Novel stabilizer/blocker formulations providing rare dual abilities to block immunoassay diagnostic surfaces and prevent protein denaturation for effective long term dried stabilityT Jentz, W NelsonSurModics, Inc., Eden Prairie, MN#474

Contact InformationTim Jentz

tjentz@surmodics.com

Objectives/GoalsDemonstrate the benefits of SurModics’

stabilizers/blockers for use in protein stabilization and

blocking applications. Benefits include; dried protein

stability, in-solution protein stability, ELISA blocking,

membrane blocking, and microarray stability.

Summary – Novel Stabilizer/Blocker Features:• Demonstrated equal or superior dried antibody stability when compared to competitors and common in-house stabilizers • Provided the greatest in-solution conjugated antibody retained activity and no significant antibody/antigen affinity loss• Provided superior coating and uniformity suggesting optimal blocking effectiveness • Demonstrated strong blocking, decreased backgrounds, and enhanced detection limits with membrane-based immunoassays• Demonstrated improved assay performance in multiple applications across the immunoassay diagnostic industry

The 24th Annual Symposium of the Protein Society

AbstractOBJECTIVES: Four novel stabilizer/blocker formulations (SurModics, Eden Prairie, MN) were analyzed to identify a product that can both stabilize coated proteins and block diagnostic surfaces. Scientists are continually searching for new products to stabilize and store proteins to provide an environment close to their native conditions. Effective and thorough blocking of remaining

binding sites across all diagnostic surfaces remains a vital immunoassay step. METHODS: A mouse anti-Troponin capture antibody was coated and stabilized with 24 different commercially available stabilizers. This study accelerated the stability conditions by challenging the captured antibody at a 37ºC storage condition, versus a 4ºC control. The retained activity of the captured

antibody was evaluated in a Troponin sandwich ELISA periodically over six months by comparing the immunoassay signal produced at 4ºC versus 37ºC. Scanning Electron Microscopy (SEM) and Vertical Scanning Interferometry (VSI) surface characterization imaging were performed to demonstrate each product’s ability to completely block a polystyrene plate and prevent an

antibody from binding to the blocked surface. CONCLUSIONS: At the six-month stability time point, 10 of 24 stabilizers demonstrated greater than 90% retained activity, including four novel SurModics formulations, as measured in the Troponin ELISA. The sustained functional activity suggests the stabilizers were able to preserve the functional conformation of the dried antibody.

To examine each product’s ability to block surface sites, SEM and VSI surface characterization images were analyzed for uniformity and smoothness of coating. SurModics’ novel formulations showed improved coating and uniformity suggesting optimal blocking effectiveness. The data presented here show that four novel SurModics formulations provide a convenient product that

both stabilizes protein/antibody structure and function while also blocking non-specific binding. The use of a superior stabilizer/blocker will lead to reliability, cost savings, and accurate and consistent assay performance.

Dried Protein Stability

Liquid Stability

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Blocking Applications

Figure #6: Blocking uniformity with StabilGuard blocker

Methods: An antibody was coated onto a polystyrene ELISA plate and blocked with StabilGuard blocker (BSA-free), a BSA-free competitor, or left blank. SEM and VSI surface characterization imaging were performed to demonstrate each product’s ability to thoroughly block a polystyrene plate.

Results: StabilGuard blocker demonstrates superior coating and uniformity suggesting optimal blocking effectiveness.

StabilGuard(BSA free)

Blank Control

SEM Images

VSI Images

Competitor (BSA free)

Figure #4a and 4b: Retained in-solution antibody activity

Methods: Anti-carcinoembryonic antigen (CEA) HRP antibodies were diluted in each of the above stabilizers. Each stabilizer was separated and stored at 4°C (Figure 4a) and 37°C (Figure 4b). Kinetic enzyme activity assays were performed by measuring the absorbance two minutes after the addition of the stabilized conjugate to TMB.

Results: Storing the anti-CEA-HRP conjugated antibody in StabilGuard stabilizer demonstrated the greatest retained conjugate activity (more than 85% after 222 days at 4°C and ~60% after 165 days at 37°C).StabilGuard stabilizer demonstrated a kinetic inactivation rate 10 times lower than PBS at 37°C, indicating minimal conjugate denaturation when stored in StabilGuard stabilizer.

↓StabilGuard Stabilizer

↑StabilZyme Select® StabilizerStabilGuard↓ Stabilizer

Legend: PBS = 0.1M Na3PO4 + 0.15 M NaCl pH 7.2 BSA = 2% bovine serum albuminTRE = 1% Trehalose SGU = StabilGuard StabilizerSSE = StabilZyme Select Stabilizer GLI = 50% glycerol in PBSDRW = dried from aqueous solution DRP = dried from PBS

Figure 4a: Residual enzymatic activity stability @ 4°C Figure 4b: Residual enzymatic activity stability @ 37°C

StabilZyme Select®↓ Stabilizer

Figures 4a, 4b and 5: Reprinted with permission from

Laboria, N., Anal Chem, Vol. 82, 1712-1719, 2010 American Chemical Society

Figure #5: Variation of conjugate affinity to antigen when stored in StabilGuard stabilizer

Methods: Storage conditions on conjugate antibody affinity for 10 ng/mL CEA were also analyzed by ELISA.

Results: Figure #5 above demonstrates no significant loss in the conjugated antibody affinity to the CEA after ~ five months at 4°C in StabilGuard stabilizer.

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.

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.

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Figure #7: StabilGuard blocker as a membrane blocker

Methods: GAPDH was absorbed to a nitrocellulose membrane and blocked with one of three blocking solutions: 1% BSA in TBS, 5% milk in TBS, or StabilGuard blocker.

Results: The dot blot above demonstrates StabilGuardblocker provides strong blocking, decreased backgrounds, and also enhanced detection limits.

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5% Milkin TBS

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Figure #3: Minimal control shift over time with StabilCoat stabilizer

Methods: Each plate throughout the stability study contained a standard curve stabilized by StabilCoat stabilizer. Each control standard curve was analyzed in the graph and tables above.

Results: The control graph and table above demonstrate the consistency, stability, and repeatability of the 4°C standard curves at each time point. A two standard deviation range was established around the mean of the EC50. All EC50 values at each time point fall within two standard deviations of the mean.

Time Point EC50Day 0 111Day 1 112

1 week 1291 month 1223 months 1396 months 1499 months 154

1 year 146mean 133

std dev 16.7%CV 12.6

2 SD range 99.6 - 166.4

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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 almost 100% retained activity.

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