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#28713178   2017/07/17 Save this To Up

Glucose transporter-4 in white blood cells of young and old sled dogs: a model for human biomarker development.

The insulin responsive glucose transporter, GLUT4 is found predominantly in muscle and adipose cells. Maratou and others (2007) reported that there is GLUT4 in white blood cells (WBC) collected from human subjects in response to insulin activation. This study was designed to validate the presence of GLUT4 in white blood cells of sled dogs and furthermore to investigate whether changes in levels of the GLUT4 protein might be associated with aging. Additionally, we examined the blood insulin concentration of two populations of dogs, young and old, before and after a meal to observe their insulin response. It is documented in skeletal muscle that GLUT4 expression is increased as a result of conditioning, making sled dogs an excellent model in the circumpolar north for studying the effects of exercise, nutrition and diabetes (Felsburg 2002; Kararli 2006). Blood was withdrawn from 11 healthy sled dogs: 6 young (1-5 years) and physically fit, conditioned for racing and 5 old (7-13 years), retired from racing. The insulin response was determined using blood plasma and ELISA. The buffy coat (containing WBC) was collected with a glass pipette after centrifugation and washed and suspended in 1x phosphate buffer. GLUT4 was measured using ELISA kits (USCN Life Sciences). The results validate that GLUT4 is present in white blood cells in sled dogs. Age had no significant effect in the concentration of GLUT4 between the populations of old and young dogs. A significant difference in insulin levels pre and post meal in young (0.13 ± 0.03 ng/mL (pre), 0.22 ± 0.04 ng/mL (post), p < 0.05) and old (0.13 ± 0.02 ng/mL (pre), 0.22 ± 0.03 ng/mL (post), p < 0.05) dogs was observed, displaying the typical postprandial insulin spike. No significant difference was found in insulin concentration comparing old versus young dogs. Our data shows that white blood cells in young (40.4 ± 2.4 ng/mL) and old (35.3 ± 8.8 ng/mL) sled dogs have quantifiable but non-significant different GLUT4 levels (p > 0.05). Detecting GLUT4 via an ELISA in white blood cells, opens up minimally invasive avenues for studying the underlying molecular mechanisms associated with insulin resistance in more complex, dynamic and physiological systems. This project was the first step in developing a protocol for this simple, technique with a potential clinical application for diagnosing insulin resistance.

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#24408313   2014/02/03 Save this To Up

Prevalence of paratuberculosis in cattle and control measures within the herd influence the performance of ELISA tests.

Commercial ELISA kits are widely used in the diagnosis of paratuberculosis of dairy cattle. It is critically important to understand the influences on these test results and their relation to faecal culture (FC) results in order to interpret the findings and to make decisions concerning serial testing and control measures. A total of 1021 cattle (423 FC positive, 598 FC negative) from 14 Mycobacterium avium subspecies paratuberculosis (MAP) positive herds were tested with four ELISA systems and FC simultaneously to calculate the kappa coefficients for the agreement of the different ELISA systems as well as find influencing factors. For the agreement of FC and ELISA, the kappa coefficients were low and ranged from 0.19 to 0.24, whereas, results of the different ELISA were consistently high (0.74-0.90). Agreement with FC was enhanced with the duration of control (P≤0.001) and the lactation number (P≤0.01), and reduced with within-herd prevalence (P≤0.001). There were substantial differences in the detection rate of low (15-24 per cent) and high (85-100 per cent) MAP shedders. In conclusion, the factors shown to influence test sensitivity, should be taken into account for validation and interpretation of ELISA tests. The benefit of serial ELISA testing is low.

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#22027187   2012/01/16 Save this To Up

Transfer of tumour necrosis factor-α via colostrum to foals.

This study aimed to determine whether TNF-α is transferred to equine neonates via colostrum and the relationship between TNF-α and IgG concentrations in the equine neonate. Colostrum, presuckle and postsuckle foal serum samples were collected from healthy mares and their foals. Equine TNF-α ELISA and IgG SRID kits were used to determine the concentrations of TNF-α and IgG, respectively. Statistical analysis was performed using the Spearman rank correlation. TNF-α concentrations in all presuckle foal serum were below the limit of detection in 15/16 foals and increased in postsuckle foal serum to a mean concentration of 7.7 x 10(4) pg/ml. TNF-α concentrations in postsuckle foal serum and colostrum showed significant correlation (rho=0.668; P=0.005). However, TNF-α and IgG concentrations in colostrum or postsuckle foal serum did not correlate (rho<-0.016; P>0.05). Ratios of TNF-α/IgG in colostrum or postsuckle foal serum showed significant correlation (rho=0.750; P=0.0008). These results indicate that TNF-α is transferred to the foal via colostrum absorption and may play a role in early immunity.

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#21257399   2011/01/24 Save this To Up

Technical and financial evaluation of assays for progesterone in canine practice in the UK.

The concentration of progesterone was measured in 60 plasma samples from bitches at various stages of the oestrous cycle, using commercially available quantitative and semi-quantitative ELISA test kits, as well as by two commercial laboratories undertaking radioimmunoassay (RIA). The RIA, which was assumed to be the 'gold standard' in terms of reliability and accuracy, was the most expensive method when analysing more than one sample per week, and had the longest delay in obtaining results, but had minimal requirements for practice staff time. When compared with the RIA, the quantitative ELISA had a strong positive correlation (r=0.97, P<0.05) and a sensitivity and specificity of 70.6 per cent and 100.0 per cent, respectively, and positive and negative predictive values of 100.0 per cent and 71.0 per cent, respectively, with an overall accuracy of 90.0 per cent. This method was the least expensive when analysing five or more samples per week, but had longer turnaround times than that of the semi-quantitative ELISA and required more staff time. When compared with the RIA, the semi-quantitative ELISA had a sensitivity and specificity of 100.0 per cent and 95.5 per cent, respectively, and positive and negative predictive values of 73.9 per cent and 77.8 per cent, respectively, with an overall accuracy of 89.2 per cent. This method was more expensive than the quantitative ELISA when analysing five or more samples per week, but had the shortest turnaround time and low requirements in terms of staff time.

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#18424848   2008/04/21 Save this To Up

Use of cardiac troponin kits for the qualitative determination of myocardial cell damage due to traumatic reticuloperitonitis in cattle.

This study was designed to investigate whether kits to measure circulating cardiac troponin-I (cTn-I) and cardiac troponin-T (cTn-T) can be used to determine myocardial cell damage in cattle with traumatic reticuloperitonitis (trp). Twenty cattle with trp were compared with 10 clinically healthy cattle. cTn-I and cTn-T were determined qualitatively and cTn-I was determined quantitatively; biochemical analyses were also performed on both groups. The mean serum concentrations of total protein, globulin, glucose and calcium, and the mean activities of creatine kinase mb, aspartate aminotransferase, lactate dehydrogenase and gamma-glutamyl transferase were higher in the cattle with trp than in the control group. The cTn-I and cTn-T kits both gave positive results in three of the cattle with trp and the quantitative measurement of cTn-I was positive in 11 of the trp cases. Both tests were negative in the healthy cattle.

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#16006640   2005/07/11 Save this To Up

Comparison of two commercial ELISAs for the serological diagnosis of salmonellosis in pigs.

Serum samples from 361 pigs (194 fattening pigs and 167 sows) were examined by means of two commercial ELISAs (Svanovir; Svanova Biotech and Salmotype; Labor Diagnostik) used for the serological diagnosis of salmonellosis in pigs; 211 of the samples came from farms of known bacteriological status and the other 150 were collected randomly from 60 farms of unknown status. The ELISAs were done according to the manufacturers' directions and the samples were categorised accordingly. The results were compared by using a linear regression analysis and by the calculation of Kappa values. To try to improve the agreement between the tests, the raw optical densities (ODS) were transformed to sample/positive (S/P) ratios by using the positive control as a reference, and cut-off values for these S/P ratios were calculated by means of a receiver operating characteristic (ROC) analysis. All but two of the known infected farms were recognised as such by both tests. However, the correlation of the raw ODS for individual pigs was poor (r=0.546) and had a Kappa value for the results categorised according to the manufacturers' recommendations of 0.191. On some farms the correlation was high (r=0.97) but on others it was low (r=0.05) with no apparent reason for the difference. The S/P ratios did not improve the agreement (Kappa=0.25).

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#14653341   2003/12/04 Save this To Up

Specificity of three ELISA-gE kits for screening pig meat for antibodies to Aujeszky's disease.

Muscle samples (20 g) from 2025 pig carcases from Aujeszky's disease-free holdings were collected at the slaughterhouse. The samples were frozen and thawed to obtain meat juice, which was then analysed by three ELISA-gE test kits in parallel, to assess their specificity. After two cycles of freezing and thawing, 2.2 per cent of the samples were dry. Three times more of the samples from the sow carcases than from the finisher carcases yielded insufficient juice (< 220 microl). To validate the results of the specificity study, the sensitivity of the test kits was evaluated on 45 samples from gE-seropositive sows. On the basis of the results from 1879 samples, the specificity of the ELISA-gE kits was between 0.995 and 1.000, depending on the classification of the doubtful results. In the case of a positive or doubtful result, it proved useful to repeat the test on the same sample, in order to limit the number of false positive results.

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#12553581   2003/01/29 Save this To Up

Diagnosis of canine parvovirus by rapid immunomigration on a membrane.

Rapid immunomigration on a membrane was applied to the diagnosis of canine parvovirus (CPV) in 128 samples of faeces containing four strains of parvovirus (two CPV-2a strains, including one vaccine strain, and two CPV-2b strains). The results were compared with the results of haemagglutination and ELISA sandwich techniques. The new test was quick and easy to use, and made it possible to identify both the CPV-2a and CPV-2b strains. Its detection thresholds per gram of faeces corresponded to specific haemagglutination titres of between 320 and 640 and a virus titre of between 10(4) and 10(5) CCID50 (dose required to infect 50 per cent of cell cultures).

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#11968049   2002/05/23 Save this To Up

Use of the EIE-recombinant-Chagas-biomanguinhos kit to monitor cure of human Chagas' disease.

We used the EIE-Recombinant-Chagas-Biomanguinhos kit (EIE-Rec kit) developed by the Oswaldo Cruz Foundation, Brazil, to monitor cure of chagasic patients who were treated during the acute phase of T. cruzi infection. Treated patients were previously studied by parasitological and serological tests and classified as cured patients (CP) (n = 10), dissociated patients (DP) (n = 6), and noncured patients (NCP) (n = 6). When sera of these patients were assayed by EIE-Rec kit all sera from NCP and all sera from CP showed positive and negative reactions, respectively. These results were in full agreement with those obtained previously by the classical tests. Two DP showed a positive reaction; the remaining four displayed a negative reaction, similar to that observed in sera from nonchagasic (NCh) individuals, and could therefore be considered CP. Our results suggest that the EIE-Rec kit could be used to monitor the efficacy of Chagas' disease treatment.

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#9881440   1999/03/16 Save this To Up

Evaluation of three tests for the detection of rabbit haemorrhagic disease virus in wild rabbits.

Two double antibody sandwich ELISA kits and the immunoblot were evaluated for the detection of rabbit haemorrhagic disease (RHD) virus in wild rabbits. Either liver alone or liver and spleen separately or a pool of liver and spleen tissues from 106 wild rabbits were tested in all three tests. They produced very similar results, except that ELISA kit A gave three doubtful results which were confirmed as negative by retesting in the same test and by the immunoblot and ELISA kit B. Both ELISA kits can be used for the rapid detection of acute RHD, and ELISA kit A can detect both acute and chronic RHD virus infection. The immunoblot is useful as a confirmatory test when ELISA-positive results do not correlate with gross and/or histopathological findings.

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