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Selection of High-Affinity Synergistic Antibodies from a Phage Display scFv Immune Library

EPA Grant Number: F5D61318
Title: Selection of High-Affinity Synergistic Antibodies from a Phage Display scFv Immune Library
Investigators: Abboud, Elizabeth R.
Institution: Tulane University of Louisiana
EPA Project Officer: Willett, Stephanie H.
Project Period: January 1, 2005 through December 1, 2006
Project Amount: $90,172
RFA: GRO Graduate Fellowships (2005)
Research Category: Academic Fellowships

Description:

Objective:

Immunoglobulin A (IgA) is present in mucosal secretions and hence is secreted in the feces. Previous studies from other laboratories have shown that the levels of IgA in wastewater correlate well with coliform contamination. It is the ultimate goal of our laboratory to develop a rapid, field-portable species-specific immunoassay for IgA using an existing handheld immunosensor already under development in my laboratory. The objective of this project is to identify combinations of synergistic antibodies from a phage display scFv immune library to immunoglobulin A and analyze the molecular binding characteristic of these antibodies.

Approach:

We will construct an scFv phage library from the RNA of rabbits immunized with human immunoglobulin A. Following completion of library construction, we will screen the resulting phage library, utilizing a previously defined epitope-blocking strategy, for combinations of antibodies that bind to human IgA with synergy. We will then perform immunoassays and equilibrium binding studies on the selected antibodies to characterize the molecular interactions between the antibodies and antigen.

Expected Results:

We expect to obtain four scFv immune libraries of approximately 10 6-10 8 unique clones each from this project. We also expect to identify phage clones that contain DNA encoding for scFvs that bind to IgA with high affinity and synergistically in combination with another scFv. We will obtain master plates of positive binding clones, which will be stored and can be used in the future if desired for other screening procedures. The project will also result in the production of soluble scFvs to be used in the subsequent immunoassays and molecular binding studies. We expect to affinity-rank and specificity-rank all of the scFvs identified as positive binders to IgA. We also plan to acquire detailed molecular binding data on the scFvs that show the highest affinity and specificity for the antigen as well as for the pairs of scFvs that appear to bind synergistically to IgA. We expect to see a higher affinity for the antigen among the pairs of scFvs that bind synergistically than among the scFvs that do not.

Supplemental Keywords:

phage display, immune library, scFv library, synergistic antibodies, Immunoglobulin A, coliform contamination, immunosensors, , Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, RFA, Environmental Microbiology, Monitoring/Modeling, Environmental Monitoring, field portable monitoring, immunoassay, field detection, field deployable, coliform bacteria, bacteria monitoring, high affinity synergistic antibodies, water monitoring, animal model

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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