Lactoferrin and immunoglobulin contents in camel's milk (Camelus bactrianus, Camelus dromedarius, and Hybrids) from Kazakhstan.

Lactoferrin (Lf) and IgG were estimated in camel's milk from Kazakhstan, where 2 species of camels (Camelus bactrianus, Camelus dromedarius) and their hybrids cohabit. The concentrations of Lf and IgG were determined according to 3 variation factors: region (n = 4), season (n = 4), and species (n = 5; sample 4 was mixed milk and sample 5 was of unknown origin). The mean values in raw camel's milk were 0.229 +/- 0.135 mg/mL for Lf concentration and 0.718 +/- 0.330 mg/mL for IgG concentration. The seasonal effect was the only significant variation factor observed, with the highest values in the spring for Lf and in the winter for IgG. The Lf concentration varied in 1-wk postpartum milk from 1.422 to 0.586 mg/mL. The range in IgG concentration was wide and decreased from 132 to 4.75 mg/mL throughout the 7 d postpartum, with an important drop after parturition. In fermented milk, the lactoproteins are generally hydrolyzed. For milk samples from undefined species, discriminant analyses did not allow the origin of the species to be determined. A slight correlation between Lf and IgG concentrations was observed in raw milk. The values were slightly higher than those reported in cow's milk, but this difference was insufficient to attribute medicinal virtues to camel's milk.

Bovine muscle 20S proteasome. III: Quantification in tissue crude extracts using ELISA and radial immunodiffusion techniques and practical applications.

The 20S proteasome is a large complex (700kDa) that exhibits endo- and exo-peptidase activities with wide specificity. In postmortem muscles, several sets of evidence suggest a possible significant contribution of proteasome to meat tenderisation. Hence, an accurate and rapid quantification procedure is needed to attest that new function during the ageing of meat. In the present work, we developed an ELISA test enabling the quantification of nM concentrations of the 20S proteasome. We further tested the radial immunodiffusion (RID) technique described as a more simple method that can quantitatively determine the concentration of an antigen in a complex mixture. The ELISA test allowed us to quantify the 20S protesome in tissue homogenates and fluids with a recovery of 100%, a coefficient of variation lower than 5% and a detection limit of 9ng/ml. Quantification of the 20S proteasome in various bovine tissue by ELISA showed the highest concentration in liver followed by spleen and kidney, with muscles exhibiting the lowest concentrations. In addition, measurement of the proteasome concentration in eight different bovine muscles with various metabolic profiles led to the conclusion that the relationship between muscle metabolic properties and proteasome concentration is rather complex. Nevertheless, heart muscle exhibited the highest proteasome content (331µg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213µg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209µg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203µg/g wet tissue), an intermediate muscle. As compared to other endogenous peptidases, muscle tissue contains relatively high amounts of proteasome. Hence this complex can be quantified using the RID, which allows quantification of protein in the µg range. Plotting the concentration values determined with both methods for all bovine tissues tested gave a straight line with a correlation coefficient of 0.99.

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.

Immunoquantitation of rat erythrocyte superoxide dismutase: its use in copper deficiency.

Two immunoassays have been developed for the determination of rat erythrocyte dismutase (Cu,Zn-SOD). An enzyme-linked immunosorbent assay (ELISA) was very sensitive down to 4 ng/ml with a coefficient of variation (CV) of 18% while the single radial immunodiffusion assay (SRID) permitted an adequate detection level (5 micrograms/ml) with far better accuracy (CV = 4.2%). The latter was thus selected for the determination of Cu,Zn-SOD in the red blood cells of normal and copper-depleted rats. The average value of Cu,Zn-SOD in normal adult rat erythrocytes was 1142 +/- 120 ng/mg hemoglobin. When compared to activity measurements, good correlation was obtained between enzyme content and enzyme activity (r = 0.803, P less than .001). In an experimental copper deficiency followed by supplementation, good correlation was observed in the course of depletion (r = 0.848, P less than .001) and repletion (r = 0.896, P less than .001). During depletion, the loss of enzyme activity was mainly related to a loss of enzyme. However, enzymatically inactive protein was formed which would be activated when copper was added. These results indicate the importance of a combined use of Cu,Zn-SOD immunoquantitation and activity measurements to enable a better understanding of changes occurring with respect to enzyme activity.

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.

Experimental Corynebacterium pseudotuberculosis infection in lambs: kinetics of bacterial dissemination and inflammation.

Infection and pyogranulomas induced by Corynebacterium pseudotuberculosis were experimentally reproduced in lambs. In two separate experiments, bacterial multiplication and dissemination were studied in 30 male lambs inoculated subcutaneously into the right ear with 1.1 or 1.5 X 10(8) viable C. pseudotuberculosis strain 19R. Infected lambs were necropsied at various times until the 28th day following inoculation. After a transient hyperthermia and a strong local inflammatory reaction, an abscess developed in the right ear from postinoculation day (PID) 6; it enlarged until PID 14 and stabilized thereafter and was associated with adenopathy of lymph nodes draining the head. Three acute phase indicators of inflammation were followed in 14 out of 30 lambs; plasma levels of copper and haptoglobin increased rapidly following inoculation whereas zinc levels decreased. The peaks were reached from PID 1 to 5, and thereafter the values came back slowly to the baseline. Antibodies against C. pseudotuberculosis exotoxin increased from PID 5 and reached a plateau on PID 21. Bacterial dissemination, assessed by the number of infected organs per lamb, was maximal on PID 16 and then stabilized until the end of the experiment. Lungs were infected in seven out of 18 lambs necropsied on PID 28. These results demonstrate a significant relationship between the clinical score of superficial lymph nodes or inoculation site and the infection level of these organs, and an early localization of pyogranulomatous lesions in regional lymph nodes. The subsequent development of the disease was related to the enlargement of these lesions and, in some animals, to a bacterial dissemination from primary sites of infection in the right prescapular lymph node and in the lung.

Antipeptide antibodies recognizing plasmin sensitive sites in bovine beta-casein sequence.

To investigate plasmin activity in cheese, we produced antibodies to bovine beta-casein with controlled specificity, suitable as markers of the integrity of the major bonds involved in its initial breakdown. Sixteen rabbits were immunized with synthetic substitutes for six plasmin-sensitive peptides. Antisera raised to the peptides (f20-39), (f40-56), (f94-113), (f184-202), and (f193-209) recognized beta-casein in ACP-ELISA, Western-blot, and biosensor assays. Casein in vitro hydrolysis by plasmin or chymosin reduced the detection of these determinants in ACP-ELISA, in agreement with the enzymatic sensitivity of bonds included within the binding sites, or in their neighborhood. Antiserum to (f20-39) in particular allowed the specific detection of plasmin cleavage at the bond generating gamma1-CN. Antisera to C-terminus preferentially detected the cleavage by chymosin. Immunoassays using these antibodies would allow in situ monitoring of significant proteolysis events without bias originated in the secondary degradation of the released peptides.

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.

Immunochemical studies on gastric and intestinal digestion of soybean glycinin and beta-conglycinin in vivo.

Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and beta-conglycinin in preruminant calves fed milk replacers containing a mixture of skim milk powder and antigenic heated soybean flour. In experiment 1, duodenal passage of immunoreactive beta-conglycinin lasted for a much longer time after the morning meal than that of glycinin. Western blotting revealed the early abomasal outflow of glycinin subunits that associated nearly intact basic polypeptides to partially degraded acidic polypeptides. Intact beta-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (M(r) = 21000) associated with partially digested acidic glycinin (7000 < M(r) < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. beta-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.

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.

Immunochemical characterisation of species-specific antigens in bovine crude heparin.

In order to develop immunoassays for the control of the species origin of crude heparins, polyclonal antisera were produced in rabbits against samples obtained from the last purification steps of bovine intestinal crude heparin. The reactivity of the antisera was analysed by agar gel double immunodiffusion, immunoelectrophoresis, crossed and line immunoelectrophoresis. Up to 13 antigenic components were detected in the effluents of the ion-exchange chromatographic step, and three in the final crude heparin. The major and most anodic antigen (Ag1) was recovered in bovine crude heparin purified by the two different industrial processes. This bovine specific antigen was found in high concentrations in lung, liver, small intestine, spleen and kidney. It displayed an apparent molecular weight of 45 kDa by size-exclusion chromatography. Though the identification of Ag1 is not yet fully elucidated, a single radial immunodiffusion assay has been developed for the quantification of this antigen, allowing the detection of 6 p 1000 bovine crude heparin in porcine heparin (50 mg/ml) after 2 h diffusion.

Immunochemical control of the species origin of porcine crude heparin and detection of ovine and caprine materials.

As a consequence of the outbreak of bovine spongiform encephalopathy (BSE), ruminants materials have been generally banned from the production of heparin. Immunochemical methods have been recently developed for the control of the raw materials used by manufacturers of materials such as porcine mucosa and for the detection of bovine crude heparins. To certify the porcine origin of crude porcine heparins and to exclude ovine or caprine materials, new ELISAs were developed. Rabbit antisera were produced against species-specific antigenic contaminants present in crude heparins or in eluted materials (EM) from the chromatographic step of the purification process. When analysed by line immunoelectrophoresis, these antisera revealed five to eleven antigenic contaminants in the EMs, the major one being the most anodic and predominant antigen in crude heparins. Using the best antisera, competitive indirect ELISAs were optimised. They allowed the detection of porcine, ovine and caprine crude heparins down to a dilution of 0.6 to 1.5 parts per 1000, with CVs ranging from 3 to 12%. These ELISAs complete the set of immunological techniques which can be routinely used by heparin manufacturers to secure their supply chain.

Early immunodiagnosis of caprine fasciolosis using the specific f2 antigen in a passive hemagglutination test.

An improved hemagglutination (HA) test using the purified specific f2 antigen of Fasciola hepatica has been evaluated with respect to its potential use in the diagnosis of caprine fasciolosis. Following experimental infection of 1-year-old goats with a single heavy infection of 300 metacercariae, f2-specific antibodies were detected 2-3 weeks after infection and increased steadily to reach a maximum titer 9 weeks after infection, after which the antibody level declined. In animals receiving multiple infections of a lower dose of 50 metacercariae given at weekly intervals for 6 weeks, f2-specific antibodies were detected 3 weeks after infection and increased to reach a plateau 11 weeks after infection which was maintained until the end of the experiment (15 weeks after infection). Depending on animals and groups, eggs appeared in the feces between 7 and 9 weeks after infection. The HA test may provide valuable information about the early detection of caprine fasciolosis, particularly during the prepatent period.

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.

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.

Immunological detection of chemotherapeutic success in bovine fasciolosis using the specific antigen f2.

An improved hemagglutination (HA) test using the purified specific f2 antigen of Fasciola hepatica has been evaluated with regard to its potential use for the prediction of chemotherapeutic success in natural bovine infections with F. hepatica. Lactating cows (n = 16) from a herd naturally infected with F. hepatica were successively treated with nitroxynil (Dovenix, Specia) and with oxyclozanide (Zanil, ICI) 1 month later. Their f2-specific antibodies were significantly lower than those of a non-treated control group (n = 15) from the second month after the first treatment, and continued to decline thereafter to negative values 5-6 months post-treatment. In a second experiment, culled and fattened cows (n = 32) of unknown fasciolosis history were treated with closantel (Janssen Pharmaceutica). Three months after treatment, f2-specific antibodies of the serologically positive animals (n = 24) were reduced nine-fold. In contrast, in the control group (n = 28), the titers of f2-specific antibodies of the serologically positive animals (n = 21) were not modified significantly. The results show that the f2-HA test is useful for the prediction of chemotherapeutic success in bovine fascioliasis.

Absorption of colostral IgG1 by the newborn lamb: influence of the length of gestation, birthweight and thyroid function.

The relationships between the length of gestation, the birthweight, thyroid and adrenocortical function, and the absorption of colostral IgG1 were studied in 18 Romanov lambs born spontaneously between 135 and 145 days of gestation. All the lambs were bottle-fed in accurately standardised conditions with a bovine colostrum pool. Negative relationships were observed between the maximum plasma IgG1 levels and the length of gestation, the birthweight, or the plasma thyreostimulin levels near birth and between the times at which the maximum plasma IgG1 levels occurred and the length of gestation, or the plasma thyroxine levels near birth. The half-lives of bovine IgG1 in the plasma of the lambs were also related to the plasma thyroxine level at birth. These results suggest that maturity of the newborn lamb, assessed by the length of gestation and the hormonal status, could greatly influence the acquisition and maintenance of colostral immunity.

Intestinal K99+ escherichia coli adhesion and absorption of colostral IgG1 in the newborn lamb: effect of fetal infusion of thyroid hormones.

Continuous infusion of thyroid hormones during the last 12 days of gestation in ovine fetuses was associated with: a reduction of the rate of appearance of blood IgG1 after colostrum feeding in the newborn lambs; an important reduction of the number of jejunal cells filled with IgG1, 12 hours post partum, suggesting a premature closure of the intestinal permeability to IgG1; and a significant reduction of the K99+ Escherichia coli adhesion on the intestinal villi. These results could partly explain relationships observed previously between perinatal thyroid function abnormalities and the occurrence of neonatal diseases.

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.

Antigenic specificity of monoclonal antibodies to beef myoglobin determined by cross-reactivity studies against myoglobins from domestic species.

Six rat monoclonal antibodies to beef myoglobin were studied to pinpoint the antigenic determinants they recognize. Their ability to bind myoglobin from beef, sheep, goat, horse, pig and chicken was compared in a competitive ELISA using biotinylated beef myoglobin. Correlation of sequence differences with relative binding allowed us to identify critical antigenic residues recognized by these antibodies. Each domain included residues previously considered not to be directly involved in the antigenic structure of myoglobin. Moreover, a possible orientation of myoglobin when adsorbed onto plastic surfaces was defined. These antibodies should be valuable tools for analysing conformational changes of the protein occurring during chemical or physical treatments.