Annie Levieux

Immunochemical quantification of heat denaturation of camel ( Camelus dromedarius ) whey proteins

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.

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.

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.

Camel ( Camelus dromedarius ) immunoglobulin G, α-lactalbumin, serum albumin and lactoferrin in colostrum and milk during the early post partum period

Colostrum and milk samples from twelve Tunisian camels were analysed for concentration of immunoglobulin G (IgG), alpha-lactalbumin (alpha-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; alpha-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 alpha-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.

Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay

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 degrees 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 degrees 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 degrees C=24.6 min) and 6.8 (D60 degrees 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 degrees 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.

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.

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.

Characterisation of Ag1, the major species-specific contaminant of bovine crude heparin, and its identification as an aprotinin/heparin complex

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.

Immunochemical control of the species origin of intestinal mucosa used for heparin purification.

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 μg/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 (1.5and 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.

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.