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.     

Development and analytic validation of an enzyme-linked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in serum

Recently, a radioimmunoassay (RIA) for measurement of canine pancreatic lipase immunoreactivity (cPLI) in serum was developed and validated. However, RIAs require frequent use of radioactive materials. Therefore, the goal of this project was to develop and validate an enzyme-linked immunosorbent assay (ELISA) for cPLI. After purifying cPL, we developed and purified antiserum against cPL in rabbits. The purified antibody was bound to microtitre plates and used to capture antigen. A portion of the purified antibody was biotinylated and used to identify the captured antigen. Streptavidin labelled with horseradish peroxidase and a horseradish peroxidase substrate were used for detection. The assay was validated by determination of sensitivity, working range, linearity, accuracy, precision, and reproducibility. The reference interval for serum cPLI was determined by the central 95th percentile in 74 clinically healthy dogs: 2.2 to 102.1 μg/L. The sensitivity and the upper limit of the working range were 0.1 and 999.2 μg/L, respectively. The ratios of observed to expected values for dilutional parallelism for 6 serum samples ranged from 0.0 to 148.8%; the ratios for spiking recovery for 4 serum samples ranged from 90.4 to 112.6%, assuming 55% recovery of the cPL. Coefficients of variation for intra- and interassay variability for 6 different serum samples were 2.4, 3.4, 4.1, 5.8, 7.4, and 10.0% and 5.9, 7.7, 11.6, 13.9, 23.5, and 46.2%, respectively. We conclude that the ELISA described here is sufficiently sensitive, linear, accurate, precise, and reproducible for clinical application. Evaluation of its clinical usefulness for the diagnosis of exocrine pancreatic disorders in dogs is under way.     

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.     

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.     

Evaluation of serum feline pancreatic lipase immunoreactivity and helical computed tomography versus conventional testing for the diagnosis of feline pancreatitis

Serum feline trypsinogen-like immunoreactivity (fTLI) concentrations and abdominal ultrasound have facilitated the noninvasive diagnosis of pancreatitis in cats, but low sensitivities (33% and 20–35%, respectively) have been reported. A radioimmunoassay has been validated to measure feline pancreatic lipase immunoreactivity (fPLI), but the assay's sensitivity and specificity have not been established. In human beings, the sensitivity of computed tomography (CT) is high (75–90%), but in a study of 10 cats, only 2 had CT changes suggestive of pancreatitis. We prospectively evaluated these diagnostic tests in cats with and without pancreatitis. In all cats, serum was obtained for fTLI and fPLI concentrations, and pancreatic ultrasound images and biopsies were acquired. Serum fPLI concentrations ( P <.0001) and ultrasound findings ( P = .0073) were significantly different between healthy cats and cats with pancreatitis. Serum fTLI concentrations ( P = .15) and CT measurements ( P = .18) were not significantly different between the groups. The sensitivity of fTLI in cats with moderate to severe pancreatitis was 80%, and the specificity in healthy cats was 75%. Feline PLI concentrations were both sensitive in cats with moderate to severe pancreatitis (100%) and specific in the healthy cats (100%). Abdominal ultrasound was both sensitive in cats with moderate to severe pancreatitis (80%) and specific in healthy cats (88%). The high sensitivities of fPLI and abdominal ultrasound suggest that these tests should play an important role in the noninvasive diagnosis of feline pancreatitis. As suggested by a previous study, pancreatic CT is not a useful diagnostic test for feline pancreatitis.     

Correlation between metabolomic profile constituents and feline pancreatic lipase immunoreactivity

BackgroundFeline pancreatic lipase immunoreactivity (fPLI) is commonly used to diagnose pancreatitis in cats (FP). Untargeted metabolomics has been extensively applied in human and veterinary medicine, but no metabolomic studies regarding FP have been conducted.ObjectivesTo identify metabolites significantly associated with increased fPLI.AnimalsForty‐nine client‐owned cats: 11 clinically healthy and 38 with various clinical conditions.MethodsAnalytical cross‐sectional study with convenience sampling. A panel of 630 metabolites belonging to 26 biochemical classes was quantified in plasma using a commercial metabolomic assay. The correlation between plasma metabolite concentrations and serum fPLI was evaluated using Spearman''s rank correlation coefficient (R _s) with Bonferroni correction. Multivariable analysis then was performed to control for glomerular filtration rate, liver damage, and blood glucose concentration. The accuracy of selected metabolites in discriminating between cats with normal (≤3.5 μg/L) and increased (>5.3 μg/L) fPLI was estimated using the area under the receiver operating characteristic curve (AUROC).ResultsFour hundred and seven of 630 metabolites (64.6%) were quantified in all cats. When controlled for potential confounders only 3 sphingolipids were significantly positively correlated with fPLI: 2 cerebrosides: HexCer(d18:1/24:0); (R _s = .56), and HexCer(d18:1/24:1); (R _s = .58) and 1 sphingomyelin: SM C18:0 (R _s = .55). Their AUROCs in identifying cats with increased fPLI were 82% (95% confidence interval [CI 95%], 70%‐94%), 84% (CI 95%, 72%‐96%), and 78% (CI 95%, 65%‐92%), respectively.Conclusions and Clinical ImportanceSelected sphingolipids are moderately positively correlated with fPLI and appear to have fair to moderate diagnostic accuracy in discriminating between cats with normal and increased fPLI.     

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.     

Sensitivity and specificity of radioimmunoassay of serum trypsin-like immunoreactivity for the diagnosis of canine exocrine pancreatic insufficiency

Concentrations of serum trypsin-like immunoreactivity (TLI) measured by radioimmunoassay were low (less than 1.9 micrograms/L) in 25 dogs with exocrine pancreatic insufficiency (EPI), compared with 100 clinically normal (control) dogs (5.2 to 34.0 micrograms/L; P less than 0.001; sensitivity, 100%). Serum TLI concentrations (5.5 to 35.0 micrograms/L) in a group of 50 dogs with small intestinal disease (SID) were not significantly different from those of control dogs, values being greater than the lower limit of the control range in all cases (specificity, 100%). Results of bentiromide (N-benzoyl-L-tyrosyl-p-aminobenzoic acid [BT-PABA]) tests and fecal proteolytic activity (determined by use of an azocasein substrate) were abnormal in 21 of 22 dogs with EPI (sensitivity, 95%). Bentiromide test results were subnormal in 13 of 35 dogs with SID (specificity, 63%), whereas fecal proteolytic activity was subnormal in 7 of 34 dogs with SID (specificity, 79%). It was concluded that assay of serum TLI is a highly sensitive and specific test for the identification of dogs with EPI.     

Serum lipase activities and pancreatic lipase immunoreactivity concentrations in dogs with exocrine pancreatic insufficiency

OBJECTIVE: To determine serum lipase activities and pancreatic lipase immunoreactivity (PLI) concentrations in dogs with exocrine pancreatic insufficiency (EPI). ANIMALS: 74 healthy dogs and 25 dogs with EPI. PROCEDURES: A diagnosis of EPI was made on the basis of clinical signs, low serum trypsin like immunoreactivity (TLI) concentration, and response to treatment with enzyme replacement. Median values for fasting serum lipase activity and serum PLI concentrations were compared between the 2 groups with a Mann-Whitney U test. RESULTS: Median fasting serum lipase activity was not significantly different between dogs with EPI (366.0 U/L) and healthy dogs (294.5 U/L), and only 1 dog with EPI had a serum lipase activity less than the lower limit of the reference range. Median serum PLI concentration was significantly lower in dogs with EPI (0.1 microg/L) than in healthy dogs (16.3 microg/L). All dogs with EPI had serum PLI concentrations less than the lower limit of the reference range. CONCLUSION AND CLINICAL RELEVANCE: Serum lipase activity is not limited to the exocrine pancreas in origin, whereas serum PLI is derived only from the exocrine pancreas. Unlike in serum TLI concentrations, there was a small degree of overlap in serum PLI concentrations between healthy dogs and dogs with EPI. Serum TLI concentration remains the test of choice for diagnosis of EPI.     

Patient-side assay of lipase activity correlating with pancreatic lipase immunoreactivity in the dog

Pancreatitis is a common exocrine pancreatic disease in dogs, and the pancreatic lipase immunoreactivity (PLI) test is used for diagnosis. Enzyme catalytic assay is thought to have low specificity, but a lipase activity assay with increased specificity has been developed in human clinical chemistry. We measured serum lipase activity of 65 client-owned dogs using the newly developed FUJI DRI-CHEM slide and compared the results with their PLI concentrations. The results showed a good correlation (r = 0.91), and the normal and pancreatitis dogs identified based on the PLI values were correctly separated based on lipase activity. The present study suggests that FUJI DRI-CHEM lipase activity would be helpful for diagnosis of pacreatitis in dogs and, in particular, that it can be used as a patient-side assay and contributes to immediate treatment.     

Development and validation of a radioimmunoassay for thyrotropin in cattle.

In mammals, thyrotropin, or thyroid-stimulating hormone (TSH), assay is used for the diagnosis of primary hypothyroidism. Hypothyroidism is the most common type of thyroid disorder in cattle. The aim of this study was to develop and validate, under physiologic and pathologic conditions, a radioimmunoassay (RIA) for bovine TSH (bTSH). Double RIA was performed with purified bTSH and specific bovine antiserum. Laboratory validation included research of minimal detection limit, accuracy, and reproducibility. The physiologic validation included a thyrotropin-releasing hormone (TRH) challenge performed on euthyroid cows and a follow-up of bTSH concentration over a 24-hour period. Furthermore, bTSH concentration was assayed in a large population of healthy dairy and beef cows to define reference interval. The pathologic validation was made by assaying bTSH and thyroid hormones on healthy and goitrous newborn calves. The minimum detection limit (MDL) for bTSH assay was 1.3 microU/ml. The recovery was 101% to 106%. The intra- and interassay coefficients of variation (CVs) ranged from 5% to 11% and 11% to 15%, respectively. The RIA covered the whole range of physiologic bTSH values, as shown by bTSH values induced by TRH-challenge. A pulsatile secretion of bTSH was observed, accompanied by a diurnal variation with lower night values than day values. Reference intervals of bTSH ranged from 1.3 to 13.0 microU/ml for beef and dairy breeds. Finally, bTSH easily discriminated goitrous newborn calves from healthy ones, leading to the definition of a cutoff value of 35 microU/ml. The bTSH assay positively reacted to physiologic and pathologic conditions. The accuracy and precision of the RIA were satisfying.     

Development and analytic validation of a radioimmunoassay for the quantification of canine calprotectin in serum and feces from dogs

OBJECTIVE: To develop and analytically validate a radioimmunoassay (RIA) for the quantification of canine calprotectin (cCP) in serum and fecal extracts of dogs. Sample Population-Serum samples (n = 50) and fecal samples (30) were obtained from healthy dogs of various breeds and ages. PROCEDURES: A competitive, liquid-phase, double-antibody RIA was developed and analytically validated by assessing analytic sensitivity, working range, linearity, accuracy, precision, and reproducibility. Reference intervals for serum and fecal cCP concentrations were determined. RESULTS: Sensitivity and upper limit of the working range were 29 and 12,774 microg/L for serum and 2.9 and 1,277.4 microg/g for fecal extracts, respectively. Observed-to-expected ratios for serial dilutions of 6 serum samples and 6 fecal extracts ranged from 95.3% to 138.2% and from 80.9% to 118.1%, respectively. Observed-to-expected ratios for spiking recovery for 6 serum samples and 6 fecal extracts ranged from 84.6% to 121.5% and from 80.3% to 132.1%, respectively. Coefficients of variation for intra-assay and interassay variability were < 3.9% and < 8.7% for 6 serum samples and < 8.5% and < 12.6% for 6 fecal extracts, respectively. Reference intervals were 92 to 1,121 microg of cCP/L for serum and < 2.9 to 137.5 microg of cCP/g for fecal extracts. CONCLUSIONS AND CLINICAL RELEVANCE: The RIA described here was analytically sensitive, linear, accurate, precise, and reproducible for the quantification of cCP in serum and fecal extracts. This assay should facilitate research into the clinical use of serum and fecal cCP measurements in dogs with inflammatory bowel disease.     

Development and validation of a specific radioimmunoassay for equine osteocalcin

This study describes for the first time the development and validation of a sensitive and specific radioimmunoassay (RIA) for equine osteocalcin (OC) quantification using purified equine OC as standard, tracer, and immunogen for antibody formation in rabbits. The assay allowed to measure equine serum OC levels with a sensitivity of 0.2 ng/mL. Immunoreactive serum OC values of clinically normal, different-aged horses ranged from 3.68 to 127.31 ng/mL. Intra- and inter-assay coefficients of variation (CV) were 6.2 and 8.2%, respectively. Serial equine serum sample dilutions were linear. The recovery of equine OC from equine serum samples ranged from 93.88 to 107.9%. There was a tight correlation between OC values measured with the equine-specific OC RIA and two commercially available bovine-specific OC RIA kits. However, highest serum OC values were obtained with the equine-specific OC RIA. In conclusion, our equine-specific OC RIA is sensitive, linear, accurate, precise, and reproducible. The assay allowed to quantify OC in equine serum samples and might, therefore, be used to monitor equine osteoblast activity associated with bone diseases, exercise, therapy forms or diet.     

Validation of a radioimmunoassay for measurement of gastrin in equine serum

A commercial radioimmunoassay kit designed for measuring gastrin in human serum was validated for use with equine serum. This nonextraction, double-antibody procedure uses an antiserum with broad specificity for molecular forms of gastrin. Synthetic human gastrin (G17-I) was added to pooled equine serum, and the observed assay values were compared with the mass added. Recovery was 99 to 115% in the gastrin concentration range of 40 to 640 pg/ml. Dilutions of postprandial serum with serum from fasted horses were assayed, and the inhibition curves were compared with those of the human gastrin kit standards, using a log-logit transformation. The slopes of the sample dilution plots were not significantly different from the slopes of the standard curves. Ethylenediamine tetraacetate and heparin adversely affected the assay, resulting in lower assayed gastrin concentration values. The intra-assay coefficient of variation (n = 10) was 3.8%, and the interassay coefficient of variation (n = 6) was 11.2%. The assay sensitivity, as reported by the manufacturer, is 8 pg/ml. Gastrin concentrations in serum from fasted horses ranged from undetectable values (less than 8 pg/ml) to 17.5 pg/ml, and peaked at a mean value (n = 6) of 70 pg/ml 3 hours after feeding. Serum cortisol values monitored during the postprandial blood collection period were in the normal range for horses.     

Evaluation of serum feline trypsin-like immunoreactivity for the diagnosis of pancreatitis in cats

OBJECTIVE: To evaluate serum feline trypsin-like immunoreactivity (fTLI) concentration and results of abdominal ultrasonography, CBC, and serum biochemical analyses for diagnosis of pancreatitis in cats. DESIGN: Prospective study. ANIMALS: 28 cats with clinical signs compatible with pancreatitis. PROCEDURE: Serum fTLI concentrations were determined, and abdominal ultrasonography, CBC, and serum biochemical analyses were performed prior to histologic evaluation of pancreatic, hepatic, and intestinal specimens. On the basis of histologic results, cats were categorized as having a normal pancreas (n = 10), pancreatic fibrosis with ongoing inflammation (9), pancreatic fibrosis without inflammation (4), and acute necrotizing pancreatitis (5). Serum fTLI concentrations and results of CBC, serum biochemical analyses, and histologic evaluation of hepatic and intestinal specimens were compared among groups. RESULTS: Significant differences in serum fTLI concentrations or any hematologic or biochemical variable were not detected among the 4 groups of cats. Median serum fTLI concentrations were 51 micrograms/L (range, 18 to 200 micrograms/L) in cats with a normal pancreas, 32 micrograms/L (range, 12 to > 200 micrograms/L) in cats with pancreatic fibrosis and ongoing inflammation, 124 micrograms/L (range, 36 to > 200 micrograms/L) in cats with pancreatic fibrosis without ongoing inflammation, and 30 micrograms/L (range, 24 to 84 micrograms/L) in cats with acute necrotizing pancreatitis. We detected a high prevalence of concurrent hepatic and intestinal tract disease in cats with pancreatitis. CONCLUSIONS AND CLINICAL RELEVANCE: In cats with clinical signs of pancreatitis, serum fTLI concentration is poorly associated with histopathologic diagnosis.     

Serum feline trypsin-like immunoreactivity in cats with exocrine pancreatic insufficiency

Exocrine pancreatic insufficiency is thought to occur rarely in cats. This assumption has been made based on the lack of a specific test for this disease in the cat. Clinical data from the 1st 20 cats with serum feline trypsin-like immunoreactivity (fTLI) concentrations < or = 8 microg/L are presented. In 17 of these 20 cats compelling evidence for a diagnosis of exocrine pancreatic insufficiency (EPI) was present and in the remaining 3 supportive evidence for a diagnosis of EPI was available. The conclusion was made that serum fTLI concentration is a specific test for EPI in the cat.     

Feline pancreatic ducts are consistently identified on CT and more likely to be dilated in the body of pancreas in cats with elevated feline pancreatic lipase immunoreactivity

Feline pancreatitis is a challenge to diagnose and no previously published study has described the CT characteristics of the pancreatic duct (PD) in cats. The current prospective analytical study was performed to identify and describe the CT characteristics of the PD in normal cats and to compare that to those cats with an elevated feline pancreatic lipase immunoreactivity (fPLI). Contrast‐enhanced CT was performed in 16 normal cats and 13 cats with an elevated fPLI. Two ACVR‐certified radiologists blinded to the fPLI status assessed whether or not the PD could be identified, contrast phase during which the PD was most conspicuous, and PD shape in the body, right and left lobes. A second‐year radiology resident blinded to the fPLI status measured maximum PD diameter and PD:parenchyma. The PD was identified in 84 of 87 pancreatic segments, which was most conspicuous in the portal phase in 28 of 29 cats. The PD shape was tubular (48/84), tapered (34/84), or beaded (2/84) with no significant difference (P = 1.0 to .1615) between groups. Mean maximal PD diameters of normal cats were 1.5‐1.7 mm, which was significantly larger in the body of the pancreas in cats with an elevated fPLI (2.4 mm, P = .0313). Mean PD:parenchyma was not significantly different between groups (P = .2001 to .949). In conclusion, the feline PD can be consistently identified on CT, for which the portal phase is preferred. Cats with an elevated fPLI are more likely to exhibit dilation of the PD in the body of the pancreas on CT.