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Monoclonal Antibodies to the Myogenic Regulatory Protein MyoD1: Epitope Mapping and Diagnostic Utility¹

Department of Biochemical and Clinical Pharmacology [P. D.,P. J. H.], Pathology and Laboratory Medicine [D. M. P.], and Hematology-Oncology [D. N. S.], St. Jude Children's Research Hospital, Memphis, Tennessee 38101; Department of Genetics, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 [S. J. T.]; and Department of Pathology, University of Tennessee, Memphis, Tennessee 38103 [D. M. P.]

² To whom requests for reprints should be addressed, at St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38101.

Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167–318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170–209 by Western analysis and immunoprecipitation of in vitro transcribed and translated MyoDl protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoDl into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent.

MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diaonnsed as undifferenriated sarCOmas.

¹ Supported by USPHS awards CA23099, 5U10CA24507, and CA21765 (CORE) from the National Cancer Institute and by the American Lebanese Syrian Associated Charities.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 8/92. Accepted 9/18/92.

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