Evaluation of a commercially available human C-reactive protein (CRP) turbidometric immunoassay for determination of canine serum CRP concentration

Background: Serum C-reactive protein (CRP) is an acute phase marker in dogs that is useful for the diagnosis and monitoring of inflammatory disease. Rapid, reliable, and automated assays are preferable for routine evaluation of canine serum CRP concentration.
Objective: The aim of this study was to evaluate whether canine serum CRP concentration could be measured reliably using an automated turbidometric immunoassay (TIA) designed for use with human serum.
Methods: A commercially available TIA for human serum CRP (Bayer, Newbury, UK) was used to measure canine serum CRP concentration. Cross-reactivity of antigen was evaluated by the Ouchterlony procedure. Intra-and interassay imprecision was investigated by multiple measurements on canine serum samples and serum pools, respectively. Assay inaccuracy was investigated by linearity under dilution and comparison of methodologies (canine CRP ELISA, Tridelta Development Ltd, Kildare, UK). Then the assay was applied to serum samples from 14 clinically healthy dogs, 11 dogs with neoplasia, 13 with infections, 8 with endocrine or metabolic diseases, and 10 with miscellaneous diseases.
Results: Cross-reactivity between canine serum CRP and the anti-human CRP antibody was found. Intra-and interassay imprecision ranged from 5.2% to 10.8% and 3.0% to 10.2%, respectively. Serum CRP concentration was measured in a linear and proportional manner. There was no significant disagreement and there was linear correlation of the results in the comparison of methodologies, except for a slight proportional discrepancy at low CRP concentrations (<10 μg/mL). Dogs with infections had a significantly higher concentration of serum CRP than did all other dogs, and dogs with neoplasia had a significantly higher concentration of serum CRP than did clinically healthy dogs.
Conclusions: Canine serum CRP concentration can be measured reliably using the commercially available TIA designed for human CRP.     

Measurement of serum interleukin-10 in the dog

The objective was to evaluate independently the reliability of a commercially available canine serum interleukin-10 (IL-10) enzyme-linked immunoassay (ELISA) and to investigate canine serum IL-10 concentrations in healthy dogs, in dogs with a naturally-occurring acute phase reaction and in dogs following surgical stimulus by assessing intra- and interassay imprecision, inaccuracy and detection limits. Median (and range) serum IL-10 concentrations (ng/L) in the various groups were as follows: healthy dogs (n=15), 18.9 (11.2-71.5); dogs with pyometra (n=9), 37.9 (12.4-201.8); dogs with angiostrongylosis (n=8), 20.29 (14.3-108.7) and values in dogs following surgical stimulus (n=15), 14.8 (10.7-65.8). The assay measured canine serum IL-10 reliably (intra- and interassay imprecision 4.9-8.3% and 9.9-10.9%, respectively; detection limit 10.7 ng/L with no significant inaccuracy). No significant increases in IL-10 were observed following surgical stimulus and no difference in IL-10 was observed between the diagnostic groups. IL-10 values showed a higher degree of variation in dogs with an inflammatory response, i.e. those with elevated serum C-reactive protein (CRP) concentrations, compared to healthy dogs. As anticipated, healthy dogs had low levels of both analytes, whereas dogs with an acute phase response had IL-10 levels with no clear relationship to CRP concentrations, with observed low IL-10 values even when there was a marked inflammatory response.     

An immunoturbidimetric assay for canine C-reactive protein

Antiserum was raised in sheep against canine C-reactive protein (CRP) and antibody, which was not specific for CRP, was removed by absorption with normal canine serum protein linked to agarose beads. The antiserum was used to develop an immunoturbidimetric assay for canine CRP on a MIRA (Roche Diagnostics) automated clinical biochemical analyser and assessed for routine analysis of CRP in canine serum samples. The assay gave standard curves with each standard having a coefficient of variance (CV) between 4.8 and 11%, interassay CVs below 11% and intra-assay CVs of less than 5%. Parallel dilution curves were obtained with purified CRP diluted in buffer and with endogenous CRP in serum diluted with buffer or with a serum with a negligible CRP content. The immunoturbidimetric assay results correlated with the results obtained using an ELISA method, r=0.88. The immunoturbidimetric assay of canine CRP proved to be suitable for the routine analysis of canine CRP.     

Use of a time-resolved immunofluorometric assay for determination of canine C-reactive protein concentrations in whole blood

OBJECTIVE: To develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for measurement of C-reactive protein (CRP) in canine whole blood. ANIMALS: 12 healthy dogs and 35 dogs with inflammatory processes. PROCEDURE: CRP was isolated from acute-phase serum by affinity chromatography and used as a standard for calibration. Analytic and functional limit of detection and intra-assay and interassay precision were calculated. Accuracy was evaluated by recovery assays and by comparison with results of a commercial ELISA. Correlation between CRP concentrations in whole blood and corresponding plasma fractions was tested by use of TR-IFMA. Stability of blood samples at 4 degrees C was assessed during a 1-month period, and effects of anticoagulants were evaluated. Measurements of CRP in blood samples from 12 healthy dogs were compared with those of 35 dogs with inflammatory diseases. RESULTS: Analytic and functional limits of detection were 0.53 and 3.26 microg/mL, respectively. Intra-assay and interassay coefficients of variation varied between 2.1% to 8.9% and 8.0% to 12.3%, respectively. Mean recoveries of added CRP were 104% and 114%. Measurements of CRP by use of TR-IFMA and ELISA were highly correlated (R2 = 0.97). Measurements of CRP in whole blood and in corresponding plasma fractions by use of TR-IFMA were also highly correlated (R2 = 0.97). Neither storage nor use of anticoagulants disturbed measurement of CRP concentrations in whole blood. Concentrations of CRP in whole blood of dogs with inflammation were significantly higher than in healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Determination of CRP concentrations in whole blood may provide a diagnostic test for inflammation in dogs.     

Serum C-reactive protein concentrations in healthy Miniature Schnauzer dogs

Background: C‐reactive protein (CRP) is a sensitive marker for inflammation in people and dogs. In people, an association between CRP concentration and atherosclerosis has been reported. Atherosclerosis is rare in dogs, but the Miniature Schnauzer breed may be at increased risk for developing this vascular disease. It is not known if CRP concentrations in Miniature Schnauzer dogs differ from those in other dog breeds. Objectives: Our objectives were to validate an automated human CRP assay for measuring CRP in dogs and compare CRP concentrations in healthy Miniature Schnauzer dogs with those in non‐Miniature Schnauzer breeds. Methods: Sera from 37 non‐Miniature Schnauzer dogs with inflammatory disease were pooled and used to validate a human CRP immunoturbidimetric assay for measuring canine CRP. Blood was collected from 20 healthy Miniature Schnauzer dogs and 41 healthy dogs of other breeds. Median serum CRP concentration of healthy Miniature Schnauzer dogs was compared with that of healthy non‐Miniature Schnauzer dogs. Results: The human CRP assay measured CRP reliably with linearity between 0 and 20 mg/L. CRP concentration for healthy Miniature Schnauzer dogs (median 4.0 mg/L, minimum–maximum 0–18.2 mg/L) was significantly higher than for the healthy non‐Miniature Schnauzer dogs (median 0.1 mg/L, minimum–maximum 0–10.7 mg/L); 17 of the 20 Miniature Schnauzer dogs had values that overlapped with those of the non‐Miniature Schnauzer dogs. Conclusions: Median CRP concentration of Miniature Schnauzer dogs was slightly higher than that of other breeds of dogs. A relationship between higher CRP concentration in Miniature Schnauzer dogs and idiopathic hyperlipidemia, pancreatitis, and possible increased risk for atherosclerosis remains to be determined.     

Validation of a species-optimized enzyme-linked immunosorbent assay for determination of serum concentrations of insulin in dogs

Background: Measurement of canine serum insulin has relied on methods developed to measure human insulin. A species‐optimized test for measurement of serum insulin in dogs is now commercially available. Objective: The purpose of this study was to validate the canine ELISA for determination of serum insulin concentration in dogs. Methods: Precision was determined by evaluating intra‐ and interassay coefficient of variation (CV), and accuracy was determined by dilution and spike recovery studies. A method comparison study with samples from 34 clinically healthy dogs and 73 dogs examined for various illnesses and disorders (“patients”) was performed using the canine ELISA and an ELISA for human insulin. Biologic relevance of the canine assay was evaluated by measuring insulin in samples collected from 8 healthy dogs after administration of glucagon. A stability study was preformed with 6 samples stored at 20°C, 4–8°C, and ?20°C. Results: For the canine ELISA, intra‐ and interassay CVs were 4.3–7.8% and 4.4–7.7%, respectively. Mean recovery after dilution was 99% and recovery after spiking with porcine insulin was 116%. The canine and human ELISAs correlated well (r²=.94 for healthy dogs, r²=.88 for patient samples). After glucagon injection serum insulin concentrations increased significantly in 8 dogs. Insulin was stable for 30 days in 6 serum samples stored at ?20°C and in most samples for 8 days at 4–8°C. Insulin was stable for <3 days at room temperature (20°C). Conclusions: The new canine serum insulin ELISA had good precision and accuracy and correlated well with the previously used assay.     

Validation of two ELISA assays for total ghrelin measurement in dogs

The aim of this study was to validate two commercially available ELISA assays for total ghrelin measurement in dogs: one canine-specific and one originally designed for measuring human ghrelin. The two assays showed intra-assay coefficient of variations (CVs) lower than 10%, while the inter-assay CVs exceeded the 15% limit. Sample dilutions resulted in linear regression equations with correlation coefficients close to 1. In order to compare methods and verify ability of the ghrelin assays to differentiate between low and high levels, ghrelin concentrations were measured in plasma samples obtained before and at different times after glucose administration in five Beagle dogs. A statistically significant changes in ghrelin after glucose administration was recorded only with assay B. In conclusion, the human ELISA validated in this study showed a good intra-assay precision, accuracy, and when applied to the glucose injection study, was better in distinguishing high and low canine ghrelin levels than the canine ELISA assay.     

Validation of a chemiluminescent enzyme immunometric assay for plasma adrenocorticotropic hormone in the dog

Background: The concentration of canine adrenocorticotropic hormone (ACTH) is usually determined by radioimmunoassay. However, chemiluminescent assay techniques have many advantages for clinical endocrine testing. Objectives: The objectives of this study were to validate a commercially available chemiluminescent assay for determination of canine ACTH concentration and to determine whether protease inhibitors are appropriate for use in the chemiluminescent assay system. Methods: Biological specificity was evaluated by treatment of 3 dogs with ovine corticotropin‐releasing hormone (CRH) followed by serial measurements of ACTH and by comparison with a previously validated immunoradiometric assay. All samples were collected both in the presence and absence of aprotinin, a protease inhibitor. The assay was further evaluated by measurement of intra‐assay precision, interassay precision, and recovery after dilution. Results: Baseline ACTH concentrations ranged from 5.6 to 15.3 pg/mL, and maximum ACTH concentrations of 158 to 1240 pg/mL were observed 30–60 minutes after CRH administration. Plasma samples collected with aprotinin had significantly lower ACTH concentrations than did samples collected without aprotinin. The intra‐assay coefficients of variance (CVs) ranged from 4.1 to 8.2%, and interassay CVs ranged from 4.6 to 14.8%. Recovery after dilution with canine plasma ranged from 93.4 to 103.0% of predicted concentration; however, inadequate recovery was observed with other diluents. There was a high correlation with the immunoradiometric assay (r= .925) but a significant negative bias (‐32.9, 95% confidence interval ?50.8 to ?14.9). Conclusions: This chemiluminescent assay is a valid technique for measurement of ACTH in canine plasma. ACTH concentration measured by chemiluminescence is lower than that measured by immunoradiometry. Aprotinin decreases the measured concentration of ACTH, and this effect should be taken into account when interpreting results. Diluents supplied with the kit should not be used for dilution of canine samples.     

C-reactive protein, tumor necrosis factor α, and interleukin-6 in dogs with pyometra and SIRS

Objective: To determine the frequency of the systemic inflammatory response syndrome (SIRS) in canine pyometra and to evaluate the relationship between C‐reactive protein (CRP), tumor necrosis factor α (TNFα), interleukin‐6 (IL‐6), and SIRS. Design: Prospective clinical study. Setting: Veterinary teaching hospital. Animals: Fifty‐three clinical cases of canine pyometra and 19 healthy control bitches. Interventions: Upon admission to the veterinary hospital, history and physical examination findings, including previously defined clinical SIRS parameters, were documented. Blood samples were obtained for hematology and biochemical tests and for CRP, TNFα, and IL‐6 analysis. The diagnosis of pyometra was confirmed by histopathology of the uterus after ovariohysterectomy. After surgery, clinical SIRS parameters, length of hospitalization, and mortality were recorded. Measurements and main results: Pyometra dogs were grouped as SIRS positive (30/53; 57%) or SIRS negative (23/53; 43%). Logistic regression showed that CRP was the only parameter that significantly related to SIRS apart from the clinical criteria that define this syndrome. The mortality rate was low (2/53; 3.8%), and conclusions regarding association with SIRS could not be drawn. A positive SIRS status, high plasma CRP concentration, and high body temperature were variables that related to increased morbidity reflected by the length of hospitalization. Conclusions: SIRS was seen in 57% of canine pyometra cases and a positive SIRS status showed a positive association with prolonged hospitalization. The mortality rate was low (3.3%) among SIRS positive dogs, indicating that progression to multiple organ dysfunction syndrome (MODS) rarely occurs in surgically treated cases of pyometra. CRP was associated with SIRS and with prolonged hospitalization. Further studies of plasma CRP may be warranted in canine intensive care cases susceptible to development of SIRS and MODS.     

Validation of a human immunoturbidimetric assay to measure canine albumin in urine and cerebrospinal fluid.

The aim of this study was to validate an automated immunoturbidimetric assay used to quantify human albumin in urine and to accurately measure canine albumin concentrations in both urine and cerebrospinal fluid. The partial homology existing between human and canine albumin limited the accuracy of the human assays in measuring canine albumin without method modifications. Thus, the assay was modified by calibrating the analyzer with calibrators made in the laboratory containing known concentrations of canine albumin. To prepare the set of calibrators, the albumin concentration of pooled sera of healthy dogs was assessed in 5 replicates using the BromocresolGreen assay. Pooled samples were aliquoted and serially diluted to obtain the expected concentrations of albumin (0.5, 1, 5, 13, and 30 mg/dl) for establishing the canine calibration curve. Thereafter, the performance was assessed by analyzing canine urine and CSF The modified assay accurately quantified canine albumin in both specimens, as indicated by the following. Intra- and interassay variability was 0.92% and 2.74%, respectively; recovery was 99.66% and 99.07% in urine and 105.02% in CSF No interference was detected when hemolysate and glucose were added to urine. The test was linear within the verified range (0-225 mg/dl). These results demonstrate that the modified human albumin immunoturbidimetric assay can be a useful tool in the veterinary diagnostic laboratory. It is accurate and tends itself to automatization on chemistry analyzers.     

Evaluation of EDTA hematology tubes for collection of blood samples for tests of secondary hemostasis in dogs

OBJECTIVE: To evaluate the use of EDTA tubes for collection of blood samples for assays of secondary hemostasis in dogs. ANIMALS: 108 dogs of various ages, breeds, and sexes (19 healthy and 89 with abnormalities of secondary hemostasis). PROCEDURES: Blood samples were collected via cephalic venipuncture and transferred to sodium citrate tubes and EDTA tubes. Plasma was harvested from each type of tube for assays of concentrations of fibrinogen and D-dimer as well as prothrombin time, activated partial thromboplastin time, and antithrombin activity. Intra-assay and interassay precision and correlation coefficients for all hemostatic tests were calculated for each type of plasma sample. The effect of storage conditions on assay results for the 2 types of plasma samples was also evaluated. RESULTS: Results of hemostatic tests were highly correlated between citrated and EDTA-treated plasma samples. Intra-assay imprecision for all hemostatic tests with the exception of D-dimer concentration was < 10% for both citrated and EDTA-treated plasma samples; interassay imprecision was higher for EDTA-treated versus citrated plasma samples. Storage of plasma samples for 1 hour did not result in significantly different assay results for either type of plasma sample, but storage for 2 hours significantly affected values for EDTA-treated plasma samples. CONCLUSIONS AND CLINICAL RELEVANCE: Although evaluation of the sensitivity and specificity of hemostatic tests that use EDTA-treated plasma samples is required, EDTA may be a suitable alternative to sodium citrate as an anticoagulant for use in hemostatic testing in conditions in which tests could be performed within 1 hour after sample collection.     

Comparison of serum amyloid A and C-reactive protein as diagnostic markers of systemic inflammation in dogs

The diagnostic performance of canine serum amyloid A (SAA) was compared with that of C-reactive protein (CRP) in the detection of systemic inflammation in dogs. Sera from 500 dogs were retrospectively included in the study. C-reactive protein and SAA were measured using validated automated assays. The overlap performance, clinical decision limits, overall diagnostic performance, correlations, and agreement in the clinical classification between these 2 diagnostic markers were compared. Significantly higher concentrations of both proteins were detected in dogs with systemic inflammation (SAA range: 48.75 to > 2700 mg/L; CRP range: 0.4 to 907.4 mg/L) compared to dogs without systemic inflammation (SAA range: 1.06 to 56.4 mg/L; CRP range: 0.07 to 24.7 mg/L). Both proteins were shown to be sensitive and specific markers of systemic inflammation in dogs. Significant correlations and excellent diagnostic agreement were observed between the 2 markers. However, SAA showed a wider range of concentrations and a significantly superior overall diagnostic performance compared with CRP.     

Evaluation of a point-of-care test for canine C-reactive protein

Background: In veterinary medicine, there is increasing interest in measuring C‐reactive protein (CRP) as a tool for diagnosis and monitoring of inflammatory diseases. Reported CRP concentrations for healthy dogs have ranged from 0 to 8.9 mg/L. Objectives: The aims of this study were to evaluate a canine‐specific point‐of‐care (POC) lateral flow immunoassay for qualitative CRP measurement in healthy and diseased dogs and to compare results with those obtained by a quantitative ELISA. Methods: Blood samples from 73 client‐owned dogs were available for testing: 16 healthy dogs and 57 dogs with a variety of infectious, inflammatory, or neoplastic diseases. CRP was measured in heparinized whole blood samples and serum with the TECOmedical Dog CRP‐visual POC test. A red line develops in the POC device if CRP is ≥5 mg/L, and results are scored as negative or positive. An ELISA validated previously for canine serum was used as the reference method. Results: For all dogs, serum CRP concentrations measured by the ELISA ranged from 0.1 to ≥350 mg/L (median=38 mg/L). Percentages of the CRP POC test results that agreed with the ELISA results were 98.6% for whole blood and 97.3% for serum samples. For serum samples, sensitivity of the POC test was 96.4% and specificity was 81.3%. For whole blood, sensitivity was 94.7% and specificity was 93.8%. Conclusions: The POC test had very good agreement with the ELISA test and had high sensitivity and specificity; therefore, it can be used as a qualitative test to screen for increases in CRP concentrations.     

C-reactive protein measurement in canine saliva.

An established time-resolved immunofluorometric assay designed for measurement of C-reactive protein (CRP) in canine blood was evaluated and validated for use in canine saliva. C-reactive protein was measured in saliva specimens from 5 healthy dogs before and after the injection of casein and in 37 dogs with different disease conditions. The analytical and functional limits of detection were 0.000053 microg/ml and 0.0091 microg/ml, respectively, and intra- and interassay coefficients of variation ranged between 6.7-9.9% and 8.5-16.5%, respectively. A recovery experiment showed no significant disagreement between detected values and expected ones, and saliva CRP concentration was measured in a linear and proportional manner. A positive correlation was found between CRP levels obtained in saliva and serum samples in the experimental (R2 = 0.76) and clinical studies (R2 = 0.70). The assay was able to detect significant differences between salivary CRP levels in healthy dogs and dogs with inflammatory processes. These results suggest that saliva can be used for CRP measurement in dogs. The use of saliva presents the advantage of an easier and less stressful sampling method for the animals, which might be performed outside of hospital environments.     

Evaluation of a chromogenic assay to measure the factor Xa inhibitory activity of unfractionated heparin in canine plasma

BACKGROUND: Unfractionated heparin (UFH) has a complex pharmacologic profile that necessitates patient monitoring to prevent inadequate anticoagulation or overdosage and hemorrhage. Factor Xa inhibitory assays (to measure anti-Xa activity) are used to adjust UFH dosage and define safe and effective regimens for specific thrombotic disorders in humans. OBJECTIVE: In this study, the accuracy, linearity, and clinical utility of a chromogenic assay were assessed for monitoring UFH anti-Xa activity in canine plasma samples. METHODS: A commercial assay (Rotachrom Heparin, Diagnostica Stago, Parsippany, NJ, USA) was used to measure anti-Xa activity in canine plasma samples spiked with different concentrations of UFH. Background absorbance and assay linearity were compared for canine and human plasmas. Percentage recovery of UFH anti-Xa activity and intra- and interassay imprecisions were investigated by multiple measurements of canine plasma to which known amounts of UFH were added. The spiked plasma samples also were used to determine the heparin sensitivity of an activated partial thromboplastin time (aPTT) test. RESULTS: Canine plasma samples were assayed at a higher dilution than were human plasma samples (3:8 versus 4:8) to eliminate higher background anti-Xa activity in canine plasma. Using this modification, the recovery of anti-Xa activity in canine plasma was linear (R2 > .9) at concentrations of 0 - 0.75 U/mL UFH. Intra- and interassay imprecisions for plasma samples containing 0.5 U/mL UFH were <10%, whereas samples containing 0.25 U/mL UFH had imprecisions of 13% and 24%, respectively. The anti-Xa activity range of 0.5 - 0.75 U/mL caused prolongation of aPTTs to 1.5 - 2.5 times the assay mean. CONCLUSION: Plasma anti-Xa activity of dogs treated with UFH can be accurately monitored using this commercially available chromogenic assay.     

Validation and diagnostic efficacy of a lipase assay using the substrate 1,2-o-dilauryl-rac-glycero glutaric acid-(6' methyl resorufin)-ester for the diagnosis of acute pancreatitis in dogs

BACKGROUND: Increased serum lipase activity has been used historically to support the diagnosis of acute pancreatitis, a common disease in dogs. Most of the lipase assays that are currently in use lack optimum sensitivity and specificity. OBJECTIVE: The objectives of this study were to 1) validate the 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester (DGGR) assay for determination of lipase activity in canine serum and 2) compare results, reference intervals, sensitivity, and specificity of the DGGR assay with a standard 1,2-diglyceride (1,2 DiG) assay for diagnosing acute pancreatitis in dogs. METHODS: Precision, linearity, and interference studies were performed for method validation on a Hitachi 911 analyzer. Lipase results from the DGGR and 1,2 DiG assays were compared by linear regression analysis. Sensitivity, specificity, and diagnostic efficacy were determined for both assays on a population of 30 dogs, 15 of which had acute pancreatitis based on history, clinical signs, and ultrasound findings. RESULTS: Within-run and within-day coefficients of variation (CVs) were low (<3%), with higher day-to-day CVs (< or =14 %). The assay was linear between 8 and 2792 U/L. No significant interference by hemolysis and lipemia was found. Poor correlation was found between the assays (r(s)=0.84). The lipase reference interval was 8-120 U/L for the DGGR assay and 30-699 U/L for the 1,2 DiG assay. Sensitivity and specificity for the diagnosis of pancreatitis were 93% and 53%, respectively, for the DGGR assay and 60% and 73% for the 1,2 DiG assay. Receiver operating characteristic curve analysis showed similar areas under the curve. CONCLUSIONS: On the basis of this study, the DGGR method is considered adequate for assaying serum lipase activity in dogs. The high sensitivity of the DGGR assay suggests it may be a useful screening test for canine pancreatitis.     

Evaluation of cystatin C as an endogenous marker of glomerular filtration rate in dogs

Cystatin C is a cysteine protease inhibitor produced by all nucleated cells. It is freely filtered by the glomerulus and is unaffected by nonrenal factors such as inflammation and gender. Because of greater sensitivity and specificity, cystatin C has been proposed to replace creatinine as a marker of glomerular filtration rate (GFR) in humans. The aims of this study were to validate an automated assay in canine plasma and to evaluate the usefulness of cystatin C as a marker of GFR in dogs. Western blotting was used to demonstrate cross-reactivity of an anti-human cystatin C antibody. An immunoturbidimetric assay was used to detect cystatin C in 25 clinically healthy dogs and 25 dogs with renal failure. Mean cystatin C concentration in the healthy dogs and the dogs with renal failure was 1.08 +/- 0.16 mg/L and 4.37 +/- 1.79 mg/L respectively. Intra- and interassay variability was <5%. The assay was linear (r = .974) between 0.14 and 7.53 mg/L. Both cystatin C and creatinine concentrations were measured in banked, frozen serum from 20 remnant kidney model dogs and 10 volume-depleted dogs for which GFR measurements by exogenous creatinine clearance had been determined previously. In the remnant kidney model, cystatin C was better correlated with GFR than creatinine (r = .79 versus .54) but was less well correlated with GFR in volume-depleted dogs (r = .54 versus .95). GFR measurements were repeated in the remnant kidney model dogs 60 days after initial GFR measurements. At this time, cystatin C and creatinine concentrations correlated equally well with GFR (r = .891 versus .894, respectively). Cystatin C concentration is a reasonable alternative to creatinine for screening dogs with decreased GFR due to chronic renal failure.     

Measurement of C‐reactive protein and Prostaglandin F2α Metabolite Concentrations in Differentiation of Canine Pyometra and Cystic Endometrial Hyperplasia/Mucometra

Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C‐reactive protein (CRP) and prostaglandin F_2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p < 0.001) in dogs with pyometra compared to those with CEH/mucometra or healthy uterus. However, no statistical difference could be detected between the groups for mean PGFM concentrations. Mean white blood cell count (WBC), alkaline phosphatase (ALP) and total protein concentrations were found significantly higher (p < 0.001) in dogs with pyometra. Escherichia coli was the most frequently isolated microorganism from dogs with pyometra (64.3%). Edwardsiella spp. was detected in a single case of pyometra for the first time. In conclusion, our results demonstrate that serum CRP concentrations were increased in dogs with pyometra and thus we conclude that serum CRP concentration but not PGFM might be useful as a marker to differentiate a case of CEH/mucometra from pyometra in female dogs. To the authors' knowledge, this is the first report in which Edwardsiella spp. has been isolated in the canine uterus.     

A protocol to guide development of a sensitive ELISA for canine erythropoietin

Background: The determination of canine erythropoietin (EPO) concentration is crucial for monitoring the effect of human recombinant (hr) EPO therapy in dogs with chronic renal failure. Current assays are not specific for canine EPO and not sensitive enough to detect physiologic EPO levels in dogs.
Objective: The objective of this study was to develop a simple and sensitive ELISA for canine EPO that could serve as a starting point for developing a commercially available assay.
Methods: The ELISA was based on a mouse monoclonal antibody (mAb) and a rabbit polyclonal antibody (pAb) using 2 different immunization techniques: gene electrotransfer (GET) to generate the pAb and multiple antigen peptides (MAPs) to generate the mAb. The ELISA was performed using both EPO obtained from HeLa cells transfected with an expression plasmid encoding canine EPO and canine plasma with known concentrations of EPO.
Results: The ELISA standard curve was linear for canine EPO concentrations of 7–66 mU/ml. Coefficients of variation were about 10%. No cross-reactivity between canine EPO and hrEPO was detected.
Conclusions: Using novel GET and MAP technology, we developed a sensitive and specific ELISA for canine EPO that can be used to guide future clinical applications for EPO detection and monitoring in dogs.     

Purification and partial characterization of canine neutrophil elastase and the development of an immunoassay for the measurement of canine neutrophil elastase in serum obtained from dogs

OBJECTIVE: To purify neutrophil elastase (NE) from dog blood and develop and validate an ELISA for the measurement of canine NE (cNE) in canine serum as a marker for gastrointestinal tract inflammation. SAMPLE POPULATION: Neutrophils from 6 dogs immediately after they were euthanatized and serum from 54 healthy dogs. PROCEDURES: cNE was purified from blood by use of dextran sedimentation, repeated cycles of freezing-thawing and sonication, cation-exchange chromatography, and continuous elution electrophoresis. Antibodies against cNE were generated in rabbits, and an ELISA was developed and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and interassay variability. A reference range was established by assaying serum samples from the 54 healthy dogs and by use of the lower 97.5th percentile. RESULTS: cNE was successfully purified from blood, and antibodies were successfully generated in rabbits. An ELISA was developed with a sensitivity of 1,100 mug/L. The reference range was established as < 2,239 mug/L. Ratios of observed-to-expected results for dilutional parallelism for 4 serum samples ranged from 85.4% to 123.1%. Accuracy, as determined by spiking recovery, ranged from 27.1% to 114.0%. Coefficient of variation for 4 serum samples was 14.2%, 16.0%, 16.8%, and 13.4%, respectively, for intra-assay variability and 15.4%, 15.0%, 10.5%, and 14.6%, respectively, for interassay variability. CONCLUSIONS AND CLINICAL RELEVANCE: The purification protocol used here resulted in rapid and reproducible purification of cNE with a high yield. The novel ELISA yielded linear results and was accurate and precise. Additional studies are needed to evaluate the clinical usefulness of this assay.