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.     

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.     

Quantitation of canine plasma von Willebrand factor antigen using a commercial enzyme-linked immunosorbent assay.

The purpose of this study was to evaluate a commercial enzyme-linked immunosorbent assay (ELISA) for human von Willebrand factor antigen (vWF:Ag) with respect to its potential value in quantitating the protein in canine plasma. The assay was a sandwich technique using F(ab')2 fragments specific for von Willebrand factor (vWF) and a peroxidase conjugated rabbit anti-vWF second antibody, with a microplate as the support surface. Canine plasmas were assayed by ELISA, and by Laurell electroimmunoassay (EIA), our reference methodology. The ELISA had a within-day variation of 1.21-4.44% and a between-day variation of 0.85-4.88% depending on the level of vWF:Ag. The sensitivity of the assay was less than 0.1% vWF:Ag. The range of vWF:Ag concentrations in plasmas from 24 clinically normal dogs compared favorably with the range for the same plasmas when assayed by EIA (ELISA = 60-152% of normal; EIA = 50-142% of normal). In 121 canine plasmas with vWF:Ag concentrations (as assessed by EIA) ranging from undetectable levels (less than 6% of normal) to 142% of normal, there was good correlation with measurements made by ELISA (correlation coefficient = 0.835). It was concluded that this commercial ELISA technique could be used to provide reliable, same-day measurements of canine plasma vWF:Ag. Since it requires no special equipment other than a microplate reader and washer it is particularly suitable for laboratories lacking the electrophoretic expertise or equipment required for EIA.     

Assessment of three automated assays for C-reactive protein determination in dogs

OBJECTIVE: To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs. Animals-56 client-owned dogs with pyometra and 11 healthy control dogs. PROCEDURES: Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs. RESULTS: By use of the ELISA, mean +/- SD plasma CRP concentration was 96.1 +/- 38.5 mg/L and 10.1 +/- 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.     

Validation of a commercially available enzyme-linked immunosorbent assay (ELISA) for the determination of cortisol in canine plasma samples

Samples were obtained from clinically normal dogs before and after ACTH stimulation and dexamethasone suppression tests. The test kit Enzymun-Test (Boehringer Mannheim) for determining cortisol concentrations in human plasma was used in connection with the analyser system Enzymun-Test (Boehringer Mannheim) System ES300 following the manufacturer's instructions. The intra-assay and inter-assay coefficients of variation were 1.28% and 5.64%, respectively. The mean recovery when assaying samples with a cortisol content of more than 100 nmol/L was 95.41%, but this percentage decreased in samples with lower cortisol levels. The sensitivity of the assay was 2.76 nmol/L. The results of the ACTH stimulation and dexamethasone suppression tests were similar to those published previously. The ELISA method evaluated allows a precise and sensitive determination of cortisol concentrations in canine plasma samples. The major drawback observed was the loss of accuracy at low cortisol concentrations. Since the assay tends then to report lower cortisol concentrations, the generally accepted concentration of 40 nmol/L may not be suitable as the cutoff value in dexamethasone suppression tests.     

Isolation and measurement of carbonic anhydrase isoenzyme III in plasma, sera, and tissues of dogs

OBJECTIVE: To purify canine carbonic anhydrase isoenzyme III (CA-III) and determine plasma, serum, and tissue concentrations of CA-III in healthy dogs and dogs with experimentally induced muscle damage. ANIMALS: 121 healthy Beagles. PROCEDURE: Muscle was obtained from 2 Beagles after euthanasia, and CA-III was purified and characterized by use of column chromatography and electrophoresis, respectively. A CA-III-specific ELISA was developed to determine concentrations of CA-III in plasma of 116 dogs and tissues of 1 dog. Serum creatine kinase (CK) activity and CA-III concentration were also determined before and after induction of muscle damage by IM injection of 2 ml of 10% lidocaine to 2 dogs. RESULTS: Canine CA-III had a molecular weight of 28 kd and an isoelectric point of 8.2. Mean (+/- SD) concentration of CA-III in plasma of healthy dogs was 16.91 +/- 9.55 ng/ml. The highest tissue concentration of CA-III was detected in skeletal muscle. Serum concentration of CA-III increased and peaked within the first 2 to 3 hours after induction of muscle damage. The increase in CA-III concentration was more rapid than that of CK activity, and concentration reached its maximum and returned to baseline sooner than did CK activity. CONCLUSIONS AND CLINICAL RELEVANCE: The CA-III ELISA we developed was a sensitive method for determining CA-III concentrations in plasma, serum samples, and tissue specimens of dogs. Use of this ELISA requires only a small volume of serum and may enable the study of changes in CA isoenzyme concentrations associated with muscle disorders in dogs.     

四川省工作犬心丝虫感染率的调查

本文调查了四川省工作犬心丝虫的感染状况,从四川省成都、德阳、绵阳、乐山、宜宾、阿坝六地的128头工作犬（包括警犬和护卫犬）身上采集128份血样,用改良Knott’s法和ELISA快速检测试剂盒分别进行犬心丝虫微丝蚴和雌性成虫的检测,结果得其感染率分别为5．47％和20．31％,证明四川是心丝虫病的流行区域。     

Accuracy of a point-of-care ELISA test kit for predicting the presence of protective canine parvovirus and canine distemper virus antibody concentrations in dogs

Canine parvovirus (CPV) and canine distemper virus (CDV) are highly infectious and often fatal diseases with worldwide distributions, and are important population management considerations in animal shelters. A point-of-care ELISA test kit is available to detect serum antibodies to CPV and CDV, and presumptively to predict protective status. The aim of this study was to determine the diagnostic accuracy of the test compared to CPV hemagglutination inhibition titers and CDV serum neutralization titers determined by a reference laboratory, using sera collected from dogs housed at animal shelters. The ELISA test was used under both field and laboratory conditions and duplicate specimens were processed using an extra wash step. The test kit yielded accurate results (CPV: sensitivity 92.3%, specificity 93.5%; CDV: sensitivity 75.7%, specificity 91.8%) under field conditions. CDV sensitivity was improved by performing the test under laboratory conditions and using an optical density (OD) meter (laboratory performed 94.0%; OD 88.1%). Point-of-care ELISA testing for serum CPV and CDV antibody titers was demonstrated to be a useful tool for determining antibody status when making decisions regarding the need for CPV and/or CDV vaccination and also in animal shelters for population management.     

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.     

Use of vitamin B12 in joint lavage for determination of dilution factors of canine synovial fluid

OBJECTIVE: To test a modified saline (0.9% NaCl) solution joint washing (lavage) technique that includes the use of vitamin B12 as an internal marker for the evaluation of synovial fluid dilution in lavage samples from canine joints. SAMPLE POPULATION: 9 plasma samples obtained from blood samples of 9 healthy dogs and 9 synovial fluid samples aspirated from stifle joints of 9 cadaveric dogs. PROCEDURE: Photometric absorbances of 25% vitamin B12 solution, canine synovial fluid, and canine plasma were measured in a spectrophotometer to establish an optimal wavelength for analysis. Canine synovial fluid and plasma samples were mixed with the 25% vitamin B12 solution to obtain 1%, 3%, 5%, 10%, 20%, and 50% solutions of synovial fluid or plasma. Diluted synovial fluid and plasma samples were used to simulate joint lavage samples and to examine the possible interference of these substances (synovial fluid or plasma) with the absorbance of the 25% vitamin B12 solution in photometric analysis. RESULTS: The optimal wavelength was found to be at 550 nm. Canine synovial fluid and plasma samples did not interfere with the absorbance measurements of the 25% vitamin B12 solution up to a 50% dilution of plasma or synovial fluid. CONCLUSIONS AND CLINICAL RELEVANCE: The modified saline solution joint lavage method with the use of a 25% vitamin B12 solution as an internal standard provides an accurate and reliable technique for the evaluation of synovial fluid dilution in lavage samples from canine joints.     

Canine heterophilic antibodies as a source of false-positive B-type natriuretic peptide sandwich ELISA results

BACKGROUND: Interference by heterophilic antibodies is a well-known cause of false-positive sandwich ELISA results in human medicine. They are considered rarely in veterinary species and have not been characterized but could become important as newer, highly sensitive sandwich immunoassay technologies are developed. OBJECTIVES: The goals of this study were to use a B-type natriuretic peptide (BNP-32) sandwich ELISA to determine the effect of heterophilic antibodies on test performance; to characterize canine heterophilic antibodies; and to develop and test a method for heterophilic antibody removal. METHODS: A sandwich ELISA was developed using a mouse IgG(1)K monoclonal and a rabbit polyclonal antibody to two synthetic peptides of canine BNP-32. The effects on false-positive results of heterophilic antibody depletion and blocking by various techniques were compared. The titers of canine heterophilic antibodies were compared with various blood antigens from other species and the relative amount of canine IgG was compared with that of IgM heterophilic antibody. RESULTS: Heterophilic antibodies in dog plasma were shown to be capable of causing false-positive ELISA results. They reacted with blood proteins from a variety of animal species at relatively low titers and consisted of both IgG and IgM. Protein A agarose antibody precipitation, in conjunction with mouse IgG(1)K blocking antibody, was effective in eliminating false-positive sandwich ELISA results while retaining adequate test performance. CONCLUSIONS: Canine heterophilic antibodies can interfere with sandwich ELISA assays and cause false-positive test results. An effective technique for their removal that has a potentially broad application was developed, and allows measurement of canine blood constituents at low picomolar concentrations.     

Evaluation of a canine D-dimer point-of-care test kit for use in samples obtained from dogs with disseminated intravascular coagulation, thromboembolic disease, and hemorrhage

OBJECTIVE: To evaluate a canine D-dimer point-of-care (cD-d POC) test kit for use in healthy dogs and dogs with disseminated intravascular coagulation (DIC), thromboembolic disease (TED), and hemorrhage. ANIMALS: 12 healthy dogs, 18 dogs with DIC, 23 dogs with TED (19 acute and 4 chronic), and 18 dogs with hemorrhage. PROCEDURE: The cD-d POC, canine D-dimer ELISA (cD-d ELISA), human D-dimer latex agglutination (hD-d LA), and fibrin degradation product (FDP) tests were performed on citrated plasma. RESULTS: All healthy dogs had negative cD-d POC test results and mean cD-d ELISA value of 0.2 U/mL. All dogs with DIC had positive cD-d POC test results and mean cD-d ELISA value of 44 U/mL. Dogs with acuteTED had a mean cD-d ELISA value of 34 U/mL, and 17 of 19 had positive cD-d POC test results. Mean cD-d ELISA value in dogs with hemorrhage was 14 units/mL, and 15 of 18 had positive cD-d POC test results. The cD-d ELISA values in dogs with hemorrhage were significantly higher than those of healthy dogs but lower than those of dogs with DIC and acute TED. The cD-d POC, cD-d ELISA, and hD-d LA tests were comparable in differentiating healthy dogs from dogs with DIC, acute TED, or hemorrhage and appeared to be superior to measurement of FDPs. CONCLUSIONS AND CLINICAL RELEVANCE: The cD-d POC test kit can be quickly and easily used and reliably detects dogs with DIC or acute TED. Positive results may also be seen in dogs with internal hemorrhage.     

Enzyme-linked immunosorbent assay for detection of canine chromogranin A by use of immunological cross-reactivity of rabbit anti-bovine chromogranin A antibody

Bovine and canine chromogranin A were extracted and purified from each specie's adrenal glands. Isolated bovine 70 kDa protein showed 100% identity to bovine CgA reported previously, whereas isolated canine 68 kDa protein showed 83.3% identity to bovine CgA by the NH(2)-terminal amino acid sequence analysis. Rabbit antibody to purified bovine protein (CgA) was found to immunologically cross-reacted with purified canine protein (CgA). In sandwich ELISA with anti-bovine CgA, concentration-dependent curves were obtained ranging from 0.3 to 20 mug/ml for canine CgA. From these findings, sandwich ELISA with anti-bovine CgA is found to be useful to determine the concentration of canine CgA.     

Effects of etomidate on adrenocortical function in canine surgical patients

Adrenocortical function in canine surgical patients given etomidate at 1 of 2 dosages (1.5 mg/kg of body weight or 3 mg/kg, IV) was evaluated and compared with that of dogs given thiopental (12 mg/kg, IV). The adrenocortical function was evaluated by use of adrenocorticotropic hormone (ACTH) stimulation tests and determination of plasma cortisol concentrations at 0 minute (base line) and 60 minutes after ACTH administration. At 24 hours before administration of either drug (ie, induction of anesthesia), each dog had an increase in plasma cortisol concentration when given ACTH. The ACTH stimulation tests were repeated 2 hours after induction of anesthesia. Dogs given thiopental had base-line plasma cortisol concentrations greater than preinduction base-line values, but did not increase plasma cortisol in response to ACTH stimulation. Postinduction ACTH stimulation tests in dogs given etomidate at either dose indicated base-line and 60-minute plasma cortisol concentrations that were not different from preinduction base-line values. Therefore, adrenocortical function was suppressed 2 and 3 hours after the administration of etomidate in canine surgical patients.     

The diagnostic accuracy of different natriuretic peptides in the investigation of canine cardiac disease

OBJECTIVES: We aimed to validate and determine the accuracy of a new sandwich ELISA for canine N-terminal pro-B-type natriuretic peptide (NT-proBNP) in the discrimination of canine patients with cardiac disease from those with respiratory disease and to determine the effect of confounding variables on NT-proBNP concentrations. METHODS: Validation studies for the new assay were undertaken. Concentrations of N-terminal atrial natriuretic peptide (NT-proANP) and NT-proBNP in both ethylenediaminetetraacetic acid (EDTA) plasma and serum were estimated in samples from 77 dogs at a laboratory blinded to the clinical status of the patient. The diagnostic accuracy of the each sample type and test was evaluated using receiver operating characteristic curves. The effect of age, gender and indicators of renal function was evaluated using a multivariate regression analysis. RESULTS: Concentrations of NT-proBNP in both serum and plasma accurately discriminated dogs with respiratory disease from those with cardiac disease, with an optimum cut-off concentration of 210 pmol/l. NT-proBNP concentrations were unaffected by sample type. Increasing creatinine concentration is associated with increasing concentration of NT-proBNP. Age and gender were not found to have significant effects on natriuretic peptide concentrations in this population. CLINICAL SIGNIFICANCE: Canine NT-proBNP appears to be a useful marker of the presence of cardiac disease, although concentrations must be interpreted in the light of the patient's renal function.     

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.     

Clinical and serological response of wild dogs (Lycaon pictus) to vaccination against canine distemper,canine parvovirus infection and rabies

Wild dogs Lycaon pictuis (n = 8) were vaccinated 4 times against canine distemper (n = 8) (initially with inactivated and subsequently with live attenuated strains of canine distemper) and canine parvovirus infection (n = 8) over a period of 360 days. Four of the wild dogs were also vaccinated 3 times against rabies using a live oral vaccine and 4 with an inactivated parenteral vaccine. Commercially-available canine distemper, canine parvovirus and parenteral rabies vaccines, intended for use in domestic dogs, were used. None of the vaccinated dogs showed any untoward clinical signs. The inactivated canine distemper vaccine did not result in seroconversion whereas the attenuated live vaccine resulted in seroconversion in all wild dogs. Presumably protective concentrations of antibodies to canine distemper virus were present in all wild dogs for at least 451 days. Canine parvovirus haemagglutination inhibition titres were present in all wild dogs prior to the administration of vaccine and protective concentrations persisted for at least 451 days. Vaccination against parvovirus infection resulted in a temporary increase in canine parvovirus haemagglutination inhibition titres in most dogs. Administration of both inactivated parenteral and live oral rabies vaccine initially resulted in seroconversion in 7 of 8 dogs. These titres, however, dropped to very low concentrations within 100 days. Booster administrations resulted in increased antibody concentrations in all dogs. It was concluded that the vaccines were safe to use in healthy subadult wild dogs and that a vaccination protocol in free-ranging wild dogs should at least incorporate booster vaccinations against rabies 3-6 months after the first inoculation.     

Measurement of myelin basic protein in the cerebrospinal fluid of dogs with degenerative myelopathy

BACKGROUND: Analysis of cerebrospinal fluid (CSF) is part of a routine clinical workup in veterinary patients when neurologic disease is suspected. However, knowledge of particular protein markers of disease in CSF is limited. The concentration of myelin basic protein (MBP) in CSF is used as a biochemical marker in humans to evaluate demyelinating lesions in the central nervous system (CNS). OBJECTIVE: The purpose of this study was to evaluate an ELISA for determination of MBP concentration in the CSF of German shepherd dogs with degenerative myelopathy (GSDM). METHODS: Cross-reactivity of the anti-human polyclonal antibody used in a commercial ELISA (Active MBP ELISA, Diagnostic Systems Laboratories Inc, Webster, TX, USA) was tested with canine MBP by immunoblotting. CSF samples were collected from both the cisterna magna and the lumbar cistern of 8 clinically healthy control dogs and 8 German shepherd dogs clinically diagnosed with GSDM. MBP concentrations were measured in all CSF samples using the ELISA. RESULTS: The mean MBP concentration in CSF from the lumbar cistern of dogs with GSDM (3.13 -/+ 0.46 ng/mL) was significantly higher than that in the cisterna magna (0.70 -/+ 0.06 ng/mL) and from both cisternal (0.47 -/+ 0.07 ng/mL) and lumbar (0.94 -/+ 0.37 ng/mL) samples from control dogs. CONCLUSION: The MBP ELISA has potential as a supplemental test of CSF to diagnose demyelinating disorders in dogs.     

Isolation and measurement of carbonic anhydrase isoenzymes in erythrocytes of dogs

OBJECTIVE: To purify canine carbonic anhydrase (CA) isoenzymes CA-I and CA-II and to determine concentrations of CA-I and CA-II in erythrocytes of Beagles and dogs native to Japan. SAMPLE POPULATION: Blood samples from 116 Beagles, including 24 pregnant Beagles, and blood samples from 29 dogs native to Japan. PROCEDURE: Canine CA-I and CA-II were purified by use of column chromatography. Concentrations of CA-I and CA-II in erythrocytes of dogs were determined, using an ELISA. RESULTS: Mean (+/- SD) concentrations of CA-I and CA-II in erythrocytes of Beagles were 3.21+/-0.86 and 1.63+/-0.39 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male Beagles than female Beagles. In contrast, mean concentration of CA-II was greater in female Beagles than male Beagles. Furthermore, concentration of CA-II was greater in pregnant female Beagles than male or nonpregnant female Beagles. Mean concentrations of CA-I and CA-II in erythrocytes of dogs native to Japan were 11.03+/-4.39 and 3.29+/-0.91 mg/g of Hb, respectively. Mean concentration of CA-I was greater in male dogs from Japan than female dogs from Japan. CONCLUSIONS AND CLINICAL RELEVANCE: The ELISA used in this study proved to be precise and sensitive for determining CA-I and CA-II concentrations in dogs. The ELISA may enable study of changes in isoenzymes associated with hereditary or metabolic disorders of blood or other body fluids, using only a small sample. Measurement of the concentrations of CA isoenzymes in dogs may be of diagnostic value.