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A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50–60°C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56°C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6·7 deg C (D60°C=24·6 min) and 6·8 (D60°C=23·0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0·994) with those obtained by the colorimetric assay. A similar high correlation (r=0·998) was obtained when industrially thermized milks (62–67°C for 20–90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by addingp-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

The 20S proteasome is a large complex (700 kDa) 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 9 ng/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 (331 μg/g wet tissue) whereas the lowest values were found for M. Tensor Fascia Latae (213 μg/g wet tissue), a fast twitch white muscle, M. Supraspinatus (209 μg/g wet tissue), a slow twitch red muscle and M. Pectoralis profondus (203 μg/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.

International audience; Emission and excitation spectra of intrinsic fluorophores present. in milk were used to evaluate changes in milk following thermal treatments in the 57-72 degreesC temperature range from 0.5 min up to 30 min. Alternatively, the concentrations of native alkaline phosphatase, lactoferrin, immunoglobulin G, bovine serum albumin, beta-lactoglobulin, and alpha-lactalbumin were determined in the same samples by enzymatic and immunochemical techniques. As principal component analysis applied to the normalized fluorescence spectra successfully discriminated different milk samples according to the temperature and time of thermal treatment, principal component regression was applied to predict the amounts of the native proteins investigated using fluorescence data. The results showed strong correlations between measured and predicted data for alkaline phosphatase and beta-lactoglobulin. This study has demonstrated that front-face fluorescence spectroscopy has a promising potential to become a rapid and nondestructive analytical technique for the evaluation of physicochemical changes in milk induced by low thermal treatment.

Colostrum and milk samples from 20 goats were analysed for concentrations of immunoglobulin G (IgG), β-lactoglobulin (β-lg), α-lactalbumin (α-la) and serum albumin (CSA) throughout the first 14 milkings post partum (7 d of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means±SD) at first milking were IgG 47·9±25·5, β-lg 30·7±10·4, α-la 2·77±0·82 and CSA 2·97±2·46 mg/ml. Large variations were recorded for IgG concentrations (19·9–94·5 mg/ml) and β-lg (9·3–49·8 mg/ml). Concentrations of IgG, β-lg and CSA dropped abruptly in the subsequent milkings and α-la concentration decreased slowly. Mean IgG concentration was

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.

A new variant of bovine β-casein was isolated from individual milk and characterized. It is proposed for nomenclature as β-CN H. The variant H differed from the variant A 2 by the substitu- tion of the residue methionine at position 93 by a leucine residue, by the substitution of the residue glutamine at position 72 by a glutamic acid residue and by another equivalent (Gln→ Glu) substitu- tion within the sequence 114-169. The leucine residue at position 93 was identified in the plasmin-in- duced peptide (f49-99) by electrospray ionization tandem mass spectrometry (ESI-MS 2 ), and confirmed by the amino acid composition of this peptide C-terminus. This mutation would corre- spond to the substitution of the codon ATG by CTG, originally reported in cDNA by Jimenez- Flores et al. (11). The molecular mass of β-casein H was measured as 23 969.1 ± 2.4 g.mol -1 .

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.

Twenty-one murine monoclonal antibodies (MAbs) were raised against bovine g -casein ( g -CN). They were discriminated by: (1) their specificity in ACP-ELISA towards g -CN fragments and g -CN from buffalo, ewe, goat, human and mare; and (2) their reactivity in BIAcore assays with bovine g -CN. The antigen sequences were compared to delineate epitopes. The MAbs recognized 14 distinct epitopes clustered in six discrete determinants (4–8, 14–24, 33–48, 49–91, 178–183, 184–209). Within the determinants, all clustered epitopes except one were found to be overlapping by BIAcore additivity assay. Epitopes were delineated at position 4–8 and 178–183. One-quarter of the residues at both termini shared equally ~ 72% of the epitopes. No MAb was bovine-specific. A single MAb discriminated bovine whole casein from that of ewe and goat. Epitopes were included in each of the plasmin-released proteose peptone and n -CN peptides, and the panel of MAbs may thus provide probes suitable for their detection.

International audience; Four diets (SMP, ML, DC and DCLC) containing 24, 18, 53 and 19% crude protein on a dry matter basis, respectively, were given to ileo-cæcal cannulated preruminant calves. In the SMP diet, protein was provided by skim milk powder. The ML diet was a mixture of raw whole milk and lactose. The DC and DCLC diets were first milking colostra diluted with water (DC) or water, lactose and UHT cream (DCLC). The apparent ileal digestibility of N was low with the DC and DCLC diets (0.71 and 0.70 vs. 0.92 with the SMP and ML). Comparisons of AA profiles suggested that could be due to: (1) an increased loss of endogenous protein ($+$ 480 and $+$ 200% for DC and DCLC, respectively), (2) an incomplete digestion of immunoglobulins G (IgG) which constituted 49 and 42% of digesta protein for DC and DCLC. The proportion of immunoreactive IgG found by radial immunodiffusion was similar for DC digesta (47% ) but lower for DCLC (6% ). Indeed, immunoblotting of non-reduced protein showed intact and/or almost intact IgG were more abundant for DC than DCLC. However, in reduced samples, intact and/or slightly degraded $\gamma$ heavy chains were abundant for both. In contrast, only traces of IgG were detected in SMP and ML digesta.; Digestion du colostrum chez le veau préruminant : digestibilité et origine des fractions indigérées à la fin de l'iléon. Quatre aliments (SMP, ML, DC et DCLC), contenant respectivement 24, 18, 53 et 19 % de matières azotées par rapport à la matière sèche, ont été distribués à des veaux préruminants munis d'une canule ré-entrante iléo-cæcale. Dans l'aliment SMP, les protéines étaient apportées par de la poudre de lait écrémé. L'aliment ML était du lait frais additionné de lactose. Les aliments DC et DCLC étaient du colostrum de première traite additionné d'eau (DC) ou d'eau, de lactose et de crème UHT (DCLC). La digestibilité apparente de l'azote a été peu élevée avec les aliments DC et DCLC (0,71 et 0,70 vs. 0,92 avec SMP et ML). Les comparaisons de profils d'acides aminés indiquent que cela pourrait être dû à : (1) une augmentation des pertes endogènes ($+$ 480 et $+$ 200 % avec les aliments DC et DCLC), (2) une digestion incomplète des immunoglobulines G (IgG) qui constituaient 49 et 42 % des protéines des digesta DC et DCLC. L'immunodiffusion radiale et les immunoempreintes confirment l'abondance des IgG dans les digesta DC et DCLC, sous des formes toutefois plus dégradées pour DCLC. En revanche, la concentration d'IgG était très faible dans les digesta SMP et ML.

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.

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.

y Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60°C) in an aqueous solution and after a treatment under a restricted water environment (50°C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non-covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β-LG. confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein.

Colostrum and milk samples from 60 Holstein–Friesian cows were analysed for concentrations and yields of immunoglobulin G (IgG), β-lactoglobulin (β-lg), α-lactalbumin (α-la) and serum albumin (BSA) throughout the first 16 milkings post partum (8 d of lactation) using a single radial immunodiffusion assay. Concentrations (mg/ml, means±SD) at first milking were IgG 59·8±28·5, β-lg 14·3±4·6, α-la 2·04±0·6, BSA 1·21±0·44. Large variations were recorded for IgG concentrations (15·3–176·2 mg/ml) and yields (0·2–925 g). Cows in their first lactation produced significantly lower concentrations and yields of colostral IgG than cows in later lactations. A colostral yield of IgG below the 100 g required to prevent calf hypo-γ-globulinaemia was found in 18·3% of the cows. The concentrations of IgG, β-lg and BSA dropped abruptly in subsequent milkings and α-la concentration decreased slowly. The mean IgG concentration was

Chez 50 vaches Holstein (15 primipares), nous avons etudie l'effet de la traite temporaire une fois par jour, a partir du velage, sur la production laitiere et l'etat nutritionnel. Les vaches primipares ont ete reparties en deux lots: un lot temoin (Pc) trait en permanence 2 fois/j et un lot experimental (P3) trait une fois/j pendant les trois premieres semaines de lactation puis deux fois par jour. Les vaches multipares ont ete reparties en trois lots: un lot temoin (Mc) trait en permanence deux fois par jour ; un lot experimental trait une fois par jour pendant les trois premieres semaines de lactation puis deux fois par jour (lot M3) ; un autre lot experimental trait une fois par jour pendant les six premieres semaines de lactation puis deux fois par jour (lot M6). L'essai s'est termine a la fin de la periode hivernale. La traite une fois par jour n'a pas semble provoquer d'inconfort chez les vaches. Elle a diminue la production laitiere de 2,7 kg.j -1 en 1 re semaine de lactation, de 8,4 kg.j -1 en 3 e semaine, et de 14,5 kg.j -1 en 6 e semaine. Son effet n'a pas ete different entre les vaches primipares et multipares (p > 10 %). Des la premiere semaine de traite deux fois.j -1 , la production laitiere des vaches experimentales a augmente de 6,8 kg.j -1 (difference non significative entre les lots P3, M3 et M6). A partir de la semaine 7 la production laitiere des lots P3 et M3 a ete, en moyenne, plus faible que celle des lots Pc et Mc de 2,4 kg.j -1 en moyenne, et celle du lot M6 a ete plus faible que celle du lot Mc de 5,4 kg.j -1 en moyenne. Le taux proteique du lait des vaches traites une fois par jour a ete plus eleve que celui des vaches temoins, de 2,4 g.kg -1 pendant les periodes experimentale et post-experimentale. Le nombre de cellules somatiques dans le lait n'a pas ete significativement different entre les lots. Les vaches traites une fois.j -1 ont perdu moins d'etat corporel et moins de poids vif en debut de lactation que les vaches temoins, et elles ont presente un bilan energetique moins negatif ou plus positif (difference significative pour le lot M6). Le profil sanguin (teneurs en glucose, acides gras libres, 3-hydroxybutyrate) a atteste un meilleur equilibre energetique des vaches traites une fois par jour en debut de lactation. Cet essai suggere que les vaches fortes productrices supportent sans inconvenient apparent d'etre traites une fois par jour pendant le debut de la lactation et qu'il pourrait etre envisageable d'alimenter ces animaux avec des regimes plus pauvres en concentres que maintenant, sans accroissement des risques sanitaires.

Acid aspartyl proteinase of Penicillium roqueforti was purified from culture filtrates using FPLC ion-exchange chromatography on Mono Q and size exclusion chromatography on Superdex 75. The purity obtained allowed us to produce, using rabbits, a specific antiserum which was then used to develop a sandwich ELISA. The test was sensitive (detection limit, 0·25 ng/ml), reproducible (CV, 3·1–6·9%) and closely correlated with enzyme activity measurement (r=0·995, P

The involvement of plasma leptin in the adaptation of dromedary camels to harsh conditions such as food or water shortages was studied through 2 experiments. In experiment 1, fourteen female camels were either fed at 68% of maintenance energy requirements (MER) during 112 d ( n = 4) or overfed at 134% of MER during the first 56 d and then underfed at 17% of MER the next 56 d (OV-UN, n = 5), or underfed and then overfed for the same durations and energy intake levels (UN-OV, n = 5). Weekly plasma samples showed that leptin, glucose and non-esterified fatty acid (NEFA) concentrations were significantly modulated by energy intake level. NEFA increased sharply but transiently in underfed camels of the UN-OV or OV-UN groups, whereas glucose and leptin concentrations decreased with underfeeding and increased with overfeeding with more significant effects in camels that were previously overfed or underfed, respectively. In experiment 2 twelve female camels were either normally watered ( n = 6) or dehydrated ( n = 6) during 23 d and then rehydrated during 4 d. Dehydration specifically increased blood hematocrit, plasma NEFA and glucose whereas leptin decreased slightly. For both experiments, leptinemia was positively related to hump adipocyte volume. Taken together these results provide new data for a better understanding of lipid and energy metabolism in camels.

Solutions of bovine myoglobin in 0.1 M‐phosphate buffer, pH 6.0, were heated over the temperature range 55–85°C for 60 min. Their immunological reactivity was tested after 0.2 μm filtration using polyclonal antibodies in the single radial immunodiffusion assay (SRID) and six monoclonal antibodies (MAbs) in a competitive and a sandwich ELISA. Reproducible results were obtained in the SRID assay (coefficient of variation (CV) = 4.0%), with a mid‐point denaturation of 79.0 ± 0.4°C. Similar results were obtained with two MAbs (mid‐point denaturation at 79.4 ± 0.5°C and 78.3 ± 0.8°C for competitive and sandwich ELISAs respectively), but the CV% values were higher (8 and 13% respectively). With the remaining four MAbs, quantification was unreliable as immunoreactivity increased during heating with large inter‐assay variations. A careful control of MAb immunoreactivity in situations covering all possible denaturation states of the test protein is an absolute prerequisite to their use in ELISAs for the immunoquan...

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.

The efficiency of colostral protein digestion was studied in nine newborn lambs fed one meal of bovine colostrum 3 h after birth. The results were compared with those obtained in two unfed lambs and four lambs fed bovine milk. The protein and peptide composition [immunoglobulins G1 and (IgG1), beta-lactoglobulin, alpha-lactalbumin, caseins and peptides resulting from casein hydrolysis] of digesta, gastrointestinal tissues, blood and urine were determined in samples taken 0.75 or 4 h after feeding. The amounts of ingested proteins in lambs fed colostrum were much higher than in those fed the milk diet, and their abomasal emptying was faster. alpha-Lactalbumin was highly degraded by abomasal and intestinal proteases, whereas beta-lactoglobulin and in particular the immunoglobulins were less sensitive. The gastric emptying of caseins was delayed in and the kinetics of appearance of peptides originating from casein hydrolysis was comparable to that observed in lambs fed milk and in 1-mo-old preruminant calves. Thirty-five percent of dietary amino acids ingested as colostrum were available within 4 h for amino acid metabolism; this percentage was 54% in the milk-fed lambs. In the lambs fed colostrum, these amino acids were provided by beta-lactoglobulin, casein and IgG1 (0.52, 0.43 and 0.30 g/kg body wt, respectively), whereas in milk-fed animals casein and beta-lactoglobulin were the most important sources of these amino acids (0.40 and 0.20 g/kg, respectively).

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 cowa ( 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 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.

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.

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.

Colostrum and milk samples from twelve Tunisian camels were analysed for concentration of immunoglobulin G (IgG), α-lactalbumin (α-la), serum albumin (CSA) and lactoferrin throughout the first 14 milkings post partum (7 days of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means±SD) at first milking were IgG, 100·7±60·4; α-la, 2·2±0·7; CSA, 8·5±3·6 and lactoferrin, 1·2±0·3. Large variations were recorded for IgG and CSA concentrations (11·8–211·1 mg/ml and 2·9–13·8 mg/ml respectively) Concentrations of IgG and CSA dropped abruptly in the subsequent milkings while α-la concentration increased until milking 5 and then decreased slowly. Lactoferrin dropped only from milking 7. Mean IgG concentrations were 3·6 and 2·5 mg/ml at milking 9 and 13 respectively. However, IgG concentration did not differ significantly, at the 1% level, from milkings 11 to 14. The contribution of CSA to the increase in whey proteins in early milks was greater than that described in the bovine and caprine species.

International audience; Gelatin is obtained from bones and hides/skin, mainly from cows and pigs using alkaline or acidic processes. The use of bovine gelatin in feed, food, and pharmaceutical products has been restricted by regulatory authorities as a consequence of the outbreak of bovine spongiform encephalopathy (BSE). On the other hand, some religions ban the porcine gelatin for human consumption. Thus, there is a need for methods able to control the species origin of gelatins. The large similarity in structure of gelatins from different origins has made unsuccessful their differentiation by physicochemical methods. Moreover, the development of immunochemical methods has been hampered by the poor immunogenicity of gelatins. We obtained high titers antibodies upon immunization of rabbits with tyrosylated bovine and porcine gelatins. Using indirect and competitive indirect ELISAs we observed large differences in titers and specificity among rabbits and during the course of immunization. Some of the antisera were not sensitive to the species origin of raw material or to the process used for gelatin production and could be used for gelatin quantitation in food. Other antisera detected the porcine acidic gelatins with 10- to 30-fold higher sensitivity than their bovine counterparts and could be used for the differentiation of the species origin of gelatins. Lastly, other antisera were highly sensitive to subtle changes in conformation of gelatins obtained by alkaline or acidic processes such as a 1,000-fold higher reactivity of bovine acid hide gelatin compared to that of its limed counterpart or a 30,000-fold higher reactivity of porcine acid bone compared to that of its limed counterpart; such antisera could be used to monitor the process induced structural changes of collagen during its transformation to gelatin.

International audience; 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 (1) 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.

The major whey proteins IgG, serum albumin and alpha-lactalbumin were purified from camel milk using gel permeation and ion-exchange chromatography. Specific antisera against each of them were raised and used to quantify their heat denaturation in early or mature milk over a range of 60-90 degrees C for 10-60 min using the single radial immunodiffusion technique. The heat denaturation midpoints for the mature milk heated 30 min were 67(.)2, 73(.)0 and 77(.)5 degrees C for IgG, albumin and alpha-lactalbumin respectively. The early milk was more sensitive to heat treatment with coagulation at low temperature and heat denaturation midpoints of 64(.)8, 71(.)6 and 72(.)6 degrees C respectively. This difference was related to the high IgG content of the early milk (12(.)6 mg/ml v. 0(.)5 mg/ml for the mature milk) and stresses the importance of monitoring the IgG level of milk to assess the absence of colostrum.

Proteolysis of casein is the principal cause of textural changes and flavour development in ripened cheese (Fox & McSweeney, 1996). Caseins are degraded into small peptides and free amino acids during a complex process, described by Grappin et al. (1985) as a two-step scheme. Caseins are initially broken down into large, well characterised fragments. This initial step, called primary proteolysis, is catalysed principally by the residual coagulant (chymosin and pepsin), and to a variable extent by endogenous milk proteases, such as plasmin, cathepsin D, and possibly somatic cell proteinases. Enzymes originating from either rennet or milk are active in most ripened cheese varieties. However, their relative contributions vary substantially depending on manufacturing practices. For instance, in Swiss-type cheeses, cooking the curd extensively inactivates the coagulant, and simultaneously enhances plasmin activity, which therefore becomes predominant (Ollikainen & Kivela, 1989).

Heparin is a potent anticoagulant polysaccharide purified for decades from ruminants or porcine tissues. However, with the emergence of bovine spongiform encephalopathy (BSE), the source of pharmaceutical heparin is currently restricted to porcine intestinal mucosa. A major species-specific contaminant, called Ag1, has recently been identified in bovine crude heparin [Rivera et al., J. Pharm. Biomed. Anal., submitted] and used to develop an enzyme-linked immunosorbent assay (ELISA) for the species origin control of crude heparins [Levieux et al., J. Immunoassay, submitted]. In this report, we describe the different investigations, which were carried out to identify Ag1. This antigen was first localised by immunohistological studies essentially in the connective tissue of the bovine small intestine. After extraction from an intestinal extract by immuno-affinity chromatography, Ag1 was isolated as a single band by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ag1 was then partly sequenced and identified as an aprotinin/heparin complex. Aprotinin, also known as the bovine pancreatic trypsin inhibitor (BPTI), is present with heparin in mast cells, and is very resistant to heat, pH, chemical treatments and proteolytic digestion. The stability of Ag1 towards the different treatments performed during heparin extraction process allows this protein to remain in sufficient amounts in crude heparin and makes it an ideal target for the immunochemical control of the absence of bovine material in crude heparins.

Heparin is an effective anticoagulant drug which has been purified for decades from bovine or porcine tissues. However, with the emergence of BSE, heparin purification is today restricted to porcine intestinal mucosa. To control the origin of crude heparins, polyclonal antibodies were raised against bovine contaminants. These antibodies were used to develop one sandwich and two competitive indirect ELISAs. Optimal results were obtained with competitive indirect ELISA, using bovine crude heparin for the coating and anti-bovine crude heparin as a detector antibody. The detection limit of the assay was 1 ppm of bovine crude heparin in a porcine heparin. Taking into account the variability of the background obtained for 10 crude and 9 pure porcine heparins from known origin, the detection level was 5 ppm. Ovine and caprine crude heparins cross-reacted slightly (6–17 ppm).

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.

Species specific antisera against bovine, ovine, and porcine serum albumin were produced in order to control the absence of bovine, ovine, or caprine tissues in the porcine intestinal mucosa used for heparin production. Two immunoassays were developed. An enzyme linked immunosorbent assay (ELISA) was very sensitive down to 1 ng/mL bovine albumin or 10 ppm bovine intestinal mucosa in porcine intestinal mucosa. For routine control, a more convenient single radial immunodiffusion assay (SRID) was found suitable to detect 2 microg/mL albumin or 3 p 1000 bovine, ovine, or caprine intestinal mucosa in porcine intestinal mucosa. Conditions of extraction of albumin from intestinal mucosa were optimized and a CV % of 4.1 was obtained for its quantitation. Due to higher albumin concentrations, detection of bovine hashed gut and lung was more sensitive (1.5 and 0.9 p 1000, respectively). Using antisera raised against porcine albumin the SRID can be applied to certify the porcine origin of the intestinal mucosa used for heparin purification and to control its adequate conservation before analysis.

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.

Colostrum, a milk particulariy rich in numerous components. Is it possible to detect its unlawful addition in milk supplies? The concentration of numerous proteins, enzymes, hormones, growth factors, vitamins, minerai and trace elements decreases abruptly during the first milk- ings post-partum and then increases in late lactation and during the udder involution before calving. Therefore, colostrum cannot be precisely defined since it is a milk ail the more abnormal than it is milked closer from calving. So its unlawful addition to milk supply may only be detected by the modification of the normal composition of the' milk. From ail the components reviewed, IgG are the most documented and sensitive indicators of an abnormai milk. They can he easily quantified using a semi-automatised single radial immunodiffusion technique and threshold values characteristic of an abnormal milk have been defined taking into account the volume of the milk supply. © Inra! Elsevier, Paris. colostrum / milk quality / IgG / automatised radial immunodiffusion

Two experiments were conducted to study gastric and small intestinal digestion of soybean glycinin and [bêta]-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 [bêta]-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 [bêta]-conglycinin was evidenced at most sampling times. In experiment 2, intact basic glycinin (Mr = 21000) associated with partially digested acidic glycinin (7000 < Mr < 25000) was demonstrated in ileal digesta up to 8-10 h after the meal. [bêta]-Conglycinin immunoreactivity could not be evidenced by Western blotting in ileal digesta.

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 foror = 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.

Immunoquantification of myoglobin, L-lactic dehydrogenases (LDH) M4 and H4, albumin, transferrin and immunoglobulin G (IgG) was performed after heat treatment of crude soluble sarcoplasmic protein extract over a range of 54-70°C for 30-780 min. Heat denaturation occurred in the following order : transferrin > IgG > LDH M4 > LDH H4 = myoglobin > albumin and could be described by first order reaction kinetics. The very low coefficient of variation (2-4%) of the single radial immunodiffusion technique resulted in a very weak calculated error ( ± 0.1-0.2°C) on temperature determination. Monitoring the concentration of LDH M4 appears appropriate for determining the minimum heating achieved in long-time, low-temperature cooking and albumin or myoglobin could be monitored for meat cooked at 69°C without holding time.

Seven rat monoclonal antibodies (MAb) for bovine myoglobin were produced. Five antibodies reacted with surface-adsorbed 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.

SummaryA sandwich ELISA (enzyme-linked immunosorbent assay) of the two-site type has been successfully developed for the detection of cows' milk in goats' or ewes' milk. The assay uses two monoclonal antibodies (MAb) raised in mice against cows' β-lactoglobulin (β-lg). These MAb recognize different epitopes of the β-lg, which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. One of the MAb recognizes a species-specific epitope of the bovine β-lg and was adsorbed to a plastic microtitration plate (capture antibody). The second MAb was labelled with peroxidase and used to detect the captured cows' β-lg. Factors affecting assay performance were investigated. The optimized assay is highly specific, reproducible (intra- and inter-assay CV were 8 and 13% respectively) and sensitive: as little as 5 ng β-lg/ml or 1 part cows' milk per 100000 parts goats' or ewes' milk can be detected. The technique is robust, cheap, rapid, reliable and suitable for high sample throughput, semi-automation and screening surveys. The MAb used guarantee the high specificity of the assay and indefinite reagent supply of constant quality once approved by collaborative national or international trials.

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.

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.

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.

Antisera specific to ovine haptoglobin were obtained by the immunnization 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.

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