An improved ferritin assay for canine sera

An improved serum ferritin assay for canine serum has been developed. It uses two monoclonal antibodies in a sandwich arrangement. Serum ferritin can be determined on undiluted canine sera with this assay. The recovery of ferritin added to canine serum ranged from 98 to 106%, the within-assay coefficient of variability was 3.3 to 4.5%, and the assay-to-assay variability was 9.8 to 10.2%. Serum ferritin from 61 apparently healthy dogs had a geometric mean of 252 ng/ml, with a range of 80 ng/ml to 800 ng/ml.     

Enzyme-linked immunosorbent assay to quantitate serum ferritin in black and white ruffed lemurs (Varecia variegata variegata).

Lemurs in captivity progressively accumulate iron deposits in a variety of organs (hemosiderosis) including duodenum, liver, and spleen throughout their lives. When excessive, the toxic effects of intracellular iron on parenchymal cells, particularly the liver, can result in clinical disease and death. The pathogenesis of excessive iron storage in these species has been attributed to dietary factors related to diets commonly fed in captivity. Tissue iron stores can be directly estimated by tissue biopsy and histologic examination, or quantitated by chemical analysis of biopsy tissue, However, expense and risk associated with anesthesia and surgery prevent routine use of tissue biopsy to assess iron status. A noninvasive means of assessing total body iron stores is needed to monitor iron stores in lemurs to determine whether dietary modification is preventing excessive iron deposition, and to monitor potential therapies such as phlebotomy or chelation. Serum ferritin concentration correlates with tissue iron stores in humans, horses, calves, dogs, cats, and pigs. Serum ferritin is considered the best serum analyte to predict total body iron stores in these species and is more reliable than serum iron or total iron binding capacity, both of which may be affected by disorders unrelated to iron adequacy or excess including hypoproteinemia, chronic infection, hemolytic anemia, hypothyroidism, renal disease, and drug administration. We have developed an enzyme-linked immunosorbent assay to measure serum ferritin in lemurs. The assay uses polyclonal rabbit anti-human ferritin antibodies in a sandwich arrangement. Ferritin isolated from liver and spleen of a black and white ruffed lemur (Varecia variegata variegata) was used as a standard. Ferritin standards were linear from 0 to 50 microg/L. Recovery of purified ferritin from lemur serum varied from 95% to 110%. The within-assay variability was 4.5%, and the assay-to-assay variability for three different samples ranged from 10% to 17%. The assay also measures serum ferritin in several other lemur species.     

An enzyme-linked immunosorbent assay (ELISA) for von Willebrand factor antigen (vWf-Ag) in canine plasma.

A quantitative enzyme-linked immunosorbent assay (ELISA) has been developed to measure canine von Willebrand factor antigen (vWf-Ag) in plasma of the dog. A vWf-Ag antiserum was raised in rabbits and purified by preabsorption with the low molecular weight vWf-Ag-deficient fraction of canine cryoprecipitate, followed by affinity chromatography on protein-A Sepharose. The rabbit anticanine vWf-Ag IgG was used to bind the vWf-Ag of the test plasmas to the solid phase and to prepare the enzyme-antibody conjugate in ELISA. Normal rat serum was used as blocking agent. The standard curve was linear (r2 greater than 0.98) and reproducible after logit-log transformation. The interassay coefficient of variation (CV) in test plasmas with various vWf-Ag concentrations was never greater than 7.7%. Assayed values in dilutions of pooled normal canine plasma added to canine vWf-Ag-deficient plasma were linear between 0 and 100% (r2 = 0.99) and indicated excellent analytical recovery of vWf-Ag. In 18 dogs with various internal diseases, including von Willebrand's disease and haemophilia A, the coefficient of correlation between the results of the ELISA and those of electroimmunodiffusion (EID) was 0.93.     

Iron content of rat serum ferritin.

The serum ferritin concentration was significantly higher in female than in male rats, reflecting higher iron stores in females than in males. The mean iron/protein ratio of serum ferritin was 0.018+/-0.008 (SD) (microg of Fe/microg of protein) in female rats and 0.011+/-0.011 in male rats, being much lower than that of liver ferritin (0.233+/-0.014 in females and 0.227+/-0.020 in males). Iron loading of rats significantly increased serum ferritin concentration, but did not influence the iron content of serum ferritin. These results indicate that rat serum ferritin contains only a small amount of iron independent of body iron stores.     

Survey and clinical application of serum iron, total iron binding capacity, transferrin saturation, and serum ferritin in captive black and white ruffed lemurs (Varecia variegata variegata).

Serum samples from 63 clinically normal captive black and white ruffed lemurs (Varecia variegata variegata) were analyzed to survey serum iron, total iron binding capacity, transferrin saturation, and serum ferritin levels. Data analysis showed no differences in these analytes attributable to sex, but significantly higher levels of serum iron, transferrin saturation, and serum ferritin in older animals. The survey data were examined in light of two black and white ruffed lemurs that were treated for iron overload with serial phlebotomies. Prior to therapy, both phlebotomized lemurs had excess hepatic iron deposition, but had serum iron, transferrin saturation, and serum ferritin below the upper limits observed in the survey animals, suggesting that some clinically normal animals included in the survey may have accumulated excess systemic iron. Serial phlebotomy therapy reduced serum iron, transferrin saturation, and serum ferritin in both animals. Three years after the conclusion of therapy in the one remaining case, serum iron and transferrin saturation have risen substantially, whereas serum ferritin has risen slightly. Serum iron, transferrin saturation, and serum ferritin may be useful predictors of systemic iron stores in this species, though the correlation between these parameters and systemic iron stores needs to be determined.     

Validation of human haptoglobin immunoturbidimetric assay for detection of haptoglobin in equine and canine serum and plasma

The Incstar(R) SPQ II human haptoglobin (Hpt) (Incstar Corporation, Stillwater, MN) immunoturbidimetric assay was validated for the determination of serum and plasma Hpt concentrations in dogs and horses. The anti-human Hpt antiserum supplied with the assay, displayed monospecificity to both dog and horse serum Hpt by immunoelectrophoresis and Western blotting techniques. The automated immunoturbidimetric assay results correlated well with the cyanmethemoglobin binding assay (r=0.953 for canine serum and r=0.941 for equine serum), and had excellent precision at both high and low serum Hpt concentrations (within run and between run coefficients of variation near or less than 5%). The assay was linear in both species by serial dilution of pooled-high serum with pooled-low serum, saline and with Hpt-free serum. Interference from hemolysis (> 25 mg/dl hemoglobin) and lipemia greater than 100 mg/dl caused a false decrease and false increase respectively in Hpt yield with the immunoturbidimetric assay. The anti-Hpt antibody supplied with the assay kit, once diluted with polymer diluent and stored at 4 degrees C, was stable for up to 6 days and gave consistent results.     

Measurement of ferritin and anti‐ferritin autoantibodies in serum and colostrum of Holstein and Japanese Black cows

Anti‐ferritin autoantibody is a ferritin‐binding protein commonly found in mammals; it is thought to form an immune complex with ferritin and thereby mediate the rapid clearance of circulating ferritin. The aim of this study is to determine concentrations of ferritin and anti‐ferritin autoantibodies (immunoglobulin (Ig)M, IgG and IgA) in serum and colostrum of Holstein (H) and Japanese Black (JB) cows within 24 h of normal calving. Blood and colostrum samples were collected from cows of various ages (2–11 years) and calving number (1–8 live births). Mean ferritin concentrations were higher in colostrum than in serum for both breeds, and higher colostrum ferritin concentrations were found in H than JB cows. IgA antibodies in serum and colostrum from both breeds had negligible ferritin‐binding activity. For both breeds, IgM and IgG antibodies had higher ferritin‐binding activity in colostrum than in serum. There was a significant correlation between IgM and IgG ferritin‐binding activities in serum and colostrum of H and JB cows. These results suggest that ferritin and IgM and IgG autoantibodies are actively transferred from the blood stream to the colostrum at prepartum or early lactation.     

A statistically standardized muscle cell culture bioassay measuring the effect of swine serum on muscle cell proliferation

We have developed a statistically standardized bioassay for quantifying the effect of swine serum on the proliferation rate of cultured L6 myogenic cells. The intra-assay coefficient of variation for this assay is 2.5%. Over 29 experiments, the relationship between the assays response to 2.5% control swine serum and its response to 2.5% serum plus 10(-7) M insulin is linear (r2 = .9). This relationship can be used to adjust experimental data obtained in different experiment to a common control value so that valid inter-assay comparisons can be made. The inter-assay coefficient of variation is reduced four- to five fold by this adjustment procedure. We believe this standardized assay provides a useful system for identifying and isolating unknown growth factors that affect muscle growth rate in an economically important species.     

Validation of an immunoturbidimetric D-dimer assay in canine citrated plasma

Abstract: D-dimer is a neoantigen formed when thrombin initiates the transformation of fibrinogen to fibrin; it is derived from plasmin digestion of cross-linked fibrin. In human medicine, the usefulness of this analyte in diagnosing disseminated intravascular coagulation (DIC) has been assessed in patients fulfilling the clinical and laboratory requirements for this disorder. In canine medicine, the use of D-dimer is relatively new. Detailed studies are needed to understand the relationship between D-dimer concentration in plasma and DIC status in dogs. We validated a D-dimer immunoturbidimetric assay (Tina-quant [a] D-Dimer, Boehringer Mannheim) in canine citrated plasma samples. Intra-assay and interassay variability (coefficient of variation) was 5.63% and 8.82%, respectively. The assay was linear, using 2 samples with low and high D-dimer concentrations (r = .996 and .998). Accuracy was 102.2% and 95.7% based on a recovery study in which 2 samples were assessed. Reference values for D-dimer were established using 70 healthy dogs that were assessed clinically and evaluated on the basis of a complete laboratory workup. The reference range was set between 0.02 and 0.28 μg/mL (chi-square test for normal distribution, P > .05).     

Cortisol and free thyroxine determination by time-resolved fluorometry in canine serum

Validation for canine serum of 2 commercially available time-resolved fluoroimmunoassays (TR-FIAs) designed for analysis of cortisol and free thyroxine (fT4) in human serum was carried out. Included was the study of interference by hemolysis, lipemia, and bilirubinemia. With the dissociation enhancement lanthanide fluoroimmunoassay kits, the intra-assay coefficient of variation (CV) ranged from 6.4% to 8.7% for cortisol and from 5.3% to 9.8% for fT4; the interassay CVs ranged from 5.8% to 10.8% and from 3.9% to 14.1%, respectively. Accuracy was evaluated by comparing cortisol and fT4 results obtained with TR-FIA and those obtained with a validated enzyme-linked immunosorbent assay (ELISA) and an equilibrium dialysis (ED) assay, respectively. The regression equations obtained were y = 0.57x + 1.18 (r2 = 0.90) for cortisol and y = 0.87x + 0.82 (r2 = 0.93) for fT4. The limits of detection for cortisol and fT4 were 4.84 nmol/L and 2.68 pmol/L, respectively. The results of adrenocorticotropin-stimulation and dexamethasone-suppression tests were similar to those published previously; likewise, serial dilution of a canine serum sample with a high cortisol content demonstrated that the TR-FIA was immunologically specific. Serial dilution of a serum sample with a high fT4 concentration showed a methodologic bias, a dependence on serum binding capacity, which indicates that the results obtained with this method should be interpreted with caution. Finally, hemolysis and lipemia significantly interfered with cortisol and fT4 measurements, whereas bilirubinemia did not affect the results.     

Relationship of serum ferritin and iron concentrations and serum total iron-binding capacity to nonheme iron stores in dogs

The relationships of various iron-related analytes were evaluated in 95 dogs. Liver and spleen nonheme iron content was determined coulometrically on acid-digested tissue specimens. Serum iron concentration and total iron-binding capacity also were measured coulometrically, whereas serum ferritin concentration was measured by ELISA. Significant (P less than 0.0002) correlation was found between serum ferritin concentration and nonheme iron stores. Significant correlation was not found between nonheme iron stores and serum iron concentration or total iron-binding capacity. Serum ferritin concentration should provide a convenient and relatively noninvasive means of estimating iron stores 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.     

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.     

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi‐quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co‐immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co‐immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin‐binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.     

Determination of canine beta2-microgloblin in plasma and urine by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay was developed for the determination of canine beta2-microglobulin (beta2-m) in plasma and urine. The detectable sensitivity for pure canine beta2-m was 0.05 microg/l and the analytical range was 0.1 to 50 microg/l. The mean analytical recovery when pure canine beta2-m was added to normal plasma was 101.9%. The mean analytical recovery in the urine was 102.1%. The intra-day variation coefficient was 3.1% in plasma, 4.3% in serum and 1.9% in urine. No difference was found between the concentration of beta2-m in plasma and serum (n=17). The concentration of beta2-m in the plasma of normal dogs was 1.82 +/- 0.57 mg/l (n=31). The mean excretion in 24 hr urine collected from normal dogs was 17.6 +/- 9.2 microg/l, 0.22 +/- 0.12 microg/kg of body weight or 14.2 +/- 9.4 microg/g of urine creatinine. The beta2-m creatinine index of random urine samples was 23.5 +/- 16.6 microg/g (n=26). There was a close correlation between the beta2-m creatinine index of 24 hr urine samples and that of random urine samples (r=0.872).     

Development of a time-resolved fluorometry based immunoassay for the determination of canine haptoglobin in various body fluids

A time-resolved immunofluorometric assay (TR-IFMA) was developed for the determination of haptoglobin (Hp) in canine serum. Haptoglobin was purified from canine acute phase serum by ammonium sulphate precipitation followed by gel filtration. This isolated dog Hp was used as the standard to calibrate the assay. Intra- and inter-assay coefficients of variation of the assay were, respectively, 5.7% and 16.6% at 0.51 mg/mL, 2.4% and 10.6% at 2.1 mg/mL and 10.5% and 11.9% at 32.5 mg/mL. The dilution of serum samples with high Hp concentrations resulted in linear regression equations with R2 of 0.99 and 0.97. A high correlation was found in serum Hp measurements by TR-IFMA and a commercial assay based on peroxidase activity of haemoglobin bound to haptoglobin (R2 = 0.96). The limit of detection for the TR-IFMA method was 0.002 microg/mL. The addition of fresh haemolysate to serum samples did not affect the haptoglobin concentration (P = 0.694). Statistical differences (P < 0.003) were found between healthy dogs and dogs with different pathological processes. In whole blood, Hp concentrations were much lower than in serum but closely related (R2 = 0.84) whereas saliva Hp concentrations were poorly related with serum concentrations (R2 = 0.53). However, the concentration of Hp in saliva was significantly (P < 0.039) higher in dogs with pathological processes compared to healthy dogs. The assay sensitivity was adequate to also be applied to whole blood and saliva specimens.     

Evaluation of an automated colorimetric assay for the measurement of lipase activity in canine sera.

An automated colorimetric method for determining lipase activity in canine sera was evaluated for precision, linearity and correlation to existing assay methods. The colorimetric method was a commercial reagent that used a series of enzymatic reactions based on the hydrolysis of 1,2 diglyceride by pancreatic lipase. Within-run and between-run coefficients of variation were < 6.8% and < 8.3%, respectively. Linearity was determined to be at least 1366 U/L. Canine serum lipase concentrations attained using the colorimetric method were compared to both titrimetric and dry-film methods for measuring serum lipase activity, resulting in significant (P < or = 0.05) correlation coefficients of 0.92 and 0.77, respectively. Canine serum lipase concentrations measured using the colorimetric assay on 2 different automated analyzers had a significant (P < or = 0.05) correlation coefficient of 0.92. A laboratory reference range using serum samples from 56 healthy dogs (0-561 U/L) was established. There were no significant (P < or = 0.05) differences in mean serum lipase concentrations comparing male and female dogs or comparing young dogs (< or = 3 y) to mature (4-7 y) and older (> 7 y) dogs using this assay. It was concluded that the automated colorimetric assay was a reliable indicator of canine serum lipase activity and offered several advantages, including small sample volume and short analysis time.     

Quantitative determination of haptoglobin (HAP) in human and bovine sera by capillary zone electrophoresis (CZE).

This study describes an original assay for serum haptoglobin determination by measuring the capacity of human haptoglobin (hHAP) and bovine haptoglobin (bHAP) to bind haemoglobin (Hb) as established by capillary zone electrophoresis (CZE). This method involves the addition of Hb in excess to the serum and the separation of the HAP-Hb complexes from free haemoglobin by CZE. Protein migration was recorded at a wavelength of 415 nm which reveals Hb alone (free or bound), and the concentration of HAP was indirectly estimated by measuring bound Hb. Different CZE conditions and the peak migration time of Hb from various species (human, equine, bovine, canine) were investigated. The electrophoretic separation of free human Hb (hHb) in excess and the hHAP-hHb complex was fully achieved by CZE, allowing a quantitative determination of hHAP. However, bovine haemoglobin (bHb) bound to bHAP and free bHb were poorly separated under the same conditions. The best detachment between bHAP-Hb complexes and free Hb was only attained in the bovine sample by use of canine haemoglobin (cHb). CZE assays performed with cHb gave very close values to those of a classic photometric method which measured the peroxidase activity of the haptoglobin-cyanmethaemoglobin complexes (y = 1.0168x - 0.072; r2 = 0.97). CZE assay was fast (< 10 min), inexpensive, did not require the use of a specific antibody and was reproducible (coefficient of variation, CV 3.6%).