Type

Journal Article

Authors

Derek Murphy
Jacqueline Cordell
David Y Mason
Maria Morgan
Jasmin Schmid
Stephanie K Seidlits
Edith Gould
Hong Chen
Richard Barry
Gregor Kijanka

Subjects

Biochemistry

Topics
nuclear proteins cos cells cell nucleus protein array analysis cercopithecus aethiops humans recombinant proteins metabolism nucleophosmin antibodies monoclonal nuclear matrix associated proteins numa1 protein human antigens nuclear immunohistochemistry immunology animals jurkat cells

Defining the molecular target of an antibody derived from nuclear extract of Jurkat cells using protein arrays. (2009)

Abstract Many diagnostic antibodies are generated by immunization with whole cells or cell extracts and are shown by screening on tissue sections to label specific cell populations. However, their target molecule then needs to be identified, and this can be technically demanding. Here we describe the use of protein arrays to define the targets of new or uncharacterized monoclonal antibodies. The technique involves screening protein arrays containing thousands of recombinant human proteins. An initial experiment showed that a well-characterized monoclonal antibody against nucleophosmin identified 22 clones on the array encoding this protein. Next, the antibody JJ166, for which the antigen had not yet been identified, was screened. This antibody was generated by immunizing with a nuclear extract of Jurkat cells and was detected in immunohistochemistry due to its distinctive nuclear staining of lymphoid tissue cells. However, its molecular target had remained unidentified using traditional approaches. A protein array screen rapidly identified the mitotic spindle-associated molecule NUMA1 (nuclear mitotic apparatus protein 1). To confirm this putative specificity, JJ166 was shown to react with COS-1 cells transiently transfected with the complementary DNA for NUMA1. Furthermore, a commercially available antibody against NUMA1 showed nearly identical staining in immunohistochemistry on human tissue and cells. Overall, this method represents an effective and quick strategy for defining the protein targets of new or uncharacterized monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.
Collections Ireland -> Royal College of Surgeons in Ireland -> PubMed

Full list of authors on original publication

Derek Murphy, Jacqueline Cordell, David Y Mason, Maria Morgan, Jasmin Schmid, Stephanie K Seidlits, Edith Gould, Hong Chen, Richard Barry, Gregor Kijanka

Experts in our system

1
Maria P Morgan
Royal College of Surgeons in Ireland
Total Publications: 27
 
2
Jasmin Schmid
Royal College of Surgeons in Ireland
Total Publications: 13
 
3
Gregor Kijanka
Dublin City University