Development and validation of an improved enzyme-linked immunosorbent assay for the detection of thyroglobulin autoantibodies in canine serum samples

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 15(6):525–536, 1998

DEVELOPMENT AND VALIDATION OF AN IMPROVED ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF THYROGLOBULIN AUTOANTIBODIES IN CANINE SERUM SAMPLES L. Iversen,1,5 A.L. Jensen,2 R. Høier,2 M. Skydsgaard,4 and F. Kristensen3 1

Central Laboratory, 2Section of Reproduction, 3Small Animal Hospital, Department of Clinical Studies, The Royal Veterinary and Agricultural University, Bhlowsvej 13, DK-1870 Frederiksberg C, Denmark, and 4 Scantox, Hestehavevej 36A, Ejby, DK-4623 Lille Skensved, Denmark Received January 31, 1998 Accepted June 29, 1998

An enzyme-linked immunosorbent assay to detect thyroglobulin autoantibodies (TGAB) in canine serum was developed and validated. The test result for each sample was derived from the optical density readings (OD) and expressed as an Ab-score(%) calculated from three in-house calibrators. The assay specifically detected TGAB as judged from lack of response in the assay after samples had been incubated with specific antigen. Intra- and interassay coefficients of variation ranged from 2.0 – 4.9% and 4.6 –9.9%, respectively. The detection limit, an Ab-score of 5.6%, was close to the median Ab-score of 10% observed in healthy dogs (n 5 132). The median Ab-score of dogs with primary hypothyroidism and lymphocytic thyroiditis (n 5 11), skin diseases (n 5 35), and non-thyroidal diseases (n 5 63) was 340%, 12%, and 8%, respectively. The prevalence of TGAB in hypothyroid dogs with lymphocytic thyroiditis (sensitivity) was 91% (95% confidence limits: 59%–99%). In dogs with dermatological diseases without lymphocytic thyroiditis the prevalence of TGAB was 3% corresponding to a specificity of 97% (95% confidence limit: 85%–100%). In dogs with non-thyroidal diseases and healthy dogs the prevalence of TGAB was 5% and 6%, respectively. The diagnostic accuracy of serum TGAB was evaluated by subjecting the data from 11 dogs with lymphocytic thyroiditis and 35 control dogs without lymphocytic thyroiditis to receiver-operating characteristic curve analysis. The area under the receiver-operating characteristic curve (W 5 0.966; 95% confidence limit 87%–100%) was significantly higher than that of a worthless test (0.5) (P , 0.0001), thereby indicating that serum TGAB measurements distinguished between dogs with and without lymphocytic thyroiditis. © Elsevier Science Inc. 1998

INTRODUCTION Primary hypothyroidism is a common endocrinopathy in dogs (1), and it is often caused by lymphocytic thyroiditis (2). Lymphocytic thyroiditis is considered to be an immunemediated disease attributed to its clinical and histologic similarities to Hashimoto’s thyroiditis in man (3,4) and because autoantibodies to thyroglobulin (TG), the major thyroid colloid glycoiodo-protein, have been detected in both humans (5) and dogs with hypothyroidism (4,6,7). Thus, detection of thyroglobulin autoantibodies (TGAB) may serve not only as an aid in the diagnosis of hypothyroidism caused by lymphocytic thyroiditis in dogs, but it has also been proposed as a screening marker for the development of lymphocytic thyroiditis and hypothyroidism in healthy dogs (7). The exact correlation between the presence of TGAB in serum samples and lymphocytic thyroiditis depicted histologically, and the value of serum TGAB as a marker of eventual develop© Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010

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ment of hypothyroidism is not known (8). The present report communicates the development and validation of an improved assay for the detection of TGAB in canine serum samples, and evaluates the usefulness of serum TGAB measurements as a marker of lymphocytic thyroiditis. MATERIALS AND METHODS Reagents. Bovine serum albumin (BSA) came from UCB Bioproducts (Brane-l 5 Alleud, Belgium). Skimmed milk powder was purchased from IRMA A/S (Rødovre, Denmark). The horseradish peroxidase-conjugated rabbit anti-canine IgG, thimerosal (sodium ethylmercurythiosalicylat), phenylmethylsulfonyl flourid (PMSF), leupeptin, Lthyroxine (T4), triiodothyronine (T3), and 3,39,5,59-tetramethylbenzidine (TMB) were from Sigma Chemical Company (St. Louis, MO, USA). 2-amino-2-(hydroxymethyl)-1,3propanediol (TRIS), TWEENt 20 (polyoxyethylenesorbitan monolaurat) and polyethylenglycol 6000 (PEG) were from Merck (Darmstadt, Germany). Other chemicals were all of at least analytical grade. Purification and Characterization of Thyroglobulin. Canine thyroglobulin was purified essentially as recommended for the purification of human thyroglobulin reference material (9) with some modifications. Thus, thyroid gland tissue was obtained from three healthy dogs immediately after euthanasia. The entire glands apart from a sample of the caudal part of the left thyroid lobe that was submitted for histologic examination, was stored at 280°C until purification. These thyroid gland specimens showed normal architecture of the follicles with no signs of inflammatory infiltration. All purification steps were performed at 4°C unless otherwise indicated. After thawing, the thyroid glands were placed in 3 ml per gram of thyroid glands in phosphate-buffered saline (PBS) (40 mM sodium phosphate, 150 mM sodium cloride, pH 7.4) to which protease inhibitors (PI) (5 mM ethylendiamin-tetraacetate, 1 mM PMSF, and leupeptin 0.2 g/liter) were added. The thyroid glands were homogenized, the suspension was filtered through sterile gaze and centrifuged at 1,500 3 g for 5 min. The lipid-free supernatant was then ultracentrifuged at 100,000 3 g for 1 hr using a Beckmann L-70 centrifuge, and the supernatant was isolated. An equal volume of 3.5 M ammonium sulfate in PBS/PI adjusted to pH 7.1 was added dropwise to the supernatant. After stirring for 2 hr, the suspension was centrifuged at 30,000 3 g for 1 hr, and the supernatant was discarded. The pellet was resuspended in 10 ml of PBS/PI and dialyzed overnight against twice 1L of PBS/PI. After sterile filtration (0.45 mm) to remove proteins not brought into solution, the thyroid protein was further purified by gel filtration at 4°C. Five ml of thyroid protein solution were added to a Hi-Prep Sephacryl S-300 HR (Pharmacia, Uppsala, Sweden), precalibrated for 48 hr with PBS. The main peak was collected using Gradifract automated collection system (Pharmacia, Uppsala, Sweden). A buffer exchange to a 25-mM ammoniumhydrogen carbonate buffer pH 6.4 was achieved by ultrafiltration. The protein content was 5.6 mg/ml as measured by ultraviolet (UV) absorbance using gammaglobulin as reference (10). After sterile filtration (0.2 mm), fractions of 40 ml were dispensed to Nunc Cryotubes (Life Technologies, Roskilde, Denmark), lyophilized overnight and stored at 253°C. The lyophilized protein, which contained 0.18% (w/w) iodine (11), was checked for purity by sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Molecular weight determination and purity check by SDS-PAGE was performed in a 5–20% gradient gel using a Protean cell (Biorad Laboratories, Copenhagen, Denmark). A panel of molecular-weight markers (HMW Electrophoresis Marker, Pharmacia, Uppsala, Sweden) was included in the run and after protein staining with Coomassie Brillant Blue R,

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a single band with an electrophoretic mobility identical to the purified porcine thyroglobulin marker with a molecular weight of 668-kDa under non-reducing conditions was identified. Canine and porcine thyroglobulins have previously been shown to have identical electrophoretical mobility in SDS-PAGE (12). After reduction of protein with 5 mM dithiotreitol, two major bands, one at 668-kDa and one at 330-kDa, corresponding to the subunits of canine TG (14) together with minor degradation products, were identified. ELISA Methodology. Preliminary experiments revealed that use of BSA as a blocking agent and carrier protein resulted in very high nonspecific binding (NSB). To overcome this problem, BSA was replaced by skimmed milk powder. Microtiter plates (NUNC Maxisorpt, Life Technologies, Roskilde, Denmark) were coated with 100 ml of lyophilized canine TG diluted to 10 mg/ml in a buffer containing 50 mM TRIS, NaCl 50 mM and thimerosal 0.001%, pH 7.4 (TBS). After incubation for 2 hr at 37°C, plates were washed three times with 350 ml 0.05% TWEEN using an automated microplate washer (Labsystems, Helsinki, Finland). Vacant binding sites were blocked for 45 min with 300 ml of 5% skimmed milk powder in TBS at room temperature. After washing five times with 350 ml of 0.05% TWEEN, 110 ml of serum samples, calibrators, and controls, in duplicates or quadruplicates, in a 100-fold dilution in TBS with 1% skimmed milk powder, were incubated for 90 min at 600 RPM on an orbital microplate shaker. Wells were washed five times with 350 ml of 0.05% TWEEN and subsequently incubated together with 120 ml horseradish peroxidase-conjugated anticanine-IgG-antibody in a 1:12000 dilution in TBS with 1% skimmed milk powder for 1 hr on rotation at room temperature. After washing six times with 350 ml 0.05% TWEEN, 150 ml of a phosphate/citrate buffer pH 5.5 containing 0.3 mM TMB and 50 ppm (W/W) hydrogenperoxide were added to each well and incubated at room temperature for 30 min. The enzyme-mediated reaction was stopped by adding 50 ml 2 M sulphuric acid to the wells. Color development was quantified using a microplate reader detecting the optical density (OD) at 450 and 620 nm (Labsystems, Helsinki, Finland) and commercial software (Genesis, Hampshire, UK). Test Results and NSB. In each run, serum from the same hypothyroid dog (Calibrator 1) with lymphocytic thyroiditis and the same two healthy dogs (Calibrator 2 and 3) were used as calibrators. The two healthy dogs were physically, hematologically, and biochemically healthy (i.e., normal routine hematologic and biochemical parameters, including normal serum total thyroxine (TT4), and thyrotropin (cTSH) concentrations) and had histologically normal thyroid glands. The hypothyroid dog was clinically and biochemically hypothyroid (low TT42, elevated cTSH3, low fT44 analyzed by equilibrium dialysis, and insufficient response in a TSH stimulation test) and it had histologically documented primary hypothyroidism attributable to lymphocytic thyroiditis. All three calibrators were measured in quadruplicates in each run. The NSB was obtained in each run from the OD resulting from incubating serum-free buffer in the analysis. OD readings for the three dogs were used to calibrate the assay by calculating a K-value for each analytical run. The K-value was calculated as follows: OD of the hypothyroid dog minus the mean of the OD of two healthy dogs, i.e., K 5 ODCalibrator 1 2 ((ODCalibrator 2 1 OD Calibrator 3)/2). The TGAB content in each serum sample measured, was then expressed as an Ab-score calculated as a percentage of the K value: Ab-scoresample (%) 5 (ODsample /K) * 100%. Assay Specificity. The assay specificity was investigated by comparing the Ab-scores achieved for serum samples from eight TGAB positive hypothyroid dogs to the Ab-scores obtained after precipitation of immunoglobulins. Immunoglobulins were precipitated by adding 40 ml of ice-cold PEG 30% (W/v) in TBS to an equal volume of serum from each of eight hypothyroid dogs. After 1 hr at 4°C, samples were centrifuged for 5 min at

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TABLE 1. SPECIFICITY INVESTIGATIONS

OF AN

ELISA METHOD FOR THE DETECTION CANINE SERUM SAMPLES

OF

THYROGLOBULIN

AUTOANTIBODIES IN

Untreated serum samplesa PEGb treated serum Thyroglobulin treated serum T3c treated serum T4d treated serum

Median (Ab-score (%))

Range (Ab-score (%))

371 0 34 335 308

110-2058 0-86 8-84 89-1641 113-1910

a

Serum samples from 8 dogs with lymphocytic thyroiditis and positive Ab-score. Polyethyleneglycol 6000. Triiodothyrononine (Liothyronine). d L-thyroxine. b c

6,000 3 g and the supernatants were then analyzed in the ELISA and compared to the genuine serum samples. Assay specificity was further investigated by preincubating serum samples from eight Ab-score positive dogs with purified TG in a concentration of 0.5 mg/mL for 1 hr at 37°C. Interference from thyroid hormones was investigated by incubating the eight serum samples with buffer to which either 0.1 mg/ml of T3 or T4 had been added before incubation for 1 hr at 37°C. The assay specificity investigations were all performed in the same analytical run. Detection Limit. The detection limit, i.e., the least detectable concentration (LDC), was defined as zero value (NSB) plus twice the standard deviation (SD). SD was obtained from 13 duplicate determinations of samples having an Ab-score below 7.5% analyzed in the same run. Analytical Imprecision. Three sera from dogs having Ab-scores representing the whole range of the TGAB positive dogs in the analysis were used to survey analytical imprecision. Imprecision in terms of the intra-assay coefficient of variation (CV) was calculated from the three specimens by analyzing the same specimen at least 15 times in the same run. The interassay CV was determined from the difference between the means of duplicates from the control dogs in 10 –15 different runs. Collection of Serum Samples. Serum samples from 241 dogs (Table 2) were collected into seven Vacutainers containing clot activator (Becton-Dickinson, Meylan Cedex, France). Serum was harvested by centrifugation (2000 3 g for 5 min) and stored at 253°C until analysis. The serum samples were collected before any therapeutic or diagnostic procedure except for one of the hypothyroid dogs that had been treated with L-thyroxine for 18 mo. Because of the lack of owner compliance, this dog had not received L-thyroxine for 3 wk and inconsistently over a period of 3 mo before collection of the serum sample. Groups of Dogs 1. Dogs with histologically confirmed lymphocytic thyroiditis. Hypothyroidism was suspected in 11 dogs on the basis of clinical signs such as alopecia, lethargy, and weight gain as well as the following laboratory findings: non-regenerative anaemia, hypercholesterolemia, low TT42, and elevated cTSH3 concentrations. In each case the definitive diagnosis of primary hypothyroidism was based on an insufficient response to a TSHstimulation test with TT4 being measured before and 6 hr after i.v. injection of 0.1 IU of bovine TSH per kg (TSH, Ferring, Sweden) and a poststimulatory TT4 concentrations not exceeding the lower limit of the laboratory reference range 2,5. Serum TT4 was measured by a commercially available radioimmunoassay for the detection of canine TT4 (CoatA-Count, Canine T4, Diagnostic Product Corporation, Los Angeles, CA), serum TSH was

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measured by a commercially available enzyme immunometric assay (Milenia, Canine TSH, Diagnostic Product Corporation) validated in our laboratory (13). Primary hypothyroidism was documented in each case by the finding of lymphocytic thyroiditis (2) on histologic examination of a thyroid biopsy from the left thyroid lobe obtained surgically as described by Jensen et al. (14), and remission of clinical signs associated with hypothyroidism after treatment with L-thyroxine (Eltroxint, Glaxo). 2. Dogs with dermatological diseases without lymphocytic thyroiditis. Thirty-five dogs with various dermatological disorders (e.g., seborrhea, atopy, pyoderma, alopecia, idiopathic dermatoses) of various breeds, sexes, and ages were included because the clinical signs of these dogs often mimic the dermatological signs of hypothyroidism in dogs. Hypothyroidism was excluded in these dogs by history, physical examination, laboratory examination (e.g., serum TT4 , 20 nmol/liter), relevant diagnostic testing being diagnostic (e.g., intradermal skin test), response to appropriate treatment, and because clinical signs could be explained by an alternative diagnosis. A thyroid biopsy was available from all 35 dogs. Histologic examination of thyroid biopsies from these dogs was inconsistent with primary hypothyroidism (2,15) and none had lymphocytic thyroiditis. 3. Dogs with various non-thyroidal diseases. TGAB was also measured in serum in a group of dogs consisting of 63 dogs with various non-thyroidal diseases (e.g., endocrine, infectious, neoplastic, autoimmune). The histologic status of the thyroid gland in these dogs was unknown. 4. Clinically healthy dogs of various breeds. TGAB was measured in 109 clinically healthy pet dogs. Serum samples were collected in six different breeding kennels. In addition, 23 clinically healthy Beagle dogs from a medical research center were included. Thyroid biopsies from these dogs were not available. Statistics. The Wilcoxon signed rank test for paired observations was used to assess the effect of interference from PEG, TG, T4, and T3 in the TGAB ELISA. Analytical imprecision and LDC were calculated using routine descriptive statistical methods (16). The Mann–Whitney U-test for unpaired observations was used to evaluate whether there was a significant difference in the Ab-score between 1) hypothyroid dogs with lymphocytic thyroiditis and 2) dogs with dermatological diseases without lymphocytic thyroiditis, and between 3) dogs with non-thyroidal diseases and 4) clinically healthy dogs. The sensitivity, i.e., the proportion of the 11 dogs with histologically determined lymphocytic thyroiditis that were positive for serum TGAB was calculated. Similarly, specificity, i.e., the proportion of the 35 dogs without histologically determined lymphocytic thyroiditis that tested negative for TGAB was calculated (17,18). Using a commercial software (MedCalct) the data from the 11 dogs with lymphocytic thyroiditis and hypothyroidism and the 35 dogs with skin diseases without histologically signs of lymphocytic thyroiditis were subjected to receiver-operating characteristic (ROC) curve analysis as previously described (19). Briefly, the ROC curve is a graphically plot of sensitivity and 1-specificity and provides a view of the whole spectrum of sensitivity/ specificity pairs obtained by continuously varying the cut-off value over the entire range of test results, hereby allowing the overall discriminative power of the test to be evaluated independently of a single chosen cut-off value (20). Thus, a test with a perfect discrimination between the two patient groups has an area under the ROC curve (W) close to 1, whereas a worthless test that does not discriminate between case and controls has an area not statistically different from 0.5. (21). Calculating the differential positive rate (DPR) ([sensitivity-(1-specificity)]) for each pair of sensitivity/specificity, the optimal cut-off value associated with the highest sensitivity and specificity was computed (19) using the MedCalc software and the data from the 11 hypothyroid dogs with lymphocytic thyroiditis and the 35 dogs with skin diseases not having lymphocytic thyroiditis. Thus, samples

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TABLE 2. MEASUREMENT OF THYROGLOBULIN AUTOANTIBODIES IN CLINICALLY HEALTHY DOGS, DOGS SKIN DISORDERS, NON-THYROIDAL DISEASES AND PRIMARY HYPOTHYROIDISM

Clinical Description of Dogs Healthy dogs Laboratory Beagles Dachshunds German Shepherds Labrador Retrievers Eng. Cocker Spaniels Poodles Yorkshire Terriers Dogs with skin disorders Dogs with non-thyroidal diseases Dogs with primary hypothyroidism Crossbreed Hovawart Labrador Retriever Siberian Husky Beagle Am. Cocker Spaniel Crossbreed Chihuahua Labrador Retrieverh Shetland Sheepdog Eurasier

No. of Positive (Ab-score .57.4%)

No. of Dogs

F/M

Age (months) Median/Range

132 21 39 23 6 11 20 12 35 63

86/46 9/12 23c/16 18/5 6c/0 6/5 14d/6e 10/2 16d/19 36d/27f

48/3-144 48/12-96 56/3-129 39/3-144 51/9-116 42/9-102 76/16-134 27/6-84 60/11-132 107/72-180

8 1 0 2 0 1 3 1 1 3

10.4 9.8 10.1 8.3 9.7 4.8 19.6 13.0 11.9 8.3

14.5 13.9 11.2 12.6 3.6 10.1 30.8 18.9 14.3 18.2

11

8d/11

61/34-103

10/11

340.4

347.3

1 1 1 1 1 1 1 1 1 1 1

F F M M Ovxg Ovx F F F M F

95 34 90 61 36 64 62 108 60 58 39

a

Median: Ab-score (%)

WITH

Q3-Q1b

454.3 2319.9 117.5 391.6 640.4 153.9 164.4 464.9 64.3 19.5 340.4

a

F, number of females; M, number of males. Q3-Q1, interquartile range, i.e., 25th–75th percentile (dispersion). Including one ovariohysterectomized female. d Including two ovariohysterectomized females. e Including one castrated male. f Including three castrated males. g Ovx, ovariohysterectomized female. h Previously treated with L-thyroxine. b c

having a Ab-score higher than the calculated cut-off value were considered positive for TGAB whereas samples having an Ab-score equal to or less than the cut-off value were considered negative for TGAB. The 95% confidence limits for sensitivity and specificity associated with the optimal cut-off value were calculated under the assumption of binomial distribution using the MedCalct software. P-values of less than 0.05 were considered to indicate statistical significance. RESULTS The results of the assay specificity investigations are shown in Table 1. Incubation of serum samples with either PEG or TG lead to a significant (P , 0.01) and almost complete reduction of signal in the assay. The effect of incubation of serum with T4 or T3, resulted in minor, but significant (P 5 0.02), changes in Ab-score with the largest changes being observed in samples incubated with T3. The LDC was 5.5% (SD 5 2.73) corresponding to an OD of 0.009. The intra-assay CV for the three control specimens with low, moderate, and high positive Ab-scores was 3.2%, 4.9%, and 2.0%, respectively. The interassay CV for the three control dogs with low, moderate, and high Ab-score was 4.6%, 7.3%, and 9.9%, respectively. The optimal cut-off value calculated from the differential positive rate was 57.4%.

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Figure 1. Relative operating characteristic (ROC) curve of canine serum thyroglobulin autoantibody measurements. Area under ROC curve (W) 5 0.966; Standard error of W(SEw) 5 0.039. The diagonal line indicate the ROC curve of a worthless test (W 5 0.5). The optimal cut-off value of the assay, an Ab-score of 57.4%, is indicated in parentheses.

Table 2 displays the Ab-scores in the 241 dogs. The median Ab-score in hypothyroid dogs with histologically confirmed lymphocytic thyroiditis was 340%. In dogs with skin diseases without lymphocytic thyroiditis the median Ab-score was 12%. In the healthy dogs and dogs with non-thyroidal diseases the median Ab-score was 10% and 8%, respectively. The Ab-score in hypothyroid dogs with lymphocytic thyroiditis was significantly different from all three groups (P , 0.0001). At the optimal cut-off (Ab-score 5 57.4%), the sensitivity of the assay in detecting lymphocytic thyroiditis was 91% (95% confidence limit: 59%–99%), i.e., 10 of 11 dogs with lymphocytic thyroiditis had Abscore of more than 57.4%. The specificity was 97% (95% confidence limit: 85%–100%), i.e., 34 of 35 control dogs without lymphocytic thyroiditis had an Ab-score less than or equal to 57.4%. The area (W) under the ROC curve (W 5 0.966; SEW 5 0.039; 95% confidence limit 87%–100%) for serum Ab-score was significantly (P , 0.0001) different from that of a worthless test (0.5) (Figure 1). In the 63 dogs with non-thyroidal diseases and the 132 clinically healthy dogs the number of dogs that tested positive for TGAB was three (5%) and eight (6%), respectively. DISCUSSION In this ELISA for the detection of TGAB in canine serum samples, skimmed milk powder was used as a blocking agent and carrier protein instead of BSA that has previously been used in ELISAs to detect TGAB in humans (22) and dogs (12,23,24). In different ELISA’s, casein, the major protein constituent of skimmed milk, has been found to be an efficient blocking agent producing low NSB (25), apparently because of its lowmolecular-weight protein fractions (26). The current assay was calibrated against a calibrator obtained from a hypothyroid dog with lymphocytic thyroiditis and the two healthy calibrators without lymphocytic thyroiditis. Test results were expressed as Ab-scores, this score being calculated as the percentage of the K-value with the K-value being calculated from the formula: OD of the hypothyroid dog minus the mean of the OD of the two healthy dogs. This secured that

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serum samples with different matrix composition were used in quantifying TGAB content. In humans, the Medical Research Council calibrators are often used to calibrate TGAB assays (27), however measurement of TGAB in dogs mainly depends on in-house calibrators (7,23,24,28 –30). This is because of the unfortunate fact that no standard serum seems to exist. Even with the use of standardized calibrators, each investigator must decide whether the autoantibodies should be quantified in terms of immunoglobulin content, antigen/epitope reactivity or binding capacity and for this reason large interlaboratory differences in TGAB measurements exists (27). Additional information on the autoantibody profile in a single dog probably could have been obtained by serial dilution of the serum samples, compared to a standard serum with expression of the autoantibody content in terms of a titer. However, TGAB in humans, and most likely also in dogs, are polyclonal antibodies that display a variety of antigen-specific immunoglobulins of different classes and subclasses with different affinity and avidity in their epitope reaction (27) leading to varying and sometimes non-linear dilution profiles (31). Thus, a titer value may be difficult to interpret, and for practical purposes when measuring large numbers of serum samples, we have found the use of an Ab-score more convenient. The incubation of serum samples with the specific antigen (TG) or precipitation of immunoglobulins by PEG prior to the analysis of serum samples in the ELISA resulted in a significant (P , 0.01) and almost complete reduction of signal in the assay, thereby confirming the assay’s specificity for TGAB (Table 1). Apparently, thyroid hormones and especially T3 had a significant (P 5 0.02) effect in the assay. The reason for this is not clear, but it could be attributable to the presence of T4 or T3 autoantibodies in some of the serum samples. T4 and T3 autoantibodies are considered as subsets of thyroglobulin autoantibodies that recognize hormonogenic sites on the TG molecule as previously described in dogs (28) and humans (32). However, we used serum to which large amounts of thyroid hormones had been added and so, the effect of matrix changes might perhaps explain the observed changes in Ab-score after addition of thyroid hormones. Earlier studies on interference from T3 in ELISA for TGAB have shown contradictory results. In a study by Gaschen et al. (28), addition of T3 to serum samples significantly reduced the binding of TGAB similar to our findings. Whereas, in a study by Young et al. (30) no such interference was observed. The assay imprecision was judged by the intra-and interassay CV in terms of Ab-score. The intra-assay CV (2.0 – 4.9%) and interassay CV (4.6 –9.9%) obtained in this study are comparable to or lower than those obtained in human and canine ELISA’s for TGAB. The obtained CV in an ELISA for human TGAB was 5.7% and 10.1% for intra- and interassay CV, respectively (22). Thacker et al. (23) reported intra- and interassay CV of 12.2% and 8.6%, respectively, in an assay for the detection of canine thyroglobulin antibodies. The LDC in the assay, in terms of Ab-score, was 5.5%. This was close to the median of the healthy dogs (Ab-score 5 10%) and many euthyroid dogs in the three control groups had serum Ab-scores comparable to LDC (Table 2). This indicates that the introduction of serum matrix into the analysis only had minor influence on the absorbance, which probably contributes to the good separation between dogs with lymphocytic thyroiditis and control dogs observed in this study. In dogs with lymphocytic thyroiditis and hypothyroidism 10 of 11 (91%) were positive for TGAB whereas in dogs with skin diseases without lymphocytic thyroiditis only 1 of 35 (3%) were positive for TGAB. In dogs with non-thyroidal diseases and healthy dogs 5% and 6% were positive for TGAB, respectively. These numbers suggest that the assay is improved compared with earlier ELISA methods. Haines et al. (7) detected TGAB in 59% of dogs with hypothyroidism, but also in 24% of dogs with skin diseases, and in 13%

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of hospitalized patients. A similar high frequency of TGAB in dogs with skin disease could not be observed in this study. The assay of Haines et al. (7) has also been used by Beale et al. (29) who found 50% of dogs with hypothyroidism were positive for TGAB. Thacker et al. (23) found 42% of dogs with clinical signs consistent with hypothyroidism to be positive for TGAB. In a recently developed ELISA (24), 38% of dogs with hypothyroidism were positive for TGAB and among clinically healthy dogs and dogs with other internal diseases, 14% and 25% were positive for TGAB, respectively. The diagnostic procedures in documenting primary hypothyroidism in these studies were different from our approach and did not include histologic evaluation of thyroid biopsies, thereby making a direct comparison of diagnostic performance difficult. Very recently, an ELISA kit for the detection of canine thyroglobulin antibodies has become commercially available (Canine Thyroglobulin Autoantibody Immunoassay Kit, Oxford Biomedical Research, Oxford, MI), and a newly reported study, using a cut-off value higher than the one recommended by the manufacturer, demonstrated that the assay presumably had a high specificity as only 5 of 112 dogs with non-thyroidal endocrine diseases had detectable auto-antibodies while the assay correctly identified TGAB in five beagle dogs with histologically confirmed lymphocytic thyroiditis (33). However, in another recent study using the same commercial kit, only 4 of 40 hypothyroid dogs were positive for TGAB (34). We have a preliminary evaluation of the performance characteristics of the commercial kit and compared them to our assay. We calculated the ratio of the maximum signal (i.e., the OD of the strongest positive control [MAX OD]) to OD of a normal dog (Normal OD) (with an Ab-score near the median of the 132 healthy dogs) and NSB (the OD obtained from incubating serum free buffer). The ratio of the MAX OD to the Normal OD corrected for NSB was 10 and 220 for the commercial kit and for our assay, respectively. The ratio of MAX OD to NSB was 34 and 96 for the two assays, respectively. The ratio of the Normal OD to NSB was 4.2 in the commercial kit and 1.4 for our assay. Compared to the commercial kit, these preliminary data suggest that our assay apparently provides a better separation between hypothyroid dogs with lymphocytic thyroiditis and healthy dogs, and that the NSB in our assay is low. In addition, the effect of introducing serum matrix into our assay is negligible. However, future studies on the diagnostic accuracy of the commercial kit in dogs with lymphocytic thyroiditis are warranted. One of the dogs with documented primary hypothyroidism due to lymphocytic thyroiditis was negative for TGAB. Another of the dogs had a low Ab-score (64%), and as described in the section of Material and Methods, this dog had previously been treated with L-thyroxine. In a recent study, TGAB titers were found to decline during L-thyroxine treatment (24). Thus, the low Ab-score of this dog, despite histologically confirmed lymphocytic thyroiditis, could perhaps be ascribed to this phenomenon. It may also be that long-lasting hypothyroidism attributable to lymphocytic thyroiditis leads to a decline in Ab-score and proposed mechanisms in this respect could be acquired peripheral tolerance resulting in reduced TGAB production attributable to either B-cell anergy or deletion (35). Alternatively, as proposed in a study with humans receiving L-thyroxine substitution therapy, there could be a reduction of antigenic substances through a decreased stimulation of thyroid tissue by circulating TSH (36). If Ab-scores decline with time, the sensitivity of the assay would be expected to be lower in dogs with long-lasting lymphocytic thyroiditis, but as the number of dogs with documented lymphocytic thyroiditis was low in this study, further studies in a larger number of dogs with documented primary hypothyroidism and lymphocytic thyroiditis of long and short duration are warranted. One dog was positive for TGAB despite the fact that lymphocytic thyroiditis was not detected on histologic examination of the thyroid gland. Measurement of TGAB and the

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finding of lymphocytic thyroiditis are unrelated tests, i.e., the histologic evaluation can be considered as a visual test that detects mononuclear cells infiltrating the thyroid gland whereas detection of TGAB can be considered as a test for the presence of TGABproducing plasma cells. For this reason it could be expected that not all dogs with lymphocytic thyroiditis will have TGAB and similarly not all dogs with TGAB will have lymphocytic thyroiditis and thus sensitivity and specificity of TGAB measurements in detecting lymphocytic thyroiditis will never be 100%. The value of serum TGAB as a marker for early detection of hypothyroidism is not known. Haines et al. (7) followed 11 antibody positive dogs over an 18-mo period and none of these developed signs consistent with hypothyroidism, although two of them had a decline in TSH-response test. Unfortunately, in the assay used by Haines et al. (7), the number of TGAB positive dogs in non-hypothyroid dogs was high, so that some of the positive dogs may have been false-positive. In humans, a 7.2% yearly incidence of clinical hypothyroidism in thyroid antibody positive (thyroglobulin and/or microsomal antibodies) patients and 26% in patients with initially raised TSH concentrations has been reported (37). It seems that the epitopic recognition of TGAB in people with thyroid autoimmune disease differs from that in healthy people with thyroid antibodies, and this difference has been proposed to be useful to predict the onset of thyroid disease in humans (38,39). As for the diagnostic accuracy (21), the obtained area under the ROC curve (W 5 0.966) was significantly higher than that of a worthless test (0.5) and very close to unity, indicating that TGAB measurements in fact did distinguish between dogs with and without lymphocytic thyroiditis. However, ROC-curve calculations must always be critically judged when the case and control groups, as in this study, have not been selected from a group of suspected animals to which identical diagnostic test procedures have been applied (21,40). It could be argued that no dogs with primary hypothyroidism due to idiopathic atrophy (15) were included in this study. Apparently, there is some controversy as to the frequency of primary hypothyroidism caused by idiopathic atrophy. Nelson et al. (41) and Lucke et al. (2) did not detect idiopathic atrophy in biopsied hypothyroid dogs but rather lymphocytic thyroiditis similar to our findings in this study. Beale and Torres (42) and Gosselin et al. (15) reported idiopathic atrophy in approximately half of their biopsies obtained from hypothyroid dogs. Gosselin et al. (15), however, reported that some of the thyroid biopsies were obtained at autopsy and did not report whether the biopsies were obtained after L-thyroxine treatment. In conclusion, the ELISA developed and validated for the measurement of thyroglobulin autoantibodies was specific and reproducible, with high sensitivity and specificity. The ROC-curve analysis showed a high diagnostic accuracy for the detection of histologically confirmed lymphocytic thyroiditis. Thus, the assay could be an important help in the diagnosis and future investigations of canine hypothyroidism due to lymphocytic thyroiditis. ACKNOWLEDGMENTS/FOOTNOTES The skillful technical assistance of E. Thomsen, A. Mehlsen, and J. Leisner is gratefully appreciated. Dr. H. D. Pedersen and Dr. A.-M. Thougaard are thanked for the participating in collection of serum samples. Dr. E. Christiansen is thanked for the analysis of iodine in thyroglobulin. The Novo Nordisk Fonden and the Vetfond are thanked for financial support. 5 Address reprint requests to: Lars Iversen, Department of Clinical Studies, Central Laboratory, The Royal Veterinary and Agricultural University, Bu¨lowsvej 13, DK-1870 Frederiksberg C, Denmark. 6 Laboratory reference range for TT4:18 – 43 nmol/liter. 7 Laboratory reference range cTSH: ,0.59 mg/liter. 8 Laboratory reference range for fT4 analyzed by equilibrium dialysis (Nichols Institute): 8 – 40 pmol/liter.

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9 In one dog the basal and the 6 hr TSH stimulated TT4 concentrations were 32 and 36 nmol/liter, respectively, because of the presence of thyroxine autoantibodies interfering in the competitive radioimmunoassay for TT4. Serum fT4 analysed by direct equilibrium dialysis was below the detection limit (,2 pmol/liter)(Laboratory reference range 8 – 40 pmol/liter). Thyroxine autoantibodies was determined by radioelectrophoresis and radioimmunoassay.

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