Determination of thyrotropin in serum by time-resolved fluoroimmunoassay evaluated

We evaluated a new, highly sensitive time-resolved fluoroimmunoassay for thyrotropin (TSH) in serum. This direct immunometric "sandwich"-type assay involves two monoclonal antibodies against TSH, one immobilized, the other labeled with europium. Extremely high specific activity of the label and the use of labeled antibody in large excess make the method sensitive enough to measure TSH values falling below the normal reference interval. The standard curve is nearly linear over a wide range of TSH concentrations (standard concentrations range from 0.25 to 324 milli-int. units/L). The lowest concentration detectable was 25 micro-int. units/L. The CV for the assay was less than 6% at 0.5 milli-int. unit/L or higher, 11.3% at 0.1 milli-int. unit/L. For a CV of 10% the lower limit of the working range would be around 0.1 milli-int. unit/L. The interassay CV was 6.7 to 11.8% for TSH concentrations of 0.31 to 19.6 milli-int. units/L. The 95% confidence interval for sera from 111 healthy persons was 0.6-3.8 (range 0.3-3.8) milli-int. units/L. For hyperthyroid patients and thyroid cancer patients treated with thyroxin after thyroidectomy, serum TSH values were all below the reference interval (most were less than 25 micro-int. units/L).     

A sensitive and practical immunoradiometric assay of thyrotropin

We describe a two-site immunoradiometric assay for thyrotropin (TSH) in serum, based on use of two monoclonal antibodies directed against two separate antigenic determinants on the TSH molecule. One antibody is immobilized on polystyrene beads; the other is radioiodinated by a modified Chloramine T method. The detection limit of the assay is 0.02 milli-int. unit/L. The working range (CV less than 10%) is from 0.1 to greater than 50 milli-int. units/L. The log mean concentration of TSH in sera collected from 100 euthyroid subjects between 08:00 and 11:00 hours was 1.9 milli-int. units/L, the range 0.4-5.4 milli-int. units/L. Values for hyperthyroid patients and thyroid-cancer patients being treated with thyroxin were much lower than those for euthyroid persons. Results by this new assay correlated excellently with those by our conventional radioimmunoassay (r = 0.99) and also with a sensitive immunofluorometric TSH method (Delfia TSH) (r = 0.99).     

Immunoenzymatic quantification of low concentrations of thyrotropin

We evaluated an immunoenzymatic assay (Abbott HTSH EIA) for thyrotropin (TSH) as a tool for detecting hyperthyroidism and for monitoring thyroid hormone suppressive therapy in patients with nodular goiter, thyroid carcinoma, and hypopituitarism. We also tested with thyroliberin (TRH), to determine the correlation between peak and basal TSH in suppressed patients. For comparison, we used a nonequilibrium radioimmunoassay optimized for maximum sensitivity (J Clin Endocrinol Metab 1975;41:676). Hyperthyroid patients with values for either or both triiodothyronine and thyroxin above the normal reference interval had Abbott assay values less than or equal to 0.2 milli-int. unit/L, clearly below the Abbott assay normal range, as determined in 116 euthyroid subjects. We detected one-third of the suppressed patients (greater than or equal to 0.3 milli-int. unit/L) with RIA, 69% with the Abbott assay (TSH greater than or equal to 0.04 milli-int. unit/L). Only 20% of patients with undetectable basal TSH values in the Abbott assay responded to TRH with a detectable peak TSH value; the peak TSH value after TRH was proportional to the basal TSH value. A single basal TSH measurement by the Abbott HTSH EIA should be adequate for monitoring the degree of thyroidal suppression in thyroid-hormone-treated patients.     

Thyrotropin as measured by a sensitive immunoradiometric assay

An immunoradiometric assay (Boots-Celltech's "Sucrosep"; IRMA) for thyrotropin (TSH) was evaluated and results were compared with those of our in-house RIA procedure. The IRMA had a sensitivity of 0.02 milli-int. unit/L. In addition it displayed excellent intra- and inter-batch precision, cross-reacted negligibly with other pituitary hormones, and appeared to be relatively free of matrix effects (although deionized water did give an apparent TSH concentration of 0.13 milli-int. unit/L). Over the range 1.2 to 64 milli-int. units/L, correlation between the IRMA (y) and RIA (x) was excellent: y = 1.16x - 1.69 (r = 0.98). The normal reference interval for the IRMA was 0.4 to 4.2 milli-int. units/L. For patients suspected of hyperthyroidism who had been subjected to the thyroliberin test, the IRMA more effectively differentiated between euthyroidism and hyperthyroidism. During testing with thyroliberin, all nonresponding hyperthyroid patients had initial baseline TSH concentrations of less than 0.02 milli-int. unit/L by IRMA. This sensitive assay represents an important advance in the ability to differentiate between euthyroidism and hyperthyroidism.     

Chemiluminescence immunoassay of thyrotropin with acridinium-ester-labeled antibody evaluated and compared with two other immunoassays

A new chemiluminometric immunoassay of thyrotropin (TSH) involves antibody labeled with acridinium ester ("Magic Lite System," Ciba Corning Diagnostic Corp.). The assay is rapid, with two incubations totaling 2.5 h, requires two standards per run, and takes 10 s per sample for the quantification step. Analytical performance, within- and between-run reproducibilities, and linearity were excellent. The detection limit is 0.04 milli-int. unit/L. Results correlated well with those obtained by immunoradiometric assay (RIA-gnost hTSH, Hoechst-Behring) and immunofluorometric assay (hTSH Delfia, LKB): r = 0.975. TSH measurements in 32 euthyroid subjects ranged from 0.4 to 4.8 milli-int. units/L (mean 1.35 milli-int. units/L). TSH values for 51 hypothyroid and subclinically hypothyroid patients ranged from 2 to 65 milli-int. units/L. TSH values for 33 hyperthyroid patients (less than 0.14 milli-int. unit/L, less than 0.04 milli-int. unit/L in 16 of the 33) were clearly lower than for most untreated euthyroid subjects. For 169 other individuals whose thyroid function was being routinely assessed. TSH ranged from 0.4 to 4.8 milli-int. units/L, three had TSH less than 0.14 milli-int. unit/L, and four had TSH between 0.14 and 0.4 milli-int. unit/L. This system is as efficient and reliable for screening for thyroid function as the two comparison systems.     

Diagnostic value of thyrotropin concentrations in serum as measured by a sensitive immunoradiometric assay

A new, highly sensitive immunoradiometric thyrotropin (TSH) assay involving solid-phase-coupled monoclonal antibodies (Boots-Celltech Sucrosep IRMA-TSH) has been evaluated in a wide variety of patients with thyroidal and nonthyroidal illnesses and the results compared with those obtained by conventional diagnostic TSH RIAs. The sensitivity of the present assay ranged from 0.036 to 0.1 milli-int. unit/L (mean 0.056). TSH, measurable in serum of each of 128 euthyroid patients, ranged from 0.1 to 6.3 milli-int. units/L (mean 1.7, SD 1.1). Similar concentrations were found in 15 healthy pregnant women. TSH was undetectable in 27 hyperthyroid patients, of whom six were tested with thyroliberin stimulation and failed to respond. The mean TSH concentration measured in 62 seriously ill hospital patients of 2.7 (SD 2.5) milli-int. units/L was significantly higher (p less than 0.05) than in the euthyroid patients. Basal values and peak TSH responses to thyroliberin testing correlated well (r = 0.63, n = 48), irrespective of clinical diagnosis. We conclude that the present assay readily discriminates between euthyroid and hyperthyroid patients and should replace conventional TSH RIAs in diagnostic laboratories.     

Specificity of sensitive assays of thyrotropin (TSH) used to screen for thyroid disease in hospitalized patients

Thyrotropin (TSH) concentrations were measured in 1580 hospitalized patients and 109 normal persons. Using the mean +/- 3 SD limits of the log values for the controls (0.35-6.7 milli-int. units/L), the proportion of abnormal TSH results in the hospitalized patients was 17.2%. TSH was undetectable (less than 0.1 milli-int. unit/L) in 3.1% of patients, suggesting hyperthyroidism, and high (greater than 20 milli-int. units/L) in 1.6%, suggesting hypothyroidism. On follow-up of 329 patients, 62% with abnormal TSH (less than 0.35 or greater than 6.7 milli-int. units/L) and 38% with normal TSH concentrations, only 24% of those with undetectable TSH had thyroid disease: 36% of them were being treated with glucocorticoids and 40% had nonthyroidal illness (NTI). Although half the patients with TSH greater than 20 milli-int. units/L had thyroid disease, 45% of patients had high TSH values associated with NTI. TSH concentrations usually returned towards normal when patients' therapy with glucocorticoids was discontinued or they recovered from NTI. TSH test sensitivity appeared good when the mean +/- 3 SD limits of the reference population were used, i.e., no cases of hyper- or hypothyroidism, as identified by free thyroxin index (FT4I), were missed. However, TSH test specificity was inferior to that of the FT4I test (90.7% vs 92.3%), although specificity could be improved to 97.0% if the wider TSH reference limits of 0.1 to 20 milli-int. units/L were used--limits considered pathological if applied to outpatients. Evidently, different reference intervals for TSH are needed for hospitalized and nonhospitalized patients. We conclude that a "sensitive TSH assay" is not a cost-effective thyroid screening test for hospitalized patients as compared with the FT4I.     

Enhanced luminescence immunoassay: evaluation of a new, more sensitive thyrotropin assay

We measured thyrotropin (TSH) with an enhanced luminometric assay ("Amerlite"; Amersham International). The detection limit of the assay is 0.02 milli-int. unit/L. Within-assay precision was 6.7 and 7.8% at 3.77 and 12.1 milli-int units/L, respectively, and between-assay precision was almost identical, whether singleton or duplicate samples were assayed. TSH measured in 132 euthyroid subjects ranged from 0.06 to 4.13 milli-int. units/L (mean 1.52, SD 0.86). Similar concentrations were found in 20 healthy pregnant women and 19 of 20 healthy post-menopausal women (one of whom had undetectable TSH). In 17 patients with primary hypothyroidism, TSH concentrations ranged from 9.34 to greater than 200 milli-int. units/L; and in 53 of 59 patients with hyperthyroidism, TSH concentrations were undetectable, ranging in the remaining six from 0.03 to 0.06 milli-int. unit/L. Results for TSH in 28 patients stimulated with thyroliberin were consonant with the results of the thyroliberin test in 25 cases. Thus, for most patients, measurement of a basal TSH concentration evidently will predict their thyroidal status and also the response to thyroliberin, but a few will require additional tests of thyroid function.     

A rapid, sensitive enzyme-linked immunoassay for human thyrotropin

In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.     

Development and utility of a monoclonal-antibody-based, highly sensitive immunoradiometric assay of thyrotropin

Using improved selection techniques, we isolated four monoclonal antibodies with high affinity for human thyrotropin (Ka = 1.6 X 10(8) to 2.6 X 10(10) L/mol). We used two of these in an immunoradiometric assay (IRMA) that also incorporates a novel phase-separation technology (Sucrosep TSH IRMA, Boots-Celltech). This assay's very low detection limit for TSH (0.03-0.08 milli-int. unit/L) and wide working range (0-250 milli-int. unit/L) allow the differential diagnosis of hypothyroid, euthyroid, and hyperthyroid patients. We compare the utility of this IRMA with that of a RIA for patients with various thyroid disorders. As determined by IRMA, a normal concentration of TSH in serum excludes hyperthyroidism or hypothyroidism, whereas an undetectable serum TSH concentration (less than 0.08 milli-int. unit/L) accurately predicts an abnormality in thyroid gland function.     

Use of a rapid, sensitive immunoradiometric assay for thyrotropin to distinguish normal from hyperthyroid subjects

Using a new three-site immunoradiometric assay for thyrotropin (TSH), we measured concentration of this hormone in the serum of 47 patients with hyperthyroidism and 46 controls. The mean and range of serum TSH concentration was significantly lower in thyrotoxic than in control subjects, and it was possible to correctly identify 96% of thyrotoxic patients on the basis of a serum TSH concentration less than 0.5 milli-int. unit/L. We conclude that such measurement is highly sensitive for distinguishing hyperthyroid from normal subjects, and that the lower limit of normal for TSH in serum is about 0.5 milli-int. unit/L.     

Clinical application of a sensitive non-isotopic immunometric assay of thyrotropin

We have evaluated an immunometric assay of thyrotropin (TSH) based on enhanced chemiluminescence signal; its detection limit is 0.06 milli-int. unit/L. Values in 101 clinically euthyroid subjects with normal thyroid hormone concentrations ranged from 0.39 to 6.83 milli-int. units/L. TSH in 15 hypothyroid patients ranged from 10.3 to greater than 200 milli-int. units/L, whereas in 31 hyperthyroid subjects with increased concentrations of free thyroxin and free triiodothyronine, TSH was undetectable serum of all but one subject. Of 32 clinically and biochemically euthyroid patients with goiter, two had undetectable serum TSH and six had values below the normal range. In 19 clinically euthyroid patients from an intensive-care unit, TSH was undetectable in two and below the normal range in another two. This immunometric chemiluminescence assay distinguishes thyrotoxic from euthyroid subjects, but caution is required in interpreting TSH values alone in subjects with goiter or nonthyroidal illness.     

High sensitivity, homogeneous particle-based immunoassay for thyrotropin (Multipact)

We describe the first homogeneous, nonradioactive, high-sensitivity assay for human thyrotropin (TSH). The assay is based on particle immunoassay techniques, wherein 800-nm particles form the basis for the immunochemistry, delivery, and the detection technologies, respectively. Our assay also is the first to involve the use of fragmented monoclonal antibodies (to eliminate serum interferences) covalently coupled to particles without loss of their binding properties. Assays are performed in a semiautomated mode with use of a new modular system (Multipact). Equilibrium is reached in less than 2 h. Precision profile, sensitivity, and clinical studies indicate that the assay is accurate, has good precision at low concentrations, and that detection-limit characteristics compare well with those of a leading commercial high-sensitivity immunoradiometric assay (IRMA) for TSH. Dilution characteristics were satisfactory down to the assay's detection limit for a range of clinical samples. Correlation studies vs a reference IRMA method yielded the regression equation, present method = 0.976 (IRMA) + 0.002 milli-int. unit/L (r = 0.98), for 223 samples with TSH concentrations in the range 0 to 30 milli-int. units/L. For 40 samples with TSH less than or equal to 1.0 milli-int. unit/L it was: present method = 0.94 (IRMA) + 0.005 milli-int. unit/L (r = 0.96).     

A laboratory and clinical evaluation of an immunochemiluminometric assay of thyrotropin in serum

We evaluated an immunochemiluminometric assay for human thyrotropin. A chemiluminescent acridinium ester is used as a label, with magnetic-particle separation. The lower limit of detection of the assay (mean + 3 SD of the zero standard) was 0.07 milli-int. unit/L, with a working range of 0.5 to greater than 60.0 milli-int. units/L. Assay accuracy was good as judged from analytical recovery, analysis of external quality-assessment samples, and comparison with an enzyme-amplified immunoassay. There were no significant interferences or cross-reactivities. Twenty-four samples assayed showed aggregation of the magnetic particles. On re-assay, four of these samples showed a significant increase in the measured TSH by the luminescence assay. Assay time for 60 tubes was approximately 3.5 h with the use of a semi-automated luminometer. The reference interval, determined from data on 144 healthy euthyroid subjects, was 0.3-4.0 milli-int. units/L. Sixteen of 19 thyrotoxic patients showed clearly suppressed concentrations of thyrotropin in serum.     

Immunoradiometric assay of thyrotropin in plasma: its value in predicting response to thyroliberin stimulation and assessing thyroid function in amiodarone-treated patients

We measured thyrotropin in plasma by an ultrasensitive immunoradiometric assay (TSH-IRMA, "Sucrosep," Boots-Celltech), before and after thyroliberin (TRH) stimulation, in 71 patients with suspected thyroid-function disorders. Thirty-three were taking amiodarone; none was receiving (anti)thyroid drugs. The patients were divided into four groups, according to their TSH response to TRH (as measured previously by conventional TSH-RIA) and the concentrations of thyroxin (T4) and triiodothyronine (T3) in their plasma. Observed ranges of plasma TSH-IRMA (milli-int. units/L) before and after TRH were: euthyroid (n = 20), 0.2-3.0 and 1.7-15.5; subclinically hypothyroid (n = 14), 4.3-18.5 and 20-75; hyperthyroid (n = 17), less than 0.09 and less than 0.09-0.4; and subclinically hyperthyroid (n = 20), less than 0.09-1.1 and less than 0.09-2.6. Evidently TSH-IRMA results for a single sample completely distinguish hyperthyroidism from euthyroidism. However, TSH-IRMA values may also be undetectable in subclinical hyperthyroidism. The TSH response to TRH can be predicted from basal TSH-IRMA results less than 0.09 or greater than or equal to 0.8 milli-int. unit/L, intermediate values can be associated with either a normal TSH response (euthyroidism) or a decreased TSH response (subclinical hyperthyroidism only). We advocate TSH-IRMA as the first diagnostic test of thyroid function for amiodarone-treated patients.     

Technical and clinical performance of six sensitive immunoradiometric assays of thyrotropin in serum

We evaluated the analytical and clinical performance of six commercial immunoradiometric assay kits for thyrotropin (TSH) in serum in 218 subjects, with and without thyroid dysfunction. Detection limits of the six kits were lower (from 0.07 to 0.25 milli-int. unit/L) than that of conventional TSH RIA (0.7 milli-int. unit/L). Precision was adequate over a wide range of concentrations, although interassay CVs at very low concentrations were good for only two kits (7.3% and 13.1%). Results by all the kits correlated to about the same degree with the TSH RIA (r = 0.92 to 0.98). All showed a positive correlation between both the basal and post-thyroliberin (TRH)-stimulation values for TSH (r = 0.78 to 0.88), and all showed similar euthyroid reference ranges for basal concentrations of TSH. With four of the six kits we could clearly distinguish most of the hyperthyroid patients from healthy euthyroids; however, basal and post-TRH TSH values were not sufficient to discriminate among groups of patients with different grades of thyroid hyperfunction.     

Detection of thyroid disorders by use of basal thyrotropin values determined with an optimized "sandwich" enzyme immunoassay

To measure the concentrations of thyrotropin (thyroid-stimulating hormone), we used the components of a commercially available two-step "sandwich" enzyme immunoassay (Enzymun-Test TSH, Boehringer Mannheim) based on the specific binding of the beta-subunit of thyrotropin by monoclonal antibodies coated on polystyrene tubes. By modifying the original assay protocol, we lowered the limit of detection to 0.18 milli-int. units/L, using a total incubation period of 22 h. With this modification we could differentiate between patients responsive to administration of thyroliberin (thyrotropin-releasing factor) and those who were non-responders, by measuring only the basal concentration of thyrotropin. Furthermore, we demonstrated a correlation between the basal concentration of thyrotropin and its increase after administration of thyroliberin (r = 0.77, n = 48).     

An automated immunoradiometric assay for human thyrotropin

In this two-site immunoradiometric assay for thyrotropin, developed for use in the "Kemtek 3000" automated radioimmunoassay system, commercially available monoclonal antibody to thyrotropin is labeled with 125I, and the solid-phase antibody is an IgG fraction of sheep antiserum to thyrotropin, covalently coupled to reprecipitated aminocellulose. There are two incubations, totalling 3 h, the sensitivity is 0.03 milli-int. unit/L. The mean thyrotropin value for 82 healthy euthyroid subjects was 1.7 milli-int. units/L (range 0.4-3.6). For 19 overtly clinically and biochemically hyperthyroid subjects the values ranged from undetectable to 0.2 milli-int. unit/L. In this assay, euthyroid and hyperthyroid subjects can be distinguished with assay of a single basal sample. The assay appears suitable for routine use as a first-line test of thyroid function.     

Thyrotroph function assessed by sensitive measurement of thyrotropin with three immunoradiometric assay kits: analytical evaluation and comparison with the thyroliberin stimulation test

We evaluated the analytical and clinical performance of three "sensitive" immunoradiometric assay (IRMA) kits (Tandem-R TSH HS, Hybritech, Inc.; EchoClonal TSH, Bio-Rad; Coat-A-Count TSH IRMA, Diagnostic Products Corp.) for measurement of thyrotropin (TSH) and compared their performance against a "regular" IRMA (ARIA-HT, Becton-Dickinson) to determine whether these assays might eliminate the need to perform the thyroliberin (TRF) stimulation test. We concluded that the Tandem and EchoClonal kits may obviate the need to perform TRF stimulation tests in some patients. Using only the basal TSH concentration to predict the TSH response to TRF, we found all three sensitive TSH assays to be useful for detecting abnormal thyrotroph function. Dose-response, linearity, analytical recovery, and specificity were acceptable for all kits, but intra-assay precision at very low TSH concentrations and analytical sensitivity differed considerably among the kits. Using the EchoClonal assay, we established a normal reference interval for TSH of 0.4-4.6 milli-int. units/L.     

Sensitive, practical bioassay of thyrotropin, with use of FRTL-5 thyroid cells and magnetizable solid-phase-bound antibodies

We have developed a new method for assessing the bioactivity of thyrotropin (TSH) in human serum by measuring cAMP production in FRTL-5 thyroid cells as an index of stimulation. To eliminate serum inhibitors of the cAMP response to TSH, we purified samples by mixing them with anti-TSH antibodies coupled to magnetizable particles. This method of immunoaffinity purification was simple and suitable for the measurement of a large number of samples. The detection limit of the bioassay was 3.13 milli-int. units of human TSH per liter. Intra-assay and interassay coefficients of variation ranged from 5.4 to 8.6% and from 12.6 to 21.5%, respectively. The concentrations of bioassayable TSH closely correlated with those of immunoassayable TSH, both in the immunoaffinity-purified samples (r = 0.946, P less than 0.001) and in the serum samples (r = 0.945, P less than 0.001) from 14 euthyroid subjects and 53 hypothyroid patients with Hashimoto's thyroiditis. The bioactivity-to-immunoactivity ratio was almost constant (0.84 +/- 0.30; mean +/- SD) over the range of concentrations of immunoassayable TSH in serum, 6.3 to 177 milli-int. units/L. We also demonstrate that the technique is applicable to measurements of bioactivities of circulating forms of immunoreactive TSH in different pathophysiological conditions.