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The X-linked form of Emery-Dreifuss muscular dystrophy (EDMD) was recently shown to be due to mutations in the STA gene on chromosome Xq28. We have demonstrated a simple test for the diagnosis of this condition, looking for altered expression of the protein, emerin, in leucocytes and skin with a monoclonal antibody. Full-length emerin is completely absent in affected boys from the EDMD families studied. The method has also enabled identification of a female carrier of the disease by reduced levels of the protein on the leucocyte Western blot and a mosaic pattern of expression by immunofluorescence microscopy of the skin biopsy.

A combination of isoform-specific, quantitative RT-PCR and site-specific monoclonal antibodies has been used to produce consistent evidence at both RNA and protein levels for the principal isoform products of SYNE1 and SYNE2, the genes encoding nesprin-1 and nesprin-2 proteins. Western blots show that both genes produce three groups of isoforms that are homologous in size and structure: giant, or full-length, isoforms of about 1000 kDa, medium isoforms of about 380 kDa (nesprin-1-beta and nesprin-2-gamma) and short isoforms of about 110 kDa (nesprin-1-alpha and nesprin-2-epsilon). SYNE2 alone produces a very short isoform at 60 kDa (nesprin-2-alpha) in skeletal muscle. Although the giant forms were the dominant isoforms in all tissues, major differences between tissues in expression of shorter isoforms were observed. From SYNE1, nesprin-1-alpha was found in skeletal and cardiac muscle only. Several cell lines, including embryonic stem cells, did not produce any detectable nesprin-1 at all. From SYNE2, we observed a developmental transition from nesprin-2-epsilon-1 at very early stages (embryonic stem cells, embryonal teratocarcinoma and ovary tissue) to nesprin-2-epsilon-2 in adult and fetal tissues. Both skeletal muscle and heart have a high proportion of short isoforms compared with other tissues, but heart expresses the nesprin-2-epsilon-2 isoform where skeletal muscle produces nesprin-2-alpha-1. This study has clarified the major nesprin isoforms expressed in cells and tissues, especially in cardiac and skeletal muscle, which are the major affected tissues in neuromuscular disease. Mutations in nesprin-1 and/or nesprin-2 can cause both Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. The operative functional unit appears to be the “LINC” complex of emerin, lamin A/C, nesprins and SUN proteins, since mutations in any single component can result in a similar pathology.

Overview: Choosing a Method for Epitope Mapping Glenn E. Morris Crystallographic Studies of Antigen-Antibody Interactions Frederick A. Saul and Pedro M. Alzari Epitope Mapping Antibody-Antigen Complexes by Nuclear Magnetic Resonance Spectroscopy Irina Kustanovich and Anat Zvi Mapping Epitopes on Antigens by Immunodiffusion in Gel Giuseppe A. Molinaro and William C. Eby A Simple Solid-Phase Competition Assay with Labeled Antigen Masahide Kuroki Epitope Mapping by Antibody Competition: Methodology and Evaluation of the Validity of the Technique Socrates J. Tzartos Epitope Mapping by Surface Plasmon Resonance in the BIAcore Berit Johne Identifying Residues in Antigenic Determinants by Chemical Modification N. Martin Young and Raymond P. Oomen Epitope Mapping by Differential Chemical Modification of Antigens Hans Rudolf Bosshard Epitope Mapping by Proteolysis of Antigen-Antibody Complexes: Protein Footprinting Ronald Jemmerson Proteolytic Fragmentation for Epitope Mapping Maria R. Mazzoni, Nickolai O. Artemyev, and Heidi E. Hamm Epitope Mapping by Chemical Fragmentation Glenn E. Morris Probing Antibody-Antigen Interactions by Mass Spectrometry Yingming Zhao and Brian T. Chait Epitope Mapping Using Multipin Peptide Synthesis Stuart J. Rodda, N. Joe Maeji, and Gordon Tribbick SPOT Synthesis: Epitope Analysis with Arrays of Synthetic Peptides Prepared on Cellulose Membranes Ronald Frank and Heike Overwin Tea Bag Synthesis of Positional Scanning Synthetic Combinatorial Libraries and Their Use for Mapping Antigenic Determinants Clemencia Pinilla, Jon R. Appel, and Richard A. Houghten Epitope Mapping Using Phage-Displayed Peptide Libraries Diane Dottavio Reiterative Screening of Phage-Display Peptide Libraries, with Antibodies Alexander Pereboev and Glenn E. Morris Homolog Scanning Lin-Fa Wang Epitope Mapping Using an Oligonucleotide Replacement Method Hannah Alexander Epitope Mapping by Region-Specified PCR Mutagenesis Takehiko Shibata and Masayuki Ikeda Random Fragment Libraries Using Yeast Expression Plasmid Serge Benichou and Genevieve Inchauspe Epitope Mapping Using Random Fragment Expression Libraries in l Phages Hartmut Porzig and Kenneth D. Philipson Random Fragment Libraries Displayed on Filamentous Phage Lin-Fa Wang and Meng Yu Epitope Mapping by Expression of Restriction Enzyme or PCR Fragments in Bacterial Plasmids Johannes A. Lenstra and Arnoud H. M. Van Vliet Epitope Mapping of Protein Antigens by Expression-PCR (E-PCR) David E. Lanar, Kevin C. Kain, and Henry B. Burch Epitope Mapping on Extracellular Domains of Cell-Surface Proteins Using Exonuclease III Thomas Brummendorf, Antonius Plagge, and Ullrich Treubert An Improved Method for Mapping Epitopes of Recombinant Antigens by Transposon Mutagenesis Steven G. Sedgwick, Brian A. Morgan, Nguyen thi Man, and Glenn E. Morris Incomplete Polypeptides of In Vitro Translation for Epitope Localization Lisa Djavadi-Ohaniance and Bertrand Friguet T-Cell Epitope Mapping with Synthetic Peptides and Peripheral Blood Mononuclear Cells Stuart J. Rodda Use of Natural or Selected Mutants and Variants for Epitope Mapping Glenn E. Morris Production of Panels of Monoclonal Antibodies by the Hybridoma Method Nguyen thi Man and Glenn E. Morris Production of Phage-Display Antibodies for Epitope Mapping Jenny Walker and George Banting Index

A large fragment of emerin cDNA was prepared by PCR and expressed as a recombinant protein in Escherichia coli. Using this as immunogen, we prepared a panel of 12 monoclonal antibodies which recognise at least four different epitopes on emerin in order to ensure that emerin can be distinguished from non-specific cross-reacting proteins. All the mAbs recognised a 34 kDa protein in all tissues tested, though minor emerin-related bands were also detected in some tissues. Immunofluorescence microscopy showed that emerin is located at the nuclear rim in all tissues examined. A muscle biopsy from an Emery-Dreifuss muscular dystrophy (EMDM) patient showed complete absence of emerin by both Western blotting and immunohistochemistry, suggesting a simple diagnostic antibody test for EDMD families. Biochemical fractionation of brain and liver tissues showed that emerin was present in nuclei purified by centrifugation through 65% sucrose and was absent from soluble fractions (post-100,000 g). From these results, together with sequence and structural homologies between emerin, thymopoietins and the nuclear lamina-associated protein, LAP2, we suggest that emerin will prove to be one member of a family of inner nuclear membrane proteins.

PCR studies have shown that exons 71-74 are spliced out in most dystrophin mRNA transcripts in the brain. We have prepared new monoclonal antibodies against the syntrophin - binding region of dystrophin encoded by exons 73-74 and examined three protein products of the dystrophin gene in brain; the widely distributed Dp71, the recently discovered, brain-specific Dp140 and dystrophin itself. Exon 73-74 m Abs bound to all three proteins in brain and the extent of binding suggests that alternatively spliced dystrophins are less prominent at the protein level than predicted by PCR data. Dp140, unlike Dp71, was found to be present at much higher levels in foetal brain than in adult brain. If lack of functional Dp140 is the cause of the cognitive impairment in some Duchenne muscular dystrophy patients, this result suggests that the effects may occur early in development, which would reduce the options for therapeutic intervention.

We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immuno-histochemistry or Western blotting with these "exon-specific" mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying "revertant" fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene.

We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies. The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure. Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion. One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary.

Using a panel of "exon-specific" monoclonal antibodies, we have examined the products of degradation of dystrophin by endogenous proteases in post-mortem human muscle. Four main sites of dystrophin digestion were identified, all of them in the C-terminal half of the molecule. Two of them correspond to "hinges" in the central rod region and a third in the C-terminal domain follows the dystroglycan binding site. The results support the Koenig and Kunkel model for the tertiary structure of dystrophin (J. Biol. Chem. 265 (1990) 4560-4566), but suggest that much of the N-terminal half of dystrophin is protected from proteolysis, possibly by interaction with the sub-sarcolemmal cytoskeleton. Although the results seem inconsistent with an anti-parallel dimer model of dystrophin in which hinge 2 and hinge 3 are close together, possible ways of reconciling them with such a model are also considered.

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.

In countries where malaria is endemic, its transmission is a hazard of blood transfusion. The microscopical and immunological methods in current use for malaria diagnosis are unsatisfactory for low levels of parasitaemia in blood donations. The polymerase chain reaction (PCR) can be 100-fold more sensitive than thick blood film examination when appropriate primers are used and can detect and distinguish Plasmodium falciparum and P. vivax in a single tube. A study of 1506 blood donations in Ho Chi Minh City (3 of which were positive) suggests that PCR can provide an effective screen for P. falciparum under local conditions. Studies in a region of Viet Nam where malaria is common showed that PCR detects many more cases of low-level parasitaemia (19/30) than thick blood films (4/30).

CK-STAR, a monoclonal antibody against human brain creatine kinase (CK), can be shown by chemical cleavage mapping and peptide synthesis to recognize an epitope at the free N-terminus of the enzyme. The epitope could be largely reproduced by a synthetic peptide based on the first 18 amino acids and could be partly formed by the first 11 amino acids. The antibody did not bind to native CK, but it did bind to CK in various partially denatured forms and to an enzymically inactive intermediate in the refolding process. Competitive binding studies have shown that the N-terminal conformations of both the refolding intermediate and the free peptide resemble that of CK partially denatured by attachment to plastic. The results suggest that the final stages of CK refolding and reactivation involve a structural change at the N-terminus or its interaction with some other part of the CK molecule, thus masking the CK-STAR epitope.

Ten monoclonal antibodies were obtained from mice immunized with a yeast recombinant hepatitis B vaccine. They were selected at an early stage for their ability to bind to native surface antigen particles (HBsAg) in human plasma. All antibodies recognized conformational epitopes which were destroyed completely or almost completely by reduction of disulphide bridges. They were divided into five epitope groups by their competition for binding to recombinant S protein, though epitopes within each group are not identical. Recombinant S protein migrated on SDS-PAGE in the absence of reducing agents as a mixture of monomers and dimers/oligomers. Sucrose gradient analysis suggests that all these forms are co-aggregated into HBsAg-Iike particles. On Western blots, all ten antibodies either bound only to dimers/oligomers or strongly preferred them over monomers. The results suggest that, of the antibodies produced in response to recombinant vaccine in mice, most of those which bind strongly to ‘native’ HBsAg particles in human plasma recognize surface structures created by interaction between two subunits.

We have located the epitopes recognized by four different monoclonal antibodies which bind to partially unfolded creatine kinase (CK) (ATP: creatine N-phosphotransferase, EC 2.7.3.2) but not to the native enzyme. The epitopes appear to be buried within the CK structure in its native, proteinase-resistant, state. When the epitopes are made accessible to antibody by mild denaturation, CK becomes enzymically-inactive and can be cleaved by proteinase V8 into two large fragments which retain the epitopes and may represent domains. Epitopes on each V8 fragment are associated with highly conserved sequences and are brought physically close to the active site of the enzyme during the later stages of CK refolding and reactivation. The results suggest a catalytic site formed at the interface between two domains which carry the epitopes on their interacting surfaces. Separation of loosely associated domains before or during immunization may account for the origin of antibodies against buried epitopes.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy gene exists known as SMN2, however, due to an aberrant splicing event, the SMN2 gene fails to produce sufficient full-length protein to protect against disease development in the absence of SMN1. While a number of compounds have recently been identified that can stimulate full-length survival motor neuron (SMN) expression from the nearly identical copy SMN2, one of the difficulties has been the lack of a highly reproducible and quantitative means to measure the levels of SMN protein. To develop a technique that allows the rapid and highly sensitive measurement of SMN protein, a Surface Plasmon Resonance (SPR) application has been developed. The ability to quantify unassociated SMN protein and monitor the binding of SMN with other proteins in solution using a SPR sensor in less than 15 min and at low ng mL(-1) levels in HEPES Buffer Saline (HBS) has been achieved. The detection limit for the specific binding of SMN in HBS pH 7.4 solution is 0.99 ng mL(-1) with non-specific binding accounting for approximately 30% of the signal. Quantification of SMN is based on an immunoassay performed on the gold surface of the SPR sensor. 16-mercaptohexadecanoic acid (MHA) was reacted with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to form a pre-activated thiol (MHA-NHS). Antibodies for SMN were then coupled to the sensor with the pre-activated thiol. Sensor specificity was examined with mixtures of myoglobin (MG) and SMN. SMN sensor response decreases by more than 60% when MG was added to SMN. The decrease in sensor response can be attributed to non-specific binding of SMN to MG, verified with a sensor for MG.

The USA and Canada are each considering measures to reduce general exposure to asbestos. Product bans and workplace exposure rules may each reduce general exposure by reducing domestic asbestos demand. The effectiveness of demand-reducing measures will depend upon the elasticity of asbestos supply to the domestic market. This paper provides calculations of this elasticity for three fibre grade groupings, using a framework that recognizes international trade in asbestos and the joint product nature of most asbestos fibre production. The results suggest that reductions in North American demand result in relatively substantial changes in asbestos use but very small changes in price.

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K ‘nicks’ native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to ‘nicked’ CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

Isolated nuclei from chick skeletal muscle cell cultures at different stages of differentiation were labelled in vitro with γ-[ 32 P]ATP. Nuclear proteins were separated into saline-soluble and non-histone (NHP) fractions and analysed by two-dimensional gel electrophoresis and autoradiography. Relatively few proteins (10–12) were phosphorylated to any extent and most of these corresponded to minor, or even undetectable, components on stained gels. 32 P incorporation into most of these proteins increased between 24 and 40 h and decreased sharply between 40 and 66 h, the changes being more marked in some phosphoproteins than in others. Pulse-chase data suggest that these overall changes in phosphorylation reflect changes in protein kinase, rather than protein phosphatase activities. One highly phosphorylated acidic protein (mol. wt 10 000 D; pI 4.2) which was undetectable on stained gels, showed a continuous increase in 32 P incorporation between 24 and 66 h in both saline-soluble and NHP fractions. This protein may be the muscle counterpart of a phosphorylated nuclear protein partially characterized in other cell types. Both nuclear protein phosphorylation and low molecular weight non-histone proteins have been particularly implicated in activation of gene transcription in other systems, although we have no direct evidence, as yet, to connect them with control of myoblast growth and differentiation.

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.

Treatment of human muscle creatine kinase (MM-CK) with glutaraldehyde produced highly aggregated forms which retained the native antigenic structure. Immunization of BALB/c mice with CK aggregates instead of untreated CK produced over ten-fold higher titres of antibody against native CK without increasing the tires of antibody against denatured enzyme. Production of high-affinity monoclonal antibodies specific for both the muscle isoenzyme and the native conformation became possible where the use of untreated CK had failed. Four monoclonal antibodies have been characterized by an epitope mapping technique and compared with a commercially available monoclonal antibody. One antibody has a much higher affinity for MM-CK than the other three and the commercial antibody. Competition studies show that it also recognizes a different epitope on the CK surface from the other three monoclonal antibodies which bind to the same surface region as the commercial antibody. Immunoassays based on the high affinity antibody can easily measure less than 1 ng of CK, a sensitivity comparable to, or better than, standard enzymatic assays.

Proteinase K cleaves a small peptide from native muscle-specific creatine kinase. We present evidence, from the binding of two monoclonal antibodies to formic acid-cleavage fragments and proteinase K-digest fragments of chick muscle creatine kinase, that the proteinase K-cleavage site is in the C-terminal region of the molecule. This specificity of proteinase K, which is not normally a highly specific enzyme, and the continued association of the two peptide fragments after cleavage suggest an unusual conformational feature in the cleavage-site region. By applying predictive methods for hydrophobicity and secondary structure to an amino acid sequence in this region, we suggest possible structural features at the cleavage site that are evidently conserved across avian and mammalian species. The most likely site is next to, or within, a beta-turn on the surface of the molecule.

Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 °C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.

Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca2+ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca2+ concentrations (0.05–0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca2+ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca2+ effects on the muscle-specific enzyme, creatine kinase.

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.