Impact/Purpose:

Our previous study, on a functionalized glass surface to covalently immobilize antibodies, was conducted to produce a more efficient means of water contamination analysis. The current methods used to detect organisms in water require up to 4 days to produce results that are indicative of only a small number of potential contaminates. Enzyme linked immunosorbent assays (ELISA) can use immunological methods to detect a wide range of biomolecules in less time than the current methods. However, traditional ELISAs produce a significant amount of solid waste and require many high-purity antibodies. The costs incurred and biologically- contaminated waste produced by ELISAs have led to the production of a smaller alternative called a microarray. Microarrays use immobilized biomolecules (proteins, lipids, antibodies, and carbohydrates) to detect conjugate biomolecules in solution. Multiple types of biomolecules can be printed onto a single solid glass or polymer substrate to detect multiple conjugate molecules in a single test. Our previous work focused on modification of a glass substrate to increase the surface area available for immobilization and the means by which antibodies were bound to the surface. Covalent immobilization of the antibodies instead of hydrophobic binding is one method that can optimize the binding concentration of antibodies onto the substrate. Our results indicated that surface modifications we made to glass substrates successfully immobilized antibodies. However, the immobilized antibodies denatured, and did not retain their functionality after the immobilization process. Stabilization of the antibodies structure is a potential solution to prevent the denaturing caused during immobilization.

Description:

We used the optimum slide treatment as determined by the previous study*: water plasma cleaning, photo-hydrolytic weathering, and silane treatment using 3-aminopropyl triethoxysilane (APS). Anti-E.coli antibodies were printed onto Corning 2947 (soda-lime-silicate) and Corning 1737 (proprietary alkaline earth aluminoborosilicate) glass slides for functionality tests. Fluorescein isothiocynate labeled, goat anti-rabbit antibodies were printed onto slides as a control to confirm the presence of immobilized antibodies. Stabilizers were added to the antibody solutions before printing on the slides. Three stabilizing agents were investigated: gluteraldehyde, trehalose, and 200 molecular weight (MW) polyethylene glycol (PEG). Functionality of the immobilized antibodies was determined by their ability to capture GFP labeled E. coli from a Blotto buffer solution. The results were measured as a positive or negative presence of E. coli based on fluorescent scanning.

None of the antibodies that had been stabilized by one of the three agents tested retained their functionality after immobilization. Fluorescent scanning of the slides confirmed the presence of immobilized antibodies, but did not detect any captured GFP labeled E. coli. XPS analysis was used to supplement the results from the functionality tests to determine if the antibodies were remaining in their native structure. The thickness of the antibody layer was determined using sputter depth profiling. Antibodies that retained their native structure would create a thicker layer than the denatured antibodies. Layer thickness was determined by the sputter time required for the concentration of carbon to decrease below the concentration of silicon, which we termed the carbon-silicon crossover. Longer sputter times indicate a thicker protein layer. The thickness of antibodies stabilized by trehalose and gluteraldehyde were compared to non-stabilized antibodies. Results from the sputter depth profiles can be seen below. The stabilized antibodies displayed slightly longer time until the carbon-silicon crossover. Also, the initial carbon concentration for stabilized antibodies is greater than the non-stabilized antibodies. Due to the greater initial carbon concentrations, we concluded the increase in sputter time was not significant enough to indicate a thicker layer.

* http://ceer.alfred.edu/Research/research_pollutants.html
http://cfpub.epa.gov/ncer_abstracts/index.cfm/fuseaction/outlinks.centers/center/145

XPS depth profile for non-stabilized antibodies. The crossover between carbon and silicon is visible at approximately 0.5 minutes and is shown by the arrow. Since no stabilizers were added, the antibodies are assumed to be denatured.

XPS depth profile for gluteraldehyde-stabilized antibodies. The carbon-silicon crossover is visible at approximately 1.2 minutes into the sputter and is shown by the arrow. The initial carbon content is greater than what was observed from the non-stabilized antibodies, leading to the conclusion that the delayed crossover was caused by residual stabilizer.

XPS depth profile for trehalose-stabilized antibodies. The carbon-silicon crossover is visible after 1 minute of sputtering and is shown by the arrow. The initial carbon content is greater than both the non-stabilized and gluteraldehyde-stabilized antibodies

Since addition of stabilizers to the predetermined process did not yield functional antibodies, the conditions used for stabilization and immobilization were modified to determine if other variables could be changed to increase the functionality of the antibodies. A comprehensive list of the variables changes and the values used is available in the table below.

Variables and Values Changed from Original Protocol

*Note: Stabilization is defined as the process of combining a stabilizing agent with antibodies before immobilization onto the substrate. Incubation is defined as the step where antibodies attach to the substrate before a wash is performed.

Variations of the conditions for stabilization and immobilization were performed changing only one variable at a time to cover the complete range of possible combinations. This would pinpoint individual steps in the process responsible for the antibodies denaturing. All of the tested processes successfully immobilized antibodies. However, we were still unable to capture E.coli under any of the new conditions. The antibodies were still denatured. Since all potential variables in the stabilizing and immobilizing processes did not yield functional antibodies, additional steps are required to prevent the denaturing of antibodies.

URLs/Downloads:

Final Progress Report

Record Details:

Record Type:PROJECT( ABSTRACT )

Start Date:09/01/2006

Completion Date:08/31/2007

Record ID: 200560

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Related Organizations:

Role :OWNER

Organization Name :ALFRED UNIVERSITY

Organization Name :CEER

Mailing Address :Saxon Dr

Citation :Alfred

State :NY

Zip Code :14802

Project IDs:

ID Code :X832541C001

Project type :Center