title: Defining the molecular target of an antibody derived from nuclear extract of Jurkat cells using protein arrays.
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.
authors.affiliation: Centre for Human Proteomics, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
authors.forename: Gregor
authors.initials: G
authors.lastname: Kijanka
authors.forename: Richard
authors.initials: R
authors.lastname: Barry
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Edith
authors.initials: E
authors.lastname: Gould
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Seidlits
authors.forename: Jasmin
authors.initials: J
authors.lastname: Schmid
authors.forename: Maria
authors.initials: M
authors.lastname: Morgan
authors.forename: David Y
authors.initials: DY
authors.lastname: Mason
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cordell
authors.forename: Derek
authors.initials: D
authors.lastname: Murphy
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000378:metabolism*; D019556:COS Cells; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000276:immunology; D002522:Chlorocebus aethiops; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D019169:Jurkat Cells; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D000090243:Nucleophosmin; D040081:Protein Array Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C000716967:NPM1 protein, human; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D000090243:Nucleophosmin