Characterization of Longissimus thoracis, Semitendinosus and Masseter muscles and relationships with technological quality in pigs. 1. Microscopic analysis of muscles.

Three porcine muscles (Longissimus thoracis, Semitendinosus, Masseter), known to have large differences in biochemical and histological traits, were fully characterized and the link between muscle structure and quality evaluated. The oxidative Masseter had more pigment, higher content of metmyoglobin, haem iron, protein and collagen, and was redder with higher fibre numbers, fibre circularity, pH and water holding capacity than the glycolytic Longissimus. Fibre type distribution showed predominance of type IIB in Longissimus and Semitendinosus white, type I in Semitendinosus red and IIA in Masseter. Type I fibres were larger than type IIB and IIA in Semitendinosus and Masseter, respectively, but not in the Longissimus, indicating that fibre size is muscle dependent. Muscle redness was positively correlated with type I fibre traits, haem iron and metmyoglobin, and negatively associated with type II fibre characteristics, non-haem iron and oxymyoglobin. Expressible juice had positive correlation with fibre size and negative with fibre number and connective tissue.

The fusion of lipid droplets is involved in fat loss during cooking of duck "foie gras".

Fat loss during cooking of duck "foie gras" is the main quality issue in processing plants. To better understand this phenomenon, a histological and ultrastructural study was conducted. The aim was to characterize changes in lipid droplets of duck "foie gras" related to fat loss during cooking. Ten fatty livers were sampled before and after cooking and prepared for optical and transmission electron microscopy. In raw livers, the lipid droplets were nearly spherical while after cooking, they were larger and lost their spherical shape. We also observed a decrease in the number of droplets after cooking, probably due to droplet fusion caused by the heat treatment. Before cooking, there were fewer lipid droplets and a higher osmium tetroxyde staining intensity in the fatty liver, which later gave a lower technological yield. Fat loss during cooking was higher when there was more fusion of lipid droplets before cooking.

Differentiation of bovine from porcine gelatines using polyclonal anti-peptide antibodies in indirect and competitive indirect ELISA.

Gelatine is a collagen derivative obtained from bones and hides/skin mainly from bovine and pigs. As a consequence of the outbreak of bovine spongiform encephalopathy (BSE), the use of bovine gelatine in feed, food and pharmaceutical products has been restricted by regulatory authorities. However, no method was presently available for its specific detection. The large similarity in amino-acid sequences of collagens from different species make their immunochemical differentiation difficult when using polyclonal antibodies raised against the whole molecule [A. Venien, D. Levieux, J. Immunoassay Immunochem., in press]. To obtain bovine-specific antibodies, we immunized rabbits against putative species-specific sequences of the bovine collagen alpha 1(I) chain. Using these antibodies, an indirect ELISA was developed to allow a quick and easy differentiation between bovine and porcine gelatines. Moreover, a competitive indirect ELISA was found suitable to detect bovine gelatine in porcine gelatine purchased from laboratory chemicals suppliers down to a dilution of 2-4 parts per 1000 with CVs ranging from 5.7 to 7.7%. When testing mixtures of the largest possible range of industrial batches of bovine and porcine gelatines (skin/hides or bones origin, acid or alkaline processes, high or low Bloom) the detection limit was down to a dilution of 8 parts per 100 bovine gelatine in porcine gelatine. These ELISAs could be routinely used by pharmaceutical and food manufacturers to secure their supply chain.

Oil/Alkanethiol Layers for the Study of Emulsified Protein Conformation by Surface Plasmon Resonance Using Monoclonal Antibodies.

A method combining surface plasmon resonance and epitope mapping was developed to study the protein conformation at the oil/water interface of an emulsion. The conformation of beta-lactoglobulin stabilizing dodecane/water and miglyol/water interfaces was investigated using five anti-beta-lactoglobulin monoclonal antibodies. The developed method allows us to specifically recognize the emulsified beta-lactoglobulin at the surface of a sensor chip with good repeatability; i.e., standard deviations range between 0.7 and 3.6%. Considering that the monoclonal antibodies, recognizing conformational epitopes, still bind to beta-lactoglobulin at oil/water interfaces, it is concluded that the protein retains a globular conformation. It is shown that the inhibition-binding values of two pairs of Mabs are different for beta-lactoglobulin stabilizing dodecane/water and miglyol/water interfaces. This indicates that the conformations of emulsified beta-lactoglobulin are slightly different according to the nature of the oil phase. Copyright 2000 Academic Press.

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: immunochemical characterization.

Bovine beta-LG was modified by glycation with lactose in a powdered state or in an aqueous solution. An immunological characterization was performed using monoclonal antibodies with defined epitopes. The results showed that the structural changes were confined to the AB loop region of the molecules when glycation was conducted in a restricted water environment and had little consequences on the association state of glycated beta-LG. The protein conformation was much more extensively modified when glycation was performed in an aqueous solution at 60 degrees C, despite a lower glycation extent. These structural changes were located at the dimer interface (AB loop, GH loop, beta-strand I, and alpha-helix). These results allowed us to establish a relationship between the conformational changes and the modification of the association state of the glycated protein (formation of disulfide bridges between the free thiol groups of two monomers), previously described.

Production and epitopic characterization of monoclonal antibodies against bovine beta-lactoglobulin.

Sixty-nine murine monoclonal antibodies were produced against the bovine beta-lactoglobulin (beta-LG) variant B. Thirty-eight specificity groups of monoclonal antibodies were defined according to the reactivity in indirect or capture ELISA with bovine beta-LG B, for bovine beta-LG B coated at pH ranging from 2.3 to 9.4, and for beta-LG with naturally occurring residue substitutions such as bovine beta-LG variant A and ovine, caprine, porcine, and equine beta-LG. Ten monoclonal antibodies appeared to be monospecific for bovine beta-LG, 58 monoclonal antibodies recognized only ruminants beta-LG, and 1 recognized all species. The specificity of the monoclonal antibodies was also studied with sequenced tryptic peptides of beta-LG variant B. Critical residues involved in the epitopes of nine classes of monoclonal antibodies were characterized. Moreover, 5 monoclonal antibodies were able to discriminate between a fresh solution of beta-LG and a solution that had been stored for > or = 2 d at 4 degrees C, and 3 monoclonal antibodies distinguished bovine beta-LG that has been purified by gel filtration from that purified by salt fractionation. This array of monoclonal antibodies could be efficient probes for characterizing conformational structures involved in biological properties of beta-LG (e.g., interaction with ligands and hypersensitivity reactions) and for studying conformational changes occurring upon physical treatments such as heat denaturation.

Tissue distribution and characterization of a 30-kDa cysteine proteinase inhibitor from bovine skeletal muscle.

Gel chromatography on a Sephadex G100 column of a crude extract obtained from bovine diaphragma muscle separated four fractions (F-I, F-II, F-III and F-IV) in the range of 12-70 kDa that were active against either papain, trypsin or both. From the F-III fraction, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatographies on Q-Sepharose and Mono Q columns. The pooled active fraction had a Mw of approximately 30 kDa, and isoelectrofocusing revealed one band with a pI of 6.7. The papain-inhibiting activity was unaffected by dithiothreital or 2-mercaptoethanol treatment, and only one band was obtained after SDS-poly acrylamide gel electrophoresis under both reducing and non-reducing conditions. These results suggest that the 30-kDa muscle cysteine proteinase inhibitor did not contain disulphide bonds essential for activity and the protein was a monomer. This proteinase inhibitor is stable between 40 and 80 degrees C and pH 5-12. Furthermore, the 30-kDa inhibitor is stable to papain proteolysis. The tissue distribution of this inhibitor was investigated using double immunodiffusion and Western blot techniques that provided evidence for its presence in bovine heart, spleen, liver and lung and its absence in bovine plasma.

Epitopic analysis and quantification of bovine myoglobin with monoclonal antibodies.

Seven rat monoclonal antibodies (MAb) for bovine myoglobin were produced. Five antibodies reacted with surface-absorbed myoglobin whereas the two remainders reacted only in a sandwich type ELISA. The ability of different antibodies to bind simultaneously to myoglobin was examined by competition and additivity experiments and three noncompeting epitope regions were found. A two-site enzyme immunoassay was developed and allowed quantification of 30 ng/ml bovine myoglobin. These antibodies should be valuable tools in comparative studies for immunological reactivity of mammalian myoglobins and for myoglobin measurement in serum and urine of myopathic animals.

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.

Early immunodiagnosis of bovine fascioliasis using the specific antigen f2 in a passive hemagglutination test.

An improved hemagglutination (HA) test using the purified specific f2 antigen of Fasciola hepatica has been evaluated with regard to its potential use for early diagnosis of infection. On experimental infection of beef calves 6-8 months of age, f2-specific antibodies were detected 2-4 weeks after inoculation and persisted at high levels during the 28 week experimental period. On natural infection of dairy calves, 6-9 months of age, allowed to graze in infected fields from April to June, 48% of the animals were positive in July although coproscopic analyses were negative. In November all the calves were HA positive but only 24% of them excreted F. hepatica eggs. Beef calves 1-3 months of age allowed to graze with their dams in infected fields continued to suckle their dams and were weakly infected according to HA results. This weak infection was not detected by coproscopical analysis. Colostral f2-specific antibodies persisted for approximately 6 months in the serum of dairy calves allowed to suckle their dams immediately after birth.

An improved passive hemagglutination test for the serological diagnosis of bovine fascioliasis using the specific antigen f2.

Optimum conditions for coupling the specific antigen f2 of Fasciola hepatica to sheep red cells and for the preparation of control cells coated with an unrelated protein are described. With a careful selection of donor sheep for erythrocytes, the problem of anti-species antibodies in bovine sera has been overcome and results may be obtained within 1 h as only one step is required in the assay system. Incorporation of a concentration of 25 mM CaCl2 in the diluent achieved increased stability of the agglutinates and enabled a more precise estimation of the end-point to be made. Analyses performed on bovine sera obtained at slaughter provided results in good agreement with the presence of flukes or fascioliasis lesions in livers at routine slaughter inspection. The developed assay is simpler, faster, more sensitive (P less than 0.01) and has a cut-off between populations of negative sera more easy to define than a recently marketed enzyme-linked immunosorbent assay.

A rapid and simple method for the preparation of a monospecific antibody to ovine haptoglobin.

Antisera specific to ovine haptoglobin were obtained by the immunization of rabbits with their own insolubilized haemoglobin after it had been allowed to react with a pool of haptoglobin-rich ovine serum. This method exploits the unique property of haptoglobin to bind with great specificity and affinity to both homologous and heterologous haemoglobin. The procedure is rapid, simple, inexpensive and could be readily applied to the production of antisera against the haptoglobins of other species.

Purification of the protein X of Streptococcus agalactiae with a monoclonal antibody.

The protein X of Steptococcus agalactiae is a surface antigen included in the typing scheme of group B streptococci (GBS). We have developed a monoclonal antibody to the protein X and used it to purify this antigen by affinity chromatography. Electrophoresis in polyacrylamide, and immunoblotting using the monoclonal antibody or a rabbit antiserum raised with the affinity purified protein X, revealed a major band in the region of 200 kDa and a smaller one at 100 kDa. The isolated protein X will make possible investigations of its potential role in virulence and protection.

[Localization, on the head of ram spermatozoa, of affinity sites for the 64kD androgen dependent epididymal prealbumin]. / Localisation sur la tête des spermatozoïdes de bélier de sites d'affinité pour la préalbumine épididymaire androgènedépendante 64 kD.

The epididymis of the ram synthesizes under androgenic control of specific 64 kD protein. The purified protein appeared as a single band in SDS polyacrylamide gel electrophoresis. It was labelled and an antiserum was prepared in mice. Results showed localization of receptors sites for 64 kD on the periacrosomal plasma membrane of testicular spermatozoa. The protein itself was found on the periacrosomal area of both epididymal and ejaculated spermatozoa.