Characterization of monoclonal antibodies directed to the amino-terminus of the WT1, Wilms' tumor suppressor protein.
We have produced and characterized three monoclonal antibodies (MAbs) directed to the amino terminus of the WT1 Wilms' tumor suppressor transcription factor and compared their properties to rabbit polyclonal sera raised to the same immunogen. A recombinant protein consisting of amino acids 1-181 of human WT1 was overexpressed in E. coli, purified, and used as the immunogen. Three MAbs designated 6F-H2, 6F-H7, and 6F-HC-17--all of the IgG1 subclass--were selected and further characterized. Each recognized all isoforms of the full-length WT1 protein in Western blot assays and immunoprecipitated WT1 in both physiologic buffers and under high detergent/high salt (RIPA) conditions. Preliminary epitope mapping suggests that all three MAbs recognize a region in the amino terminal 84 amino acids of WT1 and that the MAbs do not recognize the polyglycine or polyproline regions of the protein. The WT1 antibodies do not recognize the structurally and functionally related early growth response (EGR)1, EGR2, EGR3, or EGR4 proteins. All WT1 MAbs recognize the murine WT1 protein and immunohistochemical staining of murine embryonic and newborn kidney sections show strong staining of condensing metanephric mesenchyme and primitive podocytes in developing glomeruli. These WT1-specific MAbs should be useful in characterizing the biochemical and developmental roles of WT1 and in defining the emerging role of WT1 as a diagnostic and/or prognostic marker in mesothelioma, leukemias, and breast cancer.
Rauscher
F J
FJ
The Wistar Institute, Philadelphia, PA 19104, USA.
Morris
J F
JF
Fredericks
W J
WJ
Lopez-Guisa
J
J
Balakrishnan
C
C
Jost
M
M
Herlyn
M
M
Rodeck
U
U
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D004268:DNA-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D051766:Early Growth Response Protein 1; D051776:Early Growth Response Protein 3; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D017874:Immediate-Early Proteins; D007150:Immunohistochemistry; D007668:Kidney / Q000737:chemistry / Q000196:embryology / Q000254:growth & development; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D025721:WT1 Proteins
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D051776:Early Growth Response Protein 3