Manipulating antibody-antigen interactions in microporous membranes for selective antibody and protein purification
Monoclonal antibodies are among the most successful and most expensive therapeutic agents. Rapid, inexpensive antibody capture should decrease the cost of both production and detection of these remarkable drugs. Affinity membranes can potentially capture protein more rapidly than traditional bead-based methods without the large pressure drops of columns.This research employed an Fc (fragment, crystallizable) binding peptide (denoted as KK12) and an Fab (fragment, antigen-binding) binding peptide (denoted as K19) for rapid antibody capture in membranes. KK12-modified membranes captured 8.40 mg of Herceptin and 9.96 mg of Avastin per mL of membrane. The antibodies eluted in 100 mM Gly (pH 2.7), but the membranes were not reusable. K19-modified membranes capture 16.3 mg of Herceptin per mL of membrane and showed minimal binding of Avastin, as expected because K19 mimics the HER2 Herceptin antigen. Furthermore, K19-modified membranes selectively captured Herceptin from human serum and showed no significant non-specific adsorption. Antibody-containing membranes may also enable rapid, high-throughput immunoprecipitation. Anti-(hemagglutinin A) (HA) antibodies successfully captured HA-tagged regulator G-protein signaling 2 (HA-RGS2) from cell lysate and HA-RGS2 subsequently eluted in 5% formic acid. Non-specific adsorption, however, is a persistent problem that must be addressed prior to downstream analyses of eluted proteins. Anti-(C-peptide) antibodies captured up to 4 pmoles of C-peptide. This is a promising result for future studies that will employ membrane-immobilized anti-(C-peptide) to selectively capture C-peptide from cell secretions to help determine the role of this peptide in diabetic complications.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Bennett, Austin Landry
- Thesis Advisors
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Bruening, Merlin L.
- Committee Members
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Spence, Dana M.
Huang, Xuefei
- Date Published
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2016
- Subjects
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Antigen-antibody reactions
Membranes (Biology)
Peptides
Proteins--Purification
Immunoglobulins
Monoclonal antibodies
Biotechnology--Research
- Program of Study
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Chemistry - Master of Science
- Degree Level
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Masters
- Language
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English
- Pages
- xii, 76 pages
- ISBN
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9781339862279
1339862271
- Permalink
- https://doi.org/doi:10.25335/tapn-xf75