Development and validation of a radioimmunoassay for the measurement of canine pancreatic lipase immunoreactivity in serum of dogs

OBJECTIVE: To develop and validate a radioimmunoassay (RIA) for measuring canine pancreatic lipase immunoreactivity (cPLI) in serum obtained from dogs. SAMPLE POPULATION: Serum samples from 47 healthy dogs. PROCEDURES: Canine pancreatic lipase (cPL) was purified from pancreatic specimens of dogs. Antibodies against cPL were raised in rabbits and purified by use of affinity chromatography. A tracer was produced by iodination of cPL with 125I. An RIA was established and validated by determination of sensitivity, working range, dilutional parallelism, spiking recovery, and intra- and interassay variability. A reference range for cPLI in serum was established by use of the central 95th percentile for samples obtained from 47 healthy dogs. RESULTS: Sensitivity and upper limit of the working range were 0.88 and 863 microg/L, respectively. Observed-to-expected ratios for serial dilutions ranged from 84.9 to 116.5% for 4 samples. Observed-to-expected ratios for spiking recovery ranged from 82.8 to 128.6% for 4 samples. Coefficients of variation for intra-assay variability for 4 serum samples were 18.3, 4.2, 3.5, and 8.9%, whereas interassay coefficients of variation were 29.2, 6.2, 3.9, and 4.4%, respectively. The reference range was 4.4 to 276.1 microg/L. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that the RIA described is sensitive, linear, accurate, precise, and reproducible, with limited accuracy in the high end of the working range and limited precision and reproducibility in the low end of the working range. Additional studies are needed to evaluate whether this degree of accuracy, precision, and reproducibility will negatively impact clinical use of this assay.     

Canine and feline pancreatic lipase immunoreactivity

The diagnosis of pancreatitis in dogs and cats can be challenging. Several diagnostic tests have been evaluated over the years, but the majority have been shown to be of limited utility owing to poor performance or limited availability or because invasive procedures are required. Assays for the measurement of pancreatic lipase immunoreactivity (cPLI for dogs and fPLI for cats) were first developed over a decade ago and now include Spec cPL and SNAP cPL for dogs and Spec fPL and SNAP fPL for cats. Owing to their high sensitivity and specificity for pancreatitis compared with those of other serum tests, concentrations of cPLI and fPLI have been demonstrated to be the serum tests of choice for evaluation of dogs and cats, respectively, suspected of having pancreatitis. False-positive and false-negative results can occur, and recognition of the limitations of pancreatic lipase immunoreactivity assays is important. As there is currently no gold standard for antemortem diagnosis of pancreatitis in dogs and cats, the combination of a complete history and physical examination, measurement of pancreatic lipase immunoreactivity, and ultrasonographic examination of the pancreas is the best approach for an accurate noninvasive diagnosis of pancreatitis.     

Analytical validation of an ELISA for measurement of canine pancreas‐specific lipase

Background: The diagnosis of canine pancreatitis is challenging. Clinical presentation often includes nonspecific clinical signs, such as vomiting, anorexia, and abdominal discomfort. Increased serum lipase activity can be indicative of pancreatitis; however, it can also be increased with other conditions. An immunoassay for measurement of canine pancreas‐specific lipase in canine serum that would be suitable for commercial application and provide rapid results would be beneficial. Objective: The goal of this study was to validate the Spec cPL assay, a commercially available ELISA for the quantitative measurement of canine pancreas‐specific lipase. Methods: Dynamic range, dilutional linearity, precision, interfering substances, assay stability, and reproducibility were investigated for analytical validation. The method was compared with the reference assay, canine pancreatic lipase immunoreactivity (cPLI), and included evaluation of a sample population of dogs and bias. Results: Analytical validation showed a dynamic range of 36–954 μg/L; good precision (intra‐ and interassay coefficient of variation <12%); absence of interference from lipid, hemoglobin, or bilirubin; 12‐month kit stability; and good reproducibility. Method comparison showed a positive bias relative to the cPLI reference method; however, the bias can be accommodated by adjustment of decision limits. The upper limit of the reference interval for Spec cPL was determined to be 216 μg/L based on the upper 97.5th percentile of results from 93 clinically healthy, kennel‐housed dogs. Conclusions: Validation data demonstrated that the Spec cPL assay provides reproducible results for canine pancreas‐specific lipase. A readily available assay for measurement of this enzyme allows broader clinical utilization of this analytical tool, generating timely results to aid in the diagnosis of canine pancreatitis.     

A pilot study evaluating changes in pancreatic lipase immunoreactivity concentrations in canines treated with L-asparaginase (ASNase), vincristine, or both for lymphoma

L-asparaginase (ASNase) is a common chemotherapy agent for the treatment of lymphoid malignancies. L-asparaginase has been reported to cause clinical pancreatitis in both humans and canines. Canine pancreatic lipase immunoreactivity (cPLI) is now a common diagnostic tool for evaluating pancreatitis in dogs. A total of 52 dogs were enrolled into this study. Canine pancreatic lipase immunoreactivity (cPLI) concentrations were evaluated before and after administration of ASNase, vincristine, or both. All dogs enrolled in the study were evaluated for signs compatible with clinical pancreatitis. No dogs receiving ASNase alone showed evidence of clinical pancreatitis after administration. Also, there was no statistically significant change in cPLI concentrations before or after treatment. Fourteen percent of dogs that received both vincristine and ASNase concurrently had elevated concentrations of cPLI after treatment. Of the 11 dogs with clinical signs compatible with pancreatitis after any chemotherapy treatment, no dog had a cPLI concentration > 400 μg/dL. In conclusion, ASNase did not cause clinical pancreatitis in this cohort of dogs but larger sample sizes are required to further validate this data.     

Increased canine pancreatic lipase immunoreactivity (cPLI) and 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase in dogs with evidence of portal hypertension and normal pancreatic histology: a pilot study

The clinical presentations of both liver disease and pancreatitis are nonspecific and overlapping, which may cause difficulty in diagnosis. In our retrospective pilot study, we assessed whether dogs with evidence of portal hypertension and absence of pancreatitis on pancreatic histology have increases in canine pancreatic lipase immunoreactivity (cPLI) and 1,2-o-dilauryl-rac-glycero-3-glutaric acid-(6′-methylresorufin) ester (DGGR) lipase. We included dogs that had been presented between 2008 and 2019 if they had normal pancreatic histology, histologically confirmed hepatopathy, and if canine pancreas-specific lipase (Spec cPL; Idexx) or DGGR lipase had been measured. Only dogs with portal hypertension were included. Six dogs fulfilled the inclusion criteria. Four of 6 and 2 of 6 dogs had Spec cPL and DGGR lipase exceeding the upper reference limit, respectively. From the 4 dogs with increased Spec cPL, 2 had concentrations of 200–400 µg/L and 2 had concentrations ≥ 400 µg/L. Our results suggest that canine portal hypertension might lead to increased Spec cPL and DGGR lipase values in the absence of pancreatitis on histology. Until more evidence in a larger number of dogs with portal hypertension is available, both tests should be interpreted cautiously in the presence of portal hypertension.     

Cellular immunolocalization of gastric and pancreatic lipase in various tissues obtained from dogs

OBJECTIVE: To determine cellular immunolocalization of canine gastric lipase (cGL) and canine pancreatic lipase (cPL) in various tissues obtained from clinically healthy dogs. SAMPLE POPULATION: Samples of 38 tissues collected from 2 climically healthy dogs. PROCEDURES: The cGL and cPL were purified from gastric and pancreatic tissue, respectively, obtained from dogs. Antisera against both proteins were developed, using rabbits, and polyclonal antibodies were purified by use of affinity chromatography. Various tissues were collected from 2 healthy dogs. Primary antibodies were used to evaluate histologic specificity. Replicate sections from the collected tissues were immunolabeled for cGL and cPL and examined by use of light microscopy. RESULTS: Mucous neck cells and mucous pit cells of gastric glands had positive labeling for cGL, whereas other tissues did not immunoreact with cGL. Pancreatic acinar cells had positive labeling for cPL, whereas other tissues did not immunoreact with cPL. CONCLUSIONS AND CLINICAL RELEVANCE: We concluded that cGL and cPL are exclusively expressed in gastric glands and pancreatic acinar cells, respectively. Also, evidence for cross-immunoreactivity with other lipases or related proteins expressed by other tissues was not found for either protein. Analysis of these data suggests that gastric lipase is a specific marker for gastric glands and that pancreatic lipase is a specific marker for pancreatic acinar cells. These markers may have clinical use in the diagnosis of gastric and exocrine pancreatic disorders, respectively.     

Evaluation of canine serum for the presence of antiretinal autoantibodies in sudden acquired retinal degeneration syndrome

OBJECTIVE: To determine the presence of serum antiretinal antibodies in sudden acquired retinal degeneration syndrome (SARDS) affected dogs and the size of the antigen to which these antibodies bind via the use of enzyme-linked immunosorbent assay (ELISA) and Western blot immunoassays. ANIMALS STUDIED: Serum was collected from 13 dogs affected by SARDS and five dogs with normal ocular examinations. PROCEDURES: All serum samples were subjected to ELISA with saline-soluble canine retinal tissue and Western blot analyses with SDS solubilized normal canine retinal tissue as the antigen. Antirecoverin (23 kDa) and antiheat shock cognate (65 kDa) antibodies were used as positive controls for both procedures. Affinity-purified goat antidog IgG and IgM labeled with horseradish peroxidase were used for all clinical samples and goat antirabbit IgG was used as the secondary antibody for the positive controls. RESULTS: ELISA demonstrated antibody reaction with all samples. Western blot immunoassays identified multiple bands in all canine serum samples, as well as in negative controls. Approximate sizes of the bands were 25 and 50 kDa, corresponding to IgG light and heavy chains, respectively. CONCLUSION: No antiretinal autoantibodies were identified in the serum of dogs affected by SARDS as compared to normal canine patients.     

Sandwich-dot enzyme-linked immunosorbent assay for the detection of canine distemper virus

A sandwich-dot enzyme-linked immunosorbent assay (dot ELISA) was developed for the detection of canine distemper virus (CDV). In 56 dogs suspected to have CD the rates of detection of CDV antigen in samples of blood lymphocytes and palpebral conjunctiva by dot ELISA and ELISA were, respectively, 91% (49/54) and 81% (44/54) for the lymphocyte samples and 88% (28/32) and 75% (24/32) for the conjunctival samples. The CDV detection limits were 10 ng/50 μL for dot ELISA and 40 ng/50 μL for ELISA. The reliability of dot ELISA relative to electron microscopy was 96% with 22 samples: all 21 samples in which CDV particles were observed by electron microscopy yielded positive results with dot ELISA; the single sample in which particles were not observed yielded false-positive results with dot ELISA. The results indicate that the dot ELISA developed can serve as a reliable rapid diagnostic test in suspected cases of CD and also be useful for epidemiologic surveillance of the disease.     

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.     

Biological variability of C-reactive protein and specific canine pancreatic lipase immunoreactivity in apparently healthy dogs

Background: C‐reactive protein (CRP) and specific canine pancreatic lipase immunoreactivity (Spec cPL) are biomarkers of generalized or nonspecific inflammation and pancreatic inflammation in dogs, respectively. The extent of inter‐ and intraindividual variation over time of these analytes is not well defined in dogs. The minimal critical difference for sequential determinations of these markers (ie, the smallest change necessary to represent physiological change rather than biological variation), has not been defined. Objectives: To determine the inter‐ and intraindividual variability (CV_G and CV_I) and minimal critical difference for sequential determinations of serum CRP and Spec cPL concentrations in apparently healthy dogs. Animals: Eleven apparently healthy dogs owned by staff or students at a veterinary teaching hospital. Methods: Blood was collected repeatedly at varying intervals over 12 weeks. CRP and Spec cPL concentrations were determined with commercially available assays. Indices of inter‐, intraindividual, and assay variability and 1‐sided minimal critical differences for sequential concentrations were calculated. Results: For CRP, CV_G was 90.8%, CV_I was 115.5%, and the analytical variability (CV_A) was 6.3%; the index of individuality was 0.74, and 1‐sided critical difference was 269.9%. For Spec cPL, CV_G= 49.48%, CV_I= 193.8%, CV_A= 8.4%, index of individuality = 0.24, and 1‐sided critical difference was 452.6%. Conclusions and Clinical Importance: A population‐based reference range is appropriate for Spec cPL, but questionable for CRP in dogs. Large changes in serial measurements of Spec cPL are necessary to infer clinical importance, more modest changes in CRP are likely to be meaningful.     

Effect of immunotherapy on the serum concentrations of allergen-specific IgG antibodies in dog sera

An ELISA assay which uses horseradish peroxidase conjugated anti-canine IgG and polystyrene microtiter wells for detection of allergen-specific IgG in the serum of dogs is described. Individual allergen blanks were used to account for the variable nonspecific binding among various allergens, and the results observed in milliunits of absorbance were normalized using four reference sera. The coefficients of variation for the intraassay and interassay variability ranged from 1.34 to 12.50% and 4.62 to 9.77%, respectively. The relationship between ELISA results and serum concentrations of allergen-specific IgG was quantified. IgG antibodies with specificity for various allergens were found in the majority of non-atopic individuals and in all atopic subjects. Specific immunotherapy resulted in a rise in the serum concentration of allergen-specific IgG.     

Development and analytical validation of an enzyme linked immunosorbent assay for the measurement of canine gastric lipase immunoreactivity in serum

The objective of this study was to develop and analytically validate an enzyme linked immunosorbent assay (ELISA) for measurement of canine gastric lipase immunoreactivity (cGLI). A sandwich ELISA was developed using canine gastric lipase (cGL) purified from canine stomachs and polyclonal antibodies directed against cGL, raised in rabbits and purified by affinity chromatography. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, reproducibility, and the upper limit of the control range by determining the 97.5th percentile of serum cGLI concentration in 74 healthy canines. Sensitivity and working range in serum were 200 ng/L and 200 to 39 160 ng/L, respectively. Observed to expected ratios for dilutional parallelism for 3 serum samples and 3 dilutions ranged from 86.1% to 244.2% (mean +/- standard deviation [s]; 125.4% +/- 48.2%). Observed to expected ratios for spiking recoveries for 3 serum samples and 6 spiking concentrations ranged from 66.4% to 152.5% (mean +/- s; 104.5% +/- 22.9%). Intra-assay and interassay variabilities for 3 different serum samples were 25.5%, 9.4%, and 13.4% and 26.0%, 17.2%, and 14.4%, respectively. The upper limit of the control range for serum cGLI was 662 ng/L. We concluded that the ELISA for cGLI described here is highly sensitive and shows a wide working range. However, the validation characteristics for this assay are suboptimal and below values of approximately 2.000 ng/L the assay is more semiquantitative in nature. Despite its limitations, whether this assay is useful for the diagnosis of canine gastric disorders remains to be determined.     

Serum concentrations of pepsinogen A in healthy dogs after food deprivation and after feeding

OBJECTIVE: To develop and validate an ELISA for measurement of serum canine pepsinogen A (cPG A) as a diagnostic marker of gastric disorders in dogs and to measure serum cPG A in healthy dogs after food deprivation and after feeding. SAMPLE POPULATION: Sera from 72 healthy dogs. PROCEDURE: A sandwich ELISA was developed and validated. The reference range for serum concentrations of cPG A was determined in 64 healthy dogs. Postprandial changes in serum concentrations of cPG A were evaluated in 8 healthy dogs. RESULTS: Assay sensitivity was 18 microg/L, and the maximum detectable concentration was 1,080 microg/L. The observed-to-expected ratio (O:E) for 3 serial dilutions of 3 serum samples ranged from 69.3 to 104.1%. The O:E for 3 serum samples spiked with 8 concentrations of cPG A ranged from 58.8 to 120.4%. Coefficients of variation for intra- and interassay variability of 3 serum samples ranged from 7.6 to 11.9% and from 10.1 to 13.1%, respectively. Mean +/- SD serum concentration of cPG A in healthy dogs was 63.8 +/- 31.0 microg/L and the reference range was 18 to 129 microg/L. Significant increases in serum concentrations of cPG A were observed between 1 and 7 hours after feeding. CONCLUSIONS AND CLINICAL RELEVANCE: The ELISA for measuring cPG A was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use. Serum concentrations of cPG A increase substantially after feeding, which should be taken into account when conducting clinical studies.     

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.     

H5 antibody detection by blocking enzyme-linked immunosorbent assay using a monoclonal antibody

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.     

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (mvv) in sheep is described, in which microtitre plates are with a partly purified preparation of mvv. The antibodies bound are detected by a horseradish peroxidase conjugate.The results obtained with ELISA on a total of 493 serum samples from several commercial flocks were compared to those of a routine agar gel precipitation test (AGPT) and a complement fixation test (CFT).All samples which scored positive in AGPT, CFT or both (20.8%) were also found positive by ELISA. In addition, with ELISA a further 11.5% of the samples were positive. Serum samples from maedi-free flocks, from sheep suffering from sheep pulmonary adenomatosis and from lambs immunized against other viruses were all negative by ELISA. The assay has been used routinely for some years and proved to be specific, sensitive and suited for screening of large numbers of serum samples.     

Development and validation of an enzyme-linked immunosorbent assay for feline trypsin-like immunoreactivity

OBJECTIVE: To develop and validate an ELISA for quantitative analysis of feline trypsin-like immunore-activity (fTLI). SAMPLE POPULATION: Purified feline cationic trypsin (fCT) and rabbit anti-fCT antiserum; blood samples from 63 healthy cats. PROCEDURES: A sandwich capture ELISA was developed, using anti-fCT antiserum purified by affinity chromatography that underwent biotinylation. Purified fCT was used for standards. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. A reference range was established by assaying serum samples from the 63 healthy cats. RESULTS: Sensitivity was 1.23 microg/L; working range was 2 to 567 microg/L. Ratios of observed versus expected results for 4 samples tested at various dilutions ranged from 90.0 to 120.7%. Ratios of observed versus expected results for 5 samples spiked with various concentrations of fCT ranged from 82.0 to 101.8%. Intra- and inter-assay coefficients of variability ranged from 9.9 to 11.1% and from 10.2 to 21.7%, respectively. The reference range for serum fTLI measured with this ELISA was 12 to 82 microg/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that an ELISA can be used to measure serum fTLI in cats. The ELISA was sufficiently sensitive, linear, accurate, precise, and reproducible for clinical use.     

酶标抗原Dot－ELISA检测鸭肝炎病毒抗体

酶标抗原Dot－ELISA检测鸭肝炎病毒抗体@杨奎@陈溥言...     

Development of a canine trypsin-like immunoreactivity assay system using monoclonal antibodies

The radioimmunoassay (RIA) for trypsin-like immunoreactivity (TLI) is one of the most sensitive and specific tests for detecting exocrine pancreatic insufficiency (EPI). An abnormally low serum TLI concentration (<2.5 ng/ml) indicates end-stage EPI. Although RIA methods can be used to detect canine serum TLI, these procedures are beyond the capabilities of most veterinary clinics and general laboratories. Using monoclonal antibodies (mAbs), we developed an enzyme-linked immunosorbent assay (ELISA) for canine TLI and incorporated it into an immunochromatographic test (ICT) for the diagnosis of EPI. The ELISA was linear over TLI concentrations of 1-100 ng/ml. Levels of intra-assay coefficients of variance (CVs) were 1.8-6.1%, inter-assay CVs were 5.1-9.8%, and the recovery of TLI added to two samples of canine serum ranged from 89 to 111 and 93 to 108%, respectively. Good correlation (correlation coefficient, 0.974) occurred between the TLI values obtained by the ELISA method and those by RIA from 56 clinical samples. Serum TLI values in clinically healthy dogs ranged from 7.8 to 29.2 ng/ml by ELISA, and those from dogs with EPI were 0.0-0.6 ng/ml. The values were 0.0-287.4 ng/ml for dogs with pancreatitis, and those from dogs with gastrointestinal disease were 5.5-58.9 ng/ml. The only statistically significant difference (P<0.01) occurred between the TLI level of healthy dogs and those with EPI. The ICT kit showed high reproducibility, and the TLI values yielding negative results differed significantly (P<0.01) from those returning positive results. The ICT kit yielded negative results (indicating EPI) from clinical serum samples with TLI concentrations of 0.0-4.1 ng/ml by ELISA. Both the ELISA and ICT kit are useful tools in the diagnosis of canine EPI.     

A preliminary study on the use of enzyme-linked immunosorbent assay (ELISA) for detection of Trichinella spiralis infections in dogs

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Trichinella spiralis infections in dogs using rabbit anti-canine IgG-horseradish peroxidase prepared according to the improved periodate method and an antigen purified from T. spiralis larvae by Sephadex G-200 chromatography. Sixty-six canine sera were tested for trichinosis by the ELISA and it showed a detection rate which was significantly higher than that by trichinoscopy. This antigen of T. spiralis appeared not to cross-react with the sera of dogs infected with Ancylostoma caninum or Taenia spp. A comparison of ortho-phenylenediamine and 5-amino-2-hydroxybenzoic acid as substrates in the ELISA did not reveal a significant difference. Pieces of filter paper saturated with a defined quantity of whole blood can be substituted for serum as a source material for the test. The relationship between worm burden and the absorbance value in ELISA is discussed.