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  • Author or Editor: Silvia Martinez-Subiela x
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Abstract

Objective—To evaluate 5 commercially available ELISAs for determination of leptin concentrations in serum samples from dogs.

Sample Population—Serum samples from overweight-obese and thin–ideal weight clientowned dogs.

Procedures—Serum samples with high and low leptin concentrations (n = 7 samples each) were used for validation of the assays. Intra- and interassay precision, linearity under dilution, spiking recovery, and limit of quantification were determined. In addition, leptin concentrations in thin–ideal weight (n = 8) and overweight-obese (37) dogs were quantified.

Results—Use of 2 of the 5 ELISAs (A and B) revealed reactivity with canine leptin. Intra-and interassay coefficients of variation were < 6.1% and 76%, respectively, for assay A and 14.0% and 13.7%, respectively, for assay B. In assays A and B, dilutions of canine serum pools were used to determine linear regression equations. Recoveries were 77% to 101% for assay A and 67% to 125% for assay B. Significant differences in leptin concentrations between thin–ideal weight and overweight-obese dogs were detected only when analyzed with assay A.

Conclusions and Clinical Relevance—Among 5 leptin ELISAs evaluated, a canine-specific leptin ELISA had adequate precision, linearity, and ability to discriminate between high and low leptin concentrations corresponding to overweight-obese and thin–ideal weight dogs, respectively.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To evaluate changes in stability of haptoglobin and C-reactive protein (CRP) concentrations caused by freezing of saliva and meat juice samples.

Animals—16 specific-pathogen-free pigs and 16 pigs with clinical signs of disease.

Procedures—Saliva and diaphragmatic muscle were collected immediately before and after slaughter, respectively. Haptoglobin and CRP concentrations of pooled samples were measured before storage (day 0) and after 7, 15, 30, 60, 120, 210, and 365 days of storage at −20°C and after repeated freezing-thawing cycles (up to 7 times). In a second experiment, addition of a protease-inhibitor cocktail to saliva and storage of saliva samples at −80°C for up to 30 days were assessed for effects on CRP concentrations.

Results—Haptoglobin concentrations in saliva did not change for up to 120 days in samples stored at −20°C, but longer storage times and multiple freezing-thawing cycles increased haptoglobin concentrations. Salivary CRP concentrations decreased significantly after 7 days of storage at −20°C, and addition of a protease-inhibitor cocktail did not improve CRP stability. Lower temperatures limited salivary CRP degradation. In meat juice, haptoglobin and CRP concentrations were stable at −20°C up to 210 days.

Conclusions and Clinical Relevance—Acute-phase protein measurements in saliva should be performed as soon as possible after sample collection. When this is not possible, storage temperature of −80°C is recommended. Acute-phase protein concentrations appeared to be more stable in meat juice samples than in saliva samples. Saliva and meat juice could be used as alternatives to serum for haptoglobin and CRP analysis.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To develop and evaluate an immunoassay based on time-resolved immunofluorometry (TR-IFM) for measurement of haptoglobin concentrations in samples of various body fluids of swine.

Animals—20 pigs without clinical signs of disease and seronegative for antibodies against major viruses that affect pigs and 30 pigs with clinical signs of disease.

Procedures—Haptoglobin concentrations were measured in samples of serum, saliva, and meat juice obtained from both groups of pigs to evaluate the ability of TR-IFM to differentiate between healthy and diseased pigs. Performance of TR-IFM was evaluated by means of its calibration curve and detection limit, analytic precision during routine operation, and linearity of results for serial dilutions for the 3 types of samples. In addition, performance of TR-IFM was compared with that of a commercial spectrophotometric assay.

Results—The TR-IFM assay involved only 1 step, and the results were obtained in 20 minutes, with good analytic sensitivity and reproducibility. The analytic limit of detection was 0.52 ng/mL. Intra-assay and interassay coefficients of variation ranged from 1.13% to 4.81% and 5.97% to 13.57%, respectively. The method yielded linear results for all sample types. Serum haptoglobin concentrations determined by use of TR-IFM and spectrophotometric assays were highly correlated (r = 0.96). Differences between healthy and diseased pigs with respect to median haptoglobin concentrations were significant for all types of samples.

Conclusions and Clinical Relevance—The 1-step TR-IFM assay accurately quantified haptoglobin concentrations in serum, saliva, and meat juice samples from swine and may be useful in laboratory and meat inspection settings.

Full access
in American Journal of Veterinary Research