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For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of untibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749–2248 or the nucleotide sequence 5456–6953 of the mRNA, which corresponds to the major deletion-prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.

All previous studies of the localization of utrophin (the dystrophin-related protein) in muscle and other tissues have been performed only with antibodies against the C-terminal region of the protein. Since several short forms of dystrophin, the apodystrophins, are produced from the 3′ end of the dystrophin gene, there is a possibility that similar short forms of utrophin exist and that these could be responsible for some of the many different localizations of ‘utrophin’ in muscle. We have produced a new panel of 15 mAbs against the N-terminal region of utrophin and we have used it together with mAbs against the C-terminal region to show that full-length utrophin is present at neuromuscular junctions, in nerves, blood vessels and capillaries in normal muscle and in the sarcolemma of patients with muscular dystrophy and dermatomyositis. However, two of the 15 mAbs also recognised rat/mouse utrophin and both of these detected an additional 62 kDa protein on Western blots of rat C6 glioma cells. This potential 62 kDa ‘apo-utrophin’ was not detected in human cerebral cortex, in rat Schwannoma cells nor in any of the non-nerve cells and tissues tested.

A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS-denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross-reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple-helical region of dystrophin (amino acids 1750–2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin-like central rod model for dystrophin.

Utrophin, the autosomal dystrophin-related protein (DRP), is expressed in HeLa cells, smooth muscle-like BC3HI cells from mouse brain, COS monkey kidney cells, the P38BD1 monocyte-macrophage cell line and untransformed human skin fibroblasts, as well as in rat C6 glioma and Schwannoma cells. It was undetectable, however, in the Sp2/O mouse myeloma cell line and in hybridoma lines derived from it. Dystrophin was not detected in any of these cell lines. Although all utrophin-containing cells were capable of forming monolayers in culture, no major effects of either attachment to substratum or length of time in culture (2–17 days) on utrophin levels were observed. After subcellular fractionation ofBC3HI or glioma cells, nearly all of the utrophin was found in the Triton-soluble fraction, suggesting an association with cell membranes.

Creatine kinase (CK) is often used as an index of differentiation in cultures of embryonic muscle cells [l] and its presence in serum is also used to monitor muscle damage in disease [2,3]. Immunological assays of CK can be made specific for any one of the CK isoenzymes and polyclonal rabbit antisera have been used for the study of muscle-specific CK (MM-CK) accumulation and distribution in differentiating myoblast cultures [4,5]. A monoclonal antibody (McAb) for these assays would have the added advantages of providing an unlimited supply of antibody with defined properties which can be selected for during McAb preparation. review), McAb CK-JOE does appear to be suitable for measuring MM-CK levels in muscle cell cultures.

We have demonstrated expression of a 6.3 kb Becker muscular dystrophy (BMD) human dystrophin cDNA following retroviral-mediated transduction of cultured myoblasts from the dystrophin-deficient mdx mouse. The truncated dystrophin protein was localised to the sarcolemma of differentiated myotubes by antibodies against the C-terminus of the molecule, and produced an identical immunostaining pattern to that observed in control myotubes expressing normal endogenous dystrophin. These results indicate that retroviral-mediated gene transfer may be useful for experimental in vivo studies on the complementation of dystrophin gene mutations.

Nineteen monoclonal antibodies which bind to native dystrophin in the plasma membrane of frozen muscle sections were obtained using a recombinant fusion protein as immunogen. On Western blots of normal mouse muscle extracts, the antibodies bind specifically to a 400 000 Mr protein which is absent from dystrophic mouse (mdx) muscle. At least four distinct epitopes have been identified by cleavage mapping methods. Although the fusion protein contained 25% of the human dystrophin sequence (Cys816-Asp1747; Mr 108 000), most of the monoclonal antibodies (15 out of 19) recognize a single fragment of Mr 27 500.