’ stabilizers were able to preserve the functional conformation of the dried antibody.

Figure #2: In-house stabilizer comparison

Methods: Using the ELISA methods described in Figure #1, StabilCoat and StabilGuard stabilizers were compared versus common in-house stabilizers.

Results: After one week at 37ºC storage, StabilCoat and StabilGuard stabilizers demonstrated nearly 100% retained activity.

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TroponinTroponin(ng/mL)(ng/mL)

TRIDIA™ Epoxide Surface Epoxysilane A Epoxysilane B

Figure #5: Analysis of Troponin on a Protein Microarray

Methods: Monoclonal antibody to Troponin I (AbCam) at 1 mg/mL was printed on all slides and incubated overnight. Slides were assembled into 16 well plates using ProPlate® slide chambers (Grace Bio-Labs) and then washed/blocked in PBS- Tween

(0.05%) (PBS-T) containing 5% BSA. Troponin I was diluted, incubated for 1.5 hours and washed. Primary antibody (Rb x hTroponin; 1 g/mL) and secondary (Gt x Rb IgG-Cy3; 1 g/mL) were used to detect Troponin I. Slides were washed, centrifuged dry and then scanned on an Axon 4200AL scanner in the 532 nm channel.

Results: The TRIDIA surface blocked non-specific binding compared to commercially available epoxysilane surfaces. The epoxysilane slides show non-specific binding at the higher Troponin levels. The TRIDIA slides display more distinct spots with consistently low background across the slide.

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Figure #4: Attachment of HUVEC in a Cell Array

Methods: RGD peptide was printed on CodeLink® slides (SurModics) and incubated overnight at 75% humidity. Slides were washed and sterilized by exposure to UV light. Slides were then placed in sterile 100 mm dishes (1 slide/dish), and human umbilical vein endothelial cells (HUVEC; Lonza) were seeded at 30,000 cells/mL in serum- containing growth media (Lonza) at 20 mL/dish. After 3 days of proliferation, HUVECs were stained with 1 M calcein AM (Molecular Probes). Slides were then fixed, mounted, and imaged using FITC and DAPI filter sets.

Results: The CodeLink® surface with bound RGD peptide promoted cell adhesion and eliminated non-specific binding of cells to the remainder of the surface. Image A is a fluorescent image of 6 RGD spots on a CodeLink® slide. Image B is a magnified spot on the array highlighting the specific attachment of the cells to the spot where RGD was printed. Image C is a phase contrast image of the same spot.

Figure #6: Detection of HRP in a Dot Blot

Methods: A serial dilution of HRP conjugated antibody (Gt x Rb IgG-HRP; Jackson Immunochemicals) was blotted onto two nitrocellulose membranes. The blotted antibody was then visualized with either BioFX® Ultra Sensitive Chemiluminescent Substrate (CHMM) or BioFX® TMB Enhanced Membrane Substrate (ESPM). The optical density or light generated was quantified using GenePix software (Molecular Devices). Data was fitted to 4PL curves in Figure #6.

Results: Colorimetric detection using SurModics ESPM had almost the same detection limit as our highest sensitivity chemiluminescent substrate CHMM. The dynamic range was slightly extended on the high end in the case of ESPM. The chemiluminescent detection was beginning to saturate at the highest concentration in this experiment.

Figure #7: Western Blot of Alpha Tubulin

Methods: NIH3T3 cell lysate was prepared and run on a 4-20% Tris-Glycine gradient gel and transferred to a nitrocellulose membrane. The membrane was blocked for one hour at room temperature. Primary antibody (Rb x alpha tubulin; 1:2000) (Rockland) was diluted into the corresponding blocker and incubated overnight at 4C. After washing 3 times with TBS/0.05% Tween-20 buffer, the secondary antibody (Gt x Rb IgG-HRP; 1:100,000) (Jackson Immunochemicals) was diluted into the corresponding blocker and incubated for one hour at room temperature. The membrane was washed twice with TBS/0.05% Tween-20 buffer and once with TBS alone. Signal was visualized using BioFX

Chemiluminescent Substrate (SurModics CHMM) and captured using a CCD camera.

Results: For each member of the StabilBlot Blocker Family, an enhancement of signal was observed over similar blocker formulations, especially for both StabilBlot Casein Blockers. StabilBlot BSA blocker had considerably less background and blotchiness than the comparable formulations.

Summary• Superior protein stability/activity in both dried and in-solution applications• Decreased HAMA false positives and demonstrated minimal dynamic range reduction• StabilBlot Family of blockers provided lower backgrounds and increased sensitivity in western blotting• Precipitating TMB reagent (ESPM) provides economical detection in blotting applications

– ESPM provided similar sensitivity to high performance chemiluminescence• TRIDIA surface chemistry provides:

– Support for cell array formats– Superior epoxy surface– Simultaneous biomolecule immobilization and passivation.

Demonstrated Improved Assay Performance Across Multiple Diagnostic Applications!

TRIDIA is a trademark of SurModics

StabilGuard and StabilCoat are registered trademarks of SurModics

StabilBlot is a trademark of SurModics

SurModics is a registered trademarks of SurModicsCodeLink is a trademark of a GE Healthcare CompanyProPlate is a registered trademark of Grace Bio-Labs, Inc.Tween is a registered trademark of Uniqema Americas LLC BioFX is a registered trademark of SurModics

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Figure #3 HAMA Interference Reduction

Methods: Serum containing human anti-mouse antibody (HAMA) produced a false positive in a commercially available hCRP sandwich ELISA. The false positive results occurred when HAMA caused bridging between the mouse monoclonal capture antibody and the mouse monoclonal detection antibody. The results shown in the figure above were obtained by adding serum containing elevated HAMA levels diluted with either PBS, SurModics® Assay Diluent (SM01), or four other protein containing products.

Results: The figure above demonstrates the SurModics® Assay Diluent (SM01) provided the greatest false positive blocking (~43%) compared to other protein containing commercially available products. Although other protein based blocking products did show some blocking, this protein based blocking does have two significant drawbacks; one, specific protein containing blockers fail to encompass all types of interference and two, protein containing diluents may interfere with integrity of the assay.

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20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

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BSA Blocker Competitor

20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

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20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

StabilBlot™ Milk Blocker (SB04)

Milk Blocker Competitor

20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

StabilBlot™ Protein Free Blocker (SB01)

Protein Free Competitor

20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

StabilBlot™ BSA Blocker (SB02)

BSA Blocker Competitor

20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

StabilBlot™ Casein Blocker - PBS (SB03)

StabilBlot™ Casein Blocker - TBS (SB05)

Casein Blocker Competitor

20 10 5 2.5 1.25 0.63 0.31 0.16 0.078

StabilBlot™ Milk Blocker (SB04)

Milk Blocker Competitor

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