The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p < 0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.     

检测牛结核抗体新型ELISA方法的建立及应用

利用分子克隆和Splicing by overlapping extension(SOE)技术,表达了重组蛋白rM70-83-E6,Western blot分析rM70-83-E6具有很好的特异性。以蛋白rM70-83-E6作为诊断抗原建立了间接ELISA方法,ELISA诊断方法的判定标准,即S/P≥0.5为阳性,S/P<0.4为阴性,0.5>S/P≥0.4为可疑。ELISA方法的特异性为96.0%、敏感性为69.4%。对67份PPD皮试阳性和50份PPD皮试阴性(117份)血清样品进行了检测,并与PPD皮试比较。67份PPD皮试阳性血清样品中有46份为ELISA检测阳性,阳性符合率为68.7%,50份PPD皮试阴性牛血清都为阴性,阴性符合率为100%,本ELISA方法与PPD皮试诊断方法总复合率为82.1%。研究表明,ELISA方法具有很好的开发和应用前景。     

奶牛乳房炎乳中结合珠蛋白含量的夹心ELISA检测方法研究

为准确检测奶牛乳房炎乳中结合珠蛋白(Hp)含量,本研究采用牛血红蛋白(Hb)包被酶标板捕获乳清中Hp,利用特异性抗血清和酶标抗体建立夹心ELISA方法,并对建立的方法进行优化,用于检测牛奶中Hp含量.结果表明,ELISA方法检测下限为0.08μg/mL,与其他几种常见乳房炎乳中急性期蛋白质无交叉反应,批内试验和批间试验变异系数分别为3.27%～4.98%和5.46%～8.31%.应用本实验建立的夹心ELISA方法和商品化试剂盒分别检测50份已知患乳房炎牛乳清中Hp含量,同时对检测结果做相关性分析.结果表明,夹心ELSIA方法与商品化试剂盒具有很好的相关性(R2=0.996 2),同时验证了乳清中体细胞数与Hp含量呈正相关.夹心ELISA方法可准确测定牛奶中Hp含量,并可为奶牛乳房炎的诊断提供一种简易有效的辅助手段.     

Evaluation of an O antigen enzyme-linked immunosorbent assay for screening of milk samples for Salmonella dublin infection in dairy herds.

Levels of antibodies to the O antigens (O:1,9,12) of Salmonella dublin were tested in 1355 serum, 1143 cow milk and 160 bulk milk samples from dairy herds using an enzyme-linked immunosorbent assay (ELISA). In order to define the background reaction, milk samples from all lactating cows and serum samples from 9 animals were collected in each of 20 salmonellosis-free herds located on the island of Bornholm, where cattle salmonellosis has not been reported. Similar samples were collected from all stalled animals in 10 herds with recent (< 6 months) outbreaks of salmonellosis located in Jutland, where salmonella infection is enzootic. Using herd history of salmonellosis, herd location and clinical status of the herds as criteria, the optimal cutoff in the milk ELISA was determined as being at least 5% of the samples having optical density > 0.5, resulting in herd sensitivity of 1.0 and herd specificity of 0.95. While none of the sera in the herds from Bornholm was ELISA positive, 2 herds had a few reactors in the milk ELISA. Using the same cutoff, all but 1 bulk milk sample from 150 herds on Bornholm was ELISA-negative, and all 10 salmonellosis-positive herds from Jutland were ELISA-positive. A significant correlation was found between ELISA reactions in milk and in serum of cows (34% and 32% respectively, rs = 0.69, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of an enzyme-linked immunosorbent assay in the diagnosis of bovine tuberculosis

The behavior of the immunoenzymatic system was tested in the serologic diagnosis of bovine tuberculosis in different populations from areas free of the disease, and in other areas with different prevalence. The specificity registered in the 4009 samples showed 94.4%, while the range of sensitivity increased proportionally to the number and distribution of lesions found in the anatomopathological test. Overall the sensitivity of the ELISA was 47%. When animals from groups with different prevalence of the disease were studied, a significant correlation (78%) between the ELISA system and the Intradermal Tuberculin Test (IT) was observed in the groups with low prevalence of the disease, animals reactive to both tests were found in all the units. The results of the work and the inherent advantages of the ELISA technique allow recommending this ELISA in programs for control and/or eradication of bovine tuberculosis.     

Milk progesterone enzyme-linked immunosorbent assay as a tool to investigate ovarian cyclicity of water buffaloes in relation to body condition score and milk production

Background

Application of assisted reproductive technologies in buffaloes is limited to some extent by farmers’ inability to detect oestrus because of its poor expression. The present study aimed at investigating reliability of a milk progesterone enzyme-linked immunosorbent assay (ELISA) to assess the ovarian cyclicity during post partum, oestrus and post-breeding periods in water buffaloes.

Methods

Progesterone concentrations were measured by an ELISA in milk of 23 postpartum buffaloes in relation to oestrus, pregnancy, body condition score (BCS) and milk production. Two milk samples were taken at 10 days intervals, every month starting from day 30 and continued to day 150 post partum. BCS and milk production were recorded during sample collection. Milk samples from bred buffaloes were collected at Day 0 (day of breeding), Days 10–12 and Days 22–24. Defatted milk was preserved at −80°C until analysis. Pregnancy was confirmed by palpation per rectum on Days 70–90.

Results

Seventeen buffaloes had 47 ovulatory cycles, one to four in each, 13 were detected in oestrus once (28 % oestrus detection rate). Progesterone concentration ≥1 ng/ml in one of the two 10-day-interval milk samples reflected ovulation and corpus luteum formation. The intervals between calving to first luteal activity and to first detected oestrus varied from 41 to 123 days (n = 17) and 83 to 135 (n = 13) days, respectively. Eight buffaloes were bred in the course of the study and seven were found pregnant. These buffaloes had a progesterone profile of low (<1 ng/ml), high (≥ 1 ng/ml) and high (≥ 1 ng/ml) on Day 0, Days 10–12 and Days 22–24, respectively. Buffaloes cycling later in the postpartum period had fewer missed oestruses (P < 0.05). Buffaloes with a superior BCS had a shorter calving to oestrus interval and produced more milk (P < 0.05).

Conclusions

Milk progesterone ELISA is a reliable tool for monitoring ovarian cyclicity and good BCS may be an indicator of resuming cyclicity in water buffalo.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows

OBJECTIVE: To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples. DESIGN: Cross-sectional study. ANIMALS: 689 lactating dairy cows in 9 Ontario herds. PROCEDURE: Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis. RESULTS: Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds.     

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

Background

Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR^®BVDV-Ab indirect ELISA. The latter has successfully been used to eradicate BVD in Sweden.Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples. The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools.

Results

During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level. Moreover, we found that when testing bulk milk, the SVANOVIR^®BVDV-Ab can detect a lower prevalence of seropositive lactating cows, compared to the Danish blocking ELISA (0.78 % vs. 50 %). Values in the SVANOVIR^®BVDV-Ab better relate to low concentrations of antibody positive milk (R² = 94-98 %), than values in the blocking ELISA (R² = 23–75 %). For sera, the two ELISAs performed equally well.

Conclusions

The SVANOVIR ELISA is recommended for analysis of bulk milk samples in the current Danish situation, since infected dairy herds e.g. due to import of infected cattle can be detected shortly after BVDV introduction, when only few lactating cows have seroconverted. In sera, the two ELISAs can be used interchangeably.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of neosporosis and leukosis in lactating dairy cows

Serum and milk samples from 1229 cows on 22 Ontario dairy farms were individually tested for antibodies specific for bovine leukosis virus (BLV) and Neospora caninum by enzyme-linked immunosorbent assay (ELISA). Antibodies against BLV were present in 361 serum samples (29.4%) and 369 milk samples (30.0%). Comparing the 2 tests, agreement was almost perfect (k = 0.86; 95% CI = 0.83 to 0.90) and the proportions of samples positive were not significantly different (P = 0.56). Both tests identified the same 3 herds free of bovine leukosis virus. Antibodies against N. caninum were detected in 138 serum samples (11.2%), and 111 milk samples (9.0%). Agreement between the 2 tests was moderate (k = 0.52; 95% CI = 0.43 to 0.59). Four herds were free of neosporosis by the serum test, while 10 herds were negative by the milk test. The ELISA on milk samples facilitates sample collection to classify herds free of BLV; the milk N. caninum ELISA was less reliable in predicting herd-level infection.     

ELISA方法检测奶牛结核病的效果评价

牛结核病是由牛分枝杆菌(Mycobacterium bovis)引起的一种慢性消耗性人畜共患病.该病可通过排泄物及分泌物等进行传播,严重威胁畜牧业的发展及城乡居民的身体健康.为保障牛奶、牛肉等畜产品市场安全和人民生命健康安全,牛结核病的防控意义不言而喻.本文以PPD变态反应为检测标准,对ELISA检测结果进行评价,论述...     

Assessment of test results when using a commercial enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in repeated samples collected from adult dairy cattle

OBJECTIVE: To determine the proportion of adult cattle that change test status when an ELISA for antibodies against Mycobacterium avium subsp paratuberculosis (MAP) is used to assay samples collected twice at variable intervals and to determine whether cows with an initial strong positive result were more likely to maintain positive status, compared with all cows with an initial positive result. DESIGN: Cross-sectional observational study. ANIMALS: 3,757 adult dairy cattle. PROCEDURE: Serum samples were obtained twice from cattle at intervals ranging from 77 to 600 days between collections. Samples were tested with an ELISA for detection of antibodies to MAP. RESULTS: Of 157 cattle with initial positive results (value for the sample divided by the value for positive-control serum [S/P] > or = 0.25), 62 (39.5%) had negative results for the second sample. Of 71 cattle with an initial S/P value > or = 0.40, 13 (18.3%) had a negative result (S/P < 0.25) for the second sample. Of 33 cattle with an initial S/P > or = 0.70, 3 (9.1%) had a negative result (S/P value < 0.25) for the second sample. Interval between collection of samples did not affect results. CONCLUSIONS AND CLINICAL RELEVANCE: Many cows changed ELISA status between samples collected at variable intervals. Cows with an initial high S/P value (> or = 0.70) were more likely to maintain positive status than cows classified as positive on the basis of cutoff values of > or = 0.25 or > or = 0.40. Veterinarians should expect variability in ELISA results when repeated testing of cattle is used as part of an MAP control program.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for the detection of allergen-specific IgE antibodies in dogs

Twenty-eight atopic dogs, 22 pruritic, non-atopic dogs and 10 healthy dogs were ELISA tested. For calculations of diagnostic specificity and sensitivity, positive ELISA test results in non-atopic dogs were considered false positive results. The absence of any positive results in the atopic dogs was considered false negative results. The atopic dogs were tested both with ELISA and an intradermal test, utilising allergen extracts from the same manufacturer, to determine the frequency of positive allergen reactions in the ELISA test compared with the intradermal test. The Prausnitz-Küstner test was performed to evaluate the significance of a positive ELISA test result. Based on cross-tabulations with clinically defined atopic dermatitis, the ELISA test showed a sensitivity of 53.6% and a specificity of 84.4%. The correlation between the ELISA and the intradermal test was poor. Positive Prausnitz-Küstner tests were not obtained using sera from dogs that were intradermal test negative for the tested allergens, even though sera had high levels of IgE as measured by the ELISA. These findings question the significance of a positive ELISA test result and indicate that the test is not measuring functional allergen-specific IgE.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to bovine virus diarrhoea virus in milk

The present study shows that milk is an appropriate source for detection of seroreactors to bovine virus diarrhoea virus (BVDV). There was close agreement between antibody titres in serum and in skim milk, as determined by an indirect enzyme-linked immunosorbent assay. The antibody titres were usually lower in skim milk than in serum, but all seropositive cows (n = 84) were also skim milk-positive and all but one seronegative cow (n = 55) proved negative in skim milk. During lactation, the level of antibodies to BVDV in milk showed an inverse relationship to the amount of milk produced. However, there was a sufficient level of antibodies in milk throughout lactation to permit an adequate determination of BVDV antibody status in dairy cows. There was a mutual good agreement between milk antibody titre in the four mammary quarters, irrespective of milk cell count. Milk can be used to detect seroreactors to BVDV. Milk is preferable to blood in large-scale epidemiological studies, since the sampling procedure is much simpler.     

Evaluation of indirect and avidin-biotin enzyme linked immunosorbent assays for detection of anti-listeriolysin O antibodies in bovine milk samples

Listeria monocytogenes is a foodborne pathogen that causes a wide spectrum of diseases in humans and animals. Enzyme linked immunosorbent assays (ELISA) [indirect and avidin-biotin (A-B)] for detecting L. monocytogenes antibodies in bovine milk samples (n = 2060) were standardized and evaluated by comparison with bacteriological examination. The tests were standardized by checker board titration. Highly purified listeriolysin O (LLO) was used as an antigen. Receiver operating characteristic (ROC) analysis was performed to decide the cut-off values. The ROC analysis revealed the sensitivities of indirect and A-B ELISA as 100% and specificities as 97.1 and 99.9% respectively. Listeria monocytogenes was isolated from 105 (5.1%) milk samples collected from 52 farms. Anti-LLO IgG antibodies were detected from 137 and 112 milk samples when tested by indirect and A-B ELISA respectively. Of the 52 farms screened, 28 (53.8%) yielded one or more isolates of L. monocytogenes and 33 (63.5%) of the farms had one or more animals simultaneously positive by one or both the assays for anti-LLO antibodies.     

Evaluation of a new antibody-based enzyme-linked immunosorbent assay for the detection of bovine leukemia virus infection in dairy cattle.

The objective of this study was to validate a new blocking enzyme-linked immunosorbent assay (ELISA) (designated M108 for milk and S108 for serum samples) for detecting bovine leukemia virus (BLV) infection in dairy cattle. Milk, serum, and ethylenediaminetetraacetic acid-blood samples were collected from 524 adult Holstein cows originating from 6 dairy herds in Central Argentina. The M108 and S108 were compared with agar gel immunodiffusion (AGID), polymerase chain reaction and a commercial ELISA. Because there is currently no reference test capable of serving as a gold standard, the test sensitivity (SE) and specificity (SP) were evaluated by the use of a latent class model. Statistical inference was performed by classical maximum likelihood and by Bayesian techniques. The maximum-likelihood analysis was performed assuming conditional independence of tests, whereas the Bayesian approach allowed for conditional dependence. No clear conclusion could be drawn about conditional dependence of tests. Results with maximum likelihood (under conditional independence) and posterior Bayes (under conditional dependence) were practically the same. Conservative estimates of SE and SP (with 95% confidence intervals) for M108 were 98.6 (96.7; 99.6) and 96.7 (92.9; 98.8) and for S108 99.5 (98.2; 99.9) and 95.4 (90.9; 98.1), respectively. The ELISA 108 using either milk or serum to detect BLV-infected animals had comparable SE and SP with the official AGID and a commercial ELISA test, which are currently the most widely accepted tests for the serological diagnosis of BLV infection. Therefore, ELISA 108 can be used as an alternative test in monitoring and control programs.     

Evaluation of a bulk-milk ELISA test for the classification of herd-level bovine leukemia virus status

The results of a commercial bulk-milk enzyme-linked immunosorbent assay (ELISA) test for herd-level bovine leukemia virus (BLV) status were compared to results obtained from individual agar-gel immunodiffussion (AGID) testing on sampled cattle. A positive herd was defined as a herd having one or more AGID-positive animals. The estimated true herd status was based on the sensitivity and specificity of the AGID test and the number of cattle sampled per herd. Ninety-seven herds were used, with a mean of 13 cows sampled per herd. The AGID test indicated an apparent herd prevalence of 70.1%. After accounting for the number of cows sampled and the sensitivity and specificity of the AGID test, the estimated true herd prevalence of BLV was 52.3%. The ELISA test identified 79.4% of herds as positive for BLV, and had an apparent sensitivity and specificity of 0.97 and 0.62, respectively. However, after accounting for the sensitivity and specificity of the AGID test in individual animals, the specificity of the ELISA test was 0.44. The ELISA test was useful for identifying BLV-negative herds (i.e., ruling out the presence of BLV infection in test negative herds). With the moderately low specificity, herds identified as positive by the ELISA test would require further testing at the individual or herd level to definitively establish their BLV status.     

Evaluation of the specificity of three enzyme-linked immunosorbent assays for detection of antibodies against Salmonella in bovine bulk milk

Background

The Swedish Salmonella control program has been running for decades and has resulted in a low prevalence of Salmonella in Swedish food producing animals. Routine bacteriology is used to detect Salmonella, however, bacteriology is time consuming, costly and has a low sensitivity. Different enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of antibodies against Salmonella Dublin and S. Typhimurium in bovine bulk milk, individual milk samples as well as in sera. Screening bulk milk for antibodies against Salmonella spp. could improve the cost-effectiveness of the surveillance in Swedish dairy cattle, but as characteristics of tests may vary in different populations, tests should always be evaluated in the specific population where they will be used. Hence, the aim of this study was to evaluate the specificities of three bovine ELISAs when used to analyse bulk milk samples from Swedish dairy cattle. A second aim was to compare the performance of the two Dublin ELISAs tested.

Methods

Bulk milk samples for analysis were randomly selected from samples collected within the Swedish bulk milk sampling scheme and analyzed with the three ELISAs; a Danish in-house Dublin ELISA, PrioCHECK® Salmonella Ab bovine Dublin ELISA and PrioCHECK® Salmonella Ab bovine ELISA (hereafter named mixed ELISA). The specificities of the ELISAs were calculated assuming a disease-free status in Sweden i.e. that all test positive samples were assumed to be false positive results. This assumption can be used when a disease is known to be infrequent.

Results

The calculated specificities of the two Dublin ELISAs and the mixed ELISA, when using the producer’s recommended cut-off value of the corrected optic-density percent (ODC%) were 99.4% (95% Confidence Interval (CI): 98.8% -99.8%), 99.4% (95% CI: 98.8% -99.8%) and 97.9% (95% CI: 96.8% -98.7%), respectively. The correlation between the ODC% values of the two Dublin ELISAs was 0.83.

Conclusions

We conclude that the evaluated ELISAs have sufficiently high specificities to be used as supplement to bacteriological examinations in the Swedish Salmonella control program in cattle as well as a primary screening test in routine surveillance for S. Dublin.     

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

A milk and a serum ELISA for detection of antibodies against Mycobacterium avium ssp. paratuberculosis (MAP) were evaluated against the complement-fixation test (CFT) and culture of faecal samples from 580 cows collected between August 1996 and December 1996. Milk and serum were obtained concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP.

A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80–96% at the cut-off values of 4, 7 and 17 OD%.     

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

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