Novel stabilizer/blocker formulations providing rare dual abilities to block immunoassay diagnostic surfaces and prevent protein denaturation

for effective long term dried stability

T Jentz, W Nelson
SurModics, Inc., Eden Prairie, MN
#474 The 24th Annual Symposium of the Protein Society

Abstract Objectives/Goals
OBJECTIVES: 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 Demonstrate the benefits of SurModics’
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 stabilizers/blockers for use in protein stabilization and
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 blocking applications. Benefits include; dried protein
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. stability, in-solution protein stability, ELISA blocking,
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 membrane blocking, and microarray stability.
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.
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Dried Protein Stability

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Day 0 7 days 1 month 6 months 1 year 3.5 Time Point EC50

100
Day 0 111

% Retained Efficacy
% Retained Efficacy

3.0 day 0
100.0 Day 1 112
80 day 1
2.5 1 week 1 week 129
80.0
% Retained Activity 60 1 month 1 month 122
2.0

OD
60.0
3 months 3 months 139
40 6 months
Average OD @ 37°C x 100 1.5 6 months 149
9 months
40.0 Average OD @ 4°C 20 9 months 154
1.0 1 year
1 year 146
20.0
0 0.5 mean 133
StabilCoat StabilGuard 1X PBS + 1X PBS + std dev 16.7
0.0 0.0
StabilCoat StabilGuard Stabilizer Stabilizer 1%BSA 1%BSA + %CV 12.6
Comp #1 Comp #2 Comp #3 Comp #4 Comp #5 Comp #6 1 10 100 1000
Stabilizer Stabilizer Stabilizer 0.05% Tween ng/mL 2 SD range 99.6 - 166.4
Stabilizer

Figure #1: Dried antibody stability with StabilCoat® and StabilGuard® stabilizers Figure #3: Minimal control shift over time with StabilCoat stabilizer
Figure #2: In-house stabilizer comparison

Methods: A capture antibody was stabilized with different commercially available stabilizers. This accelerated stability study challenged Methods: Each plate throughout the stability study contained a standard curve stabilized by StabilCoat stabilizer.
Methods: Using the ELISA methods described in Figure #1, StabilCoat and
the captured antibody at 37ºC. The retained activity of the captured antibody was evaluated in a sandwich ELISA over one year by Each control standard curve was analyzed in the graph and tables above.
StabilGuard stabilizers were compared versus common in-house stabilizers.
comparing the immunoassay signal produced by the 4ºC control versus 37ºC.
Results: The control graph and table above demonstrate the consistency, stability, and repeatability of the 4°C
Results: After one week at 37ºC storage, StabilCoat and StabilGuard
Results: At the one-year stability time point, StabilCoat and StabilGuard stabilizers demonstrated greater than 90% retained activity. standard curves at each time point. A two standard deviation range was established around the mean of the EC50.
stabilizers demonstrated almost 100% retained activity.
The sustained functional activity suggests SurModics’ stabilizers were able to preserve the functional conformation of the dried antibody. All EC50 values at each time point fall within two standard deviations of the mean.

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Liquid Stability Blocking Applications
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↓StabilGuard Stabilizer

StabilGuard Blank StabilGuard Competitor

↑StabilZyme Select® Stabilizer ↓ Stabilizer
Control (BSA free) (BSA free) 1000 250 62.5 15.6 GAPDH (ngs)

1% BSA
SEM in TBS
Images
StabilZyme Select®
↓ Stabilizer
5% Milk
in TBS

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

VSI
Figure 4a: Residual enzymatic activity stability @ 4°C
Figure #5: Variation of conjugate affinity to antigen Images StabilGuard
Legend: when stored in StabilGuard stabilizer Blocker
PBS = 0.1M Na3PO4 + 0.15 M NaCl pH 7.2 BSA = 2% bovine serum albumin
TRE = 1% Trehalose SGU = StabilGuard Stabilizer
SSE = StabilZyme Select Stabilizer GLI = 50% glycerol in PBS Methods: Storage conditions on conjugate antibody
DRW = dried from aqueous solution DRP = dried from PBS
affinity for 10 ng/mL CEA were also analyzed by ELISA.
Figure #7: StabilGuard blocker as a membrane blocker
Figure #4a and 4b: Retained in-solution antibody activity Figure #6: Blocking uniformity with StabilGuard blocker
Results: Figure #5 above demonstrates no significant
Methods: GAPDH was absorbed to a nitrocellulose
Methods: Anti-carcinoembryonic antigen (CEA) HRP antibodies were diluted in each of the above stabilizers. loss in the conjugated antibody affinity to the CEA after Methods: An antibody was coated onto a polystyrene ELISA plate and blocked membrane and blocked with one of three blocking solutions:
Each stabilizer was separated and stored at 4°C (Figure 4a) and 37°C (Figure 4b). Kinetic enzyme activity ~ five months at 4°C in StabilGuard stabilizer. with StabilGuard blocker (BSA-free), a BSA-free competitor, or left blank. SEM 1% BSA in TBS, 5% milk in TBS, or StabilGuard blocker.
assays were performed by measuring the absorbance two minutes after the addition of the stabilized conjugate to and VSI surface characterization imaging were performed to demonstrate each
TMB. product’s ability to thoroughly block a polystyrene plate. Results: The dot blot above demonstrates StabilGuard
Figures 4a, 4b and 5: blocker provides strong blocking, decreased backgrounds,
Results: Storing the anti-CEA-HRP conjugated antibody in StabilGuard stabilizer demonstrated the greatest Reprinted with permission from Results: StabilGuard blocker demonstrates superior coating and uniformity and also enhanced detection limits.
retained conjugate activity (more than 85% after 222 days at 4°C and ~60% after 165 days at 37°C). Laboria, N., Anal Chem, Vol. 82, 1712-1719, 2010 suggesting optimal blocking effectiveness.
StabilGuard stabilizer demonstrated a kinetic inactivation rate 10 times lower than PBS at 37°C, indicating American Chemical Society
minimal conjugate denaturation when stored in StabilGuard stabilizer.
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Summary – Novel Stabilizer/Blocker Features:

• Demonstrated equal or superior dried antibody stability when compared to competitors and common in-house stabilizers Contact Information
• Provided the greatest in-solution conjugated antibody retained activity and no significant antibody/antigen affinity loss Tim Jentz
tjentz@surmodics.com

• 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