Chapter title |
Analysis of Antibody Clonotype by Affinity Immunoblotting
|
---|---|
Chapter number | 15 |
Book title |
Western Blotting
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2694-7_15 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2693-0, 978-1-4939-2694-7
|
Authors |
Biji T. Kurien Ph.D., R. Hal Scofield M.D., Biji T. Kurien, R. Hal Scofield |
Editors |
Biji T. Kurien, R. Hal Scofield |
Abstract |
A sensitive and specific method to analyze specific antibody clonotype changes in a lupus patient who developed autoantibodies to the Ro 60 autoantigen under observation is described. Patient sera, collected over several years, were separated by flatbed isoelectric focusing (IEF) and analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane. When the Ro 60-coated nitrocellulose was laid over the surface of the IEF gel, the antibodies present on the surface of the acrylamide gel bound the Ro antigen on the nitrocellulose. Tween-20 was used to prevent nonspecific binding. The bound IgG clonotypes were detected using alkaline phosphatase conjugated anti-IgG. The patient's sera demonstrated an oligoclonal response to the Ro 60 autoantigen that increased in complexity and affinity over time. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 6 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 3 | 50% |
Student > Ph. D. Student | 1 | 17% |
Unknown | 2 | 33% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 2 | 33% |
Agricultural and Biological Sciences | 1 | 17% |
Immunology and Microbiology | 1 | 17% |
Unknown | 2 | 33% |