Novel assay reagent formulations for protein stability and blocking applications in the diagnostic industryT Jentz, G. OppermanSurModics, Inc., Eden Prairie, MN

Contact InfoTim 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 – SurModics Stabilizer/Blocker Features:• Demonstrated equal or superior dried antibody stability when compared to competitors and

common in-house stabilizers • Provided the greatest in-solution antibody retained activity• Demonstrated improved signal-to-noise ratios and decreased non-specific binding on three

nitrocellulose surfaces• Provided superior coating and uniformity, suggesting optimal blocking effectiveness • Demonstrated improved assay performance in multiple applications from across the

immunoassay diagnostic industry

Cambridge Healthtech Institute’s. PEPTALK, Protein Science week January 10-14, 2011

AbstractNovel IVD assay components were evaluated for their respective diagnostic application. In-house competitor testing and outside customer data were utilized to analyze novel formulations in the following applications: dried protein stability, in-solution protein stability, and blocking of non-specific interactions. Dried and in-solution protein stability efficacy was demonstrated by measurement of retained activity of a targeted antibody. Blocking formulations were evaluated for strong blocking, decreased backgrounds, and enhanced detection limits with membrane-based immunoassays. The data presented will demonstrate that novel formulations provide a convenient product to both stabilize antibody structure and protein function, while also blocking non-specific binding. The use of a superior assay reagent formulation can lead to reliability, cost savings, and consistent assay performance.

In-Solution Protein Stability

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Dried Protein Stability________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

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Figure #5a and 5b: Retained in-solution antibody activity

Methods: Anti-carcinoembryonic antigen (CEA) HRP antibodies were diluted in each of the above stabilizers. Each protein / stabilizer solution was separated and stored at 4°C (Figure 5a) and 37°C (Figure 5b). 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® Stabilizer

StabilGuard↓ 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 5a: Residual enzymatic activity stability @ 4°C Figure 5b: Residual enzymatic activity stability @ 37°C

StabilZyme Select®↓ Stabilizer

Figures 5a, 5b: Reprinted with permission from Laboria, N.Anal Chem, Vol. 82, 1712-1719, 2010 American Chemical Society

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 stabilizersdemonstrated almost 100% retained activity.

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Figure #6: Analysis of GAPDH in a Dot Blot

Methods:

Slide Printing: GAPDH was prepared at various dilutions in PBS and printed on nitrocellulose slides from three manufacturers (NC-A, NC-B, and NC-C). The printing pins used deliver ~10 nl in each spot. Slideswere stored desiccated overnight.

Assay: Slides were washed/blocked with the respective blocking buffer. Slides were then incubated in either StabilGuard buffer or TBS-Tween (0.05%) (TBS-T) containing rabbit anti-GAPDH antibody at 1.2 g/mL (Sigma) for 1.5 hour. Slides were washed with TBS-T three times and then incubated with biotin-labeled goat anti-rabbit antibody (Jackson Labs) at 0.2 g/mL in either TBS-T or StabilGuard buffer for 1.5 hour. Slides were again washed three times with TBS-T and incubated in streptavidin-Cy5 (0.2 g/mL; GE/Amersham) in the either StabilGuard buffer or TBS-T for one hour. Slides were finally washed three times with TBS-T and twice with water before being centrifuged dry. Slides were then scanned on an Axon 4200AL scanner in the 635 nm channel.

Results: The images demonstrate the ability of StabilGuard buffer to block non-specific binding of assay components on three nitrocellulose surfaces. Backgrounds, on all types of nitrocellulose, were maintained at the raw nitrocellulose level. Comets were reduced by the use ofStabilGuard buffer and signal-to-noise was maintained.

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Tween-20 BlockerNC-A NC-B NC-C

Blocking Applications

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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 table 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 (mid point on the standard curve). All EC50 values at each time point fall within two standard deviations of the mean.

Time Point EC50

Day 0 111

Day 1 1121 week 1291 month 1223 months 1396 months 1499 months 154

1 year 146

mean 133

std dev 16.7%CV 12.6

2 SD range 99.6 - 166.4

Standard Curve Consistency over 1 year

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Figure #7: 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 characterizationimaging were performed to demonstrate each product’s ability to maintain uniformity across the surface of 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)

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Figure #4: Troponin In-Solution Stability

Methods: Native Human Cardiac Troponin-I was diluted to 10 µg/mL in a MOPS buffer, a commercially available in-solution competitor and SurModics stabilizer in development. The stabilized Troponin was tested in a Troponin sandwich ELISA over time compared to a freshly prepared control.

Results:. SurModics in-development stabilizer demonstrated ~ 100% retained activity compared to the freshly prepared control after 59 days at 4C.

Troponin In-Solution Stability

SurModics Stabilizer

MOPS buffer

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Fresh Control

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