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dIN. CHEM.31/4, 634-636(1985) Highly Sensitive lmmunoenzymometricAssay for Human Thyrotropin Yuklhiko Watanabe, Nobuyukl Amino,1 Haruo Tamaki, Mleko Aozasa, Junko Tachl, Yulchl Endo, and Kiyoshi MIyaI A sensitive assay procedure for immunoenzymometricassay of serumthyrotropin(TSH) was developedby makingseveral modifications of the Enzymun-Test#{174} TSH kit (Boehringer, Mannhelm GmbH). Serum samples were first incubated in plastic tubes precoated with monoclonalantibodiesspecific to the beta subunit of human TSH. After the tubes were washed, the TSH bound to the tubes was detected with peroxidase-conjugated polyclonal antibodies to TSH. The sensitivity of the assay was 0.2 milli-int.unit/L,and the intraand interassayCVs were <10%. Analytical recoverywas 96 to 106%. The normalbasal rangeof TSH was 0.5 to 4.8 millimt.units/L. The basal levels of TSH in all but one of 48 thyrotoxic patients with Graves’ disease were less than 0.2 milli-int. unit/L, clearly different from those of normal subjects. Thyrotoxicpatients in early normal pregnancy showed TSH concentrationsof 1.7 to 2.9 milli-int.units/Lby conventional double-antibodyradioimmunoassay,possiblyfrom cross reactivity with human choriogonadotropin,but undetectable TSH by this method. Measurement of basal TSH by this sensitive assay can be used as an initial screening test for thyroid dysfunction. Addftlonal K.yphraees: “kit” method thyroid status nancy . Graves’ disease monoclonal antibodies . preg- Measurement of serum thyrotropin (TSH)2 is useful for detection of various thyroid conditions, especially mild or latent primary hypothyroidism (1-3), in which TSH is increased in serum and thus easily detected with currently routine RIA or enzyme immunoassay kits. However, in patients with thyrotoxicosis, serum TSH is suppressed by excess thyroid hormones through a negative-feedback mechanism (1, 4, 5). This makes it difficult to differentiate the basal concentrations of TSH in sera of euthyroid subjects from those of thyrotoxic patients by using commercially available kits with low sensitivity. Therefore, tests with thyroliberin (thyrotropin-releasing hormone) have been widely used to diagnose borderline or mild cases of thyrotoxicosis (4, 5). In this study we developed a sensitive enzyme immunoassay for TSH in which we made several modifications of a commercially available kit (Enzymun-Test#{174}TSH) (6). We also report here the clinical application of the procedure. available Materials and Methods Subjects: We measured basal TSH concentrations in sera of 100 normal subjects (69 women, 21-43 years old; 31 men, 22-35 years old) and 48 untreated thyrotoxic patients with Graves’ disease, including five patients in early normal pregnancy. Cases of subclinical autoimmune thyroiditis Department of Laboratory Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan. ‘Author to whom correspondence should be addressed. 2Nonstandard abbreviations: TSH, thyrotropin; T4, thyroxin; T3, T3U, T3 uptake. Received October 29, 1984; accepted January 28, 1985. friiodothyrornne; 634 CLINICAL CHEMISTRY, Vol. 31, No.4, 1985 were excluded from the group of normal subjects by measurements of anti-thyroid antibodies (7). Graves’ disease was diagnosed on the basis of clinical symptoms of thyrotoxicity and laboratory findings of increased thyroxin (‘F4) and triiodothyronine (T3) in serum, and macroaggregated albumin T3 uptake (8). Conventional radioii’nmunoassay: A commercially available double-antibody RIA kit (Daichi Radioisotope Laboratory, Tokyo, Japan) was used, with minor modifications. In brief, 100 pL of serum sample or standard TSH solution was mixed with 1004 of anti-human TSH rabbit antibody, and incubated for 3 h at room temperature. Then 1004 of i251 labeled TSH was added and the mixture was incubated for 20 h at room temperature. Finally, 1004 of anti-rabbit yglobulin goat serum was added and the mixture was incubated for 5 h at room temperature. This was centrifuged at 2000 x g for 30 mm and the radioactivity in the precipitate was counted. The concentration of standard hTSH was calibrated with wiso standard MRC 68/38. The sensitivity of this assay was 1.0 milli-int. unit of TSH per liter. Immunoenzymoassay: We used a commercially available enzyme immunoassay kit (Enzymun-Test#{174} TSH; Boshringer Mannheim GmbH, Mannheim, F.R.G.), with several modifications. We incubated 2004 of serum test sample or standard ‘NH serum (calibrated with wiio Standard MRC 68/38) with 1 mL of phosphate buffer (16 mmol/L, pH 6.9) for 17 h at 4#{176}C (instead of 1 h at room temperature as in the manufacturer’s protocol), in a plastic tube precoated with monoclonal antibodies specific to the beta subunit of the human TSH molecule. We washed the tube once with Tris HC1 buffer (50 mmol/L, pH 7.4) instead of with tap water, and we incubated the tube with 1 mL of horseradish peroxidase-conjugated polyclonal antibodies against human TSH for 2 h (instead of 1 h) at room temperature. The antibody-peroxidase conjugate had been diluted 200-fold (instead of 100-fold) with phosphate buffer (36 mmol/L, pH 6.9) 1 h before the assay. After incubation, we rinsed the tube three times with the Tris HC1 buffer containing 0.5 mol of NaC1 and 0.5 mL of Tween 20 per liter, instead of rinsing once with tap water as the manufacturer recommended. After adding 1 mL of substrate solution, we incubated the mixture for 1 h at room temperature in the dark. For the substrate we used ophenylenediamine instead of the kit-supplied ABTS#{174} [diammonium 2,2’-azino-di-(3-ethylbenzthiazoline).6-sulfonate)]. The substrate solution consisted of 10 mg of ophenylenediamine, 104 of H2O2 (300 milL solution), and 25 mL of citrate buffer (0.1 mol/L, pH 5.0). After this incubation, we stopped the reaction by adding 0.2 mL of 4 mol/L sulfuric acid and measured the absorbance of the solution at 492 nm, in a spectrophotometer. To measure the lower concentrations of TSH, we prepared fresh TSH standard in sera at concentrations of 0.2 and 0.5 milli-int. unitlL by mixing TSH-free serum with standard TSH serum (1.0 mill-mt. unitfL) from the manufacturer. Measurement of thyroid hormones: Serum T4 and T3 were measured by radioimmunoassay as described previously (8). T3 uptake (T3U) was determined with a kit for measuring macro-aggregated albumin uptake (Amersham International, Amersham, Bucks., U.K.). The free-T4 index and free-T3 index were calculated as T4 x T3U/mean normal T3U and T3 x T3U/mean normal T3U, respectively, according to the modified method of Clark and Horn (9). 7.0 Results 6.0 Figure 1 shows the standard curve for sensitive immunoenzymometric assay for human TSH. The absorbance corresponding to a TSH concentration of 0.2 milli-int. unit/L was significantly different (p <0.001) from that at 0 milli-int. unitlL; thus the sensitivity of this assay is 0.2 milli-int. unit/L. The within-assay CVs for samples containing TSH at 1.0 and 15.0 mill-mt. unitsfL were 8.0% and 5.0%, respectively. The between-assay CVs for TSH at 1.0 and 14.0 milli-int. unitsfL were 10.0% and 5.8%, respectively. Analytical recovery in the assay was 96% to 106%. As shown in Figure 2, all but one normal subject had detectable basal concentrations of TSH. TSH values in normal subjects showed a normal logarithmic distribution; the reference interval (95% confidence limits) was 0.5-4.8 milli-int. units/L. All but one patient with thyrotoxic Graves’ disease had a low (<0.2 milli-int. unit/L) basal concentration of TSH. Thus, by using this sensitive enzyme immunometric assay to measure basal TSH one can clearly distinguish normal subjects from thyrotoxic patients with Graves’ disease. The one exception we saw was a patient with Graves’ disease whose TSH was 0.9 milli-int. unit/L. Table 1 summarizes the results for this assay when applied to five thyrotoxic patients with Graves’ disease who were also pregnant. All five patients had clinical symptoms -J 00 0 5.0 0 8 0 E 4.0 0 0 (0 E 3.0 000 IU) 1- 0 #{149}#{149}#{149}#{149}#{149}SS#{149}#{149}#{149}#{149}S Normal Untreated Subjects Graves’ disease Fig.2. Basal values for TSH in serum of normal untreated patients with Graves’ disease 20A subjects and of U,’int. unit E of thyrotoxicosis C hormones, (N a C .0 0.5/ double-antibody of thyroid radioim- Discussion “0 5 10 15 20 25 T S H (miUI-Int.units/L) B E C (N a, V U C V .0 0 concentrations munoassay measured detectable TSH in their serum. However, the TSH beta-subunit-specific sensitive enzyme immunoassay clearly showed that the TSH concentrations in all these patients were <0.2 milli-int. unitfL. U .0 0 and increased but the conventional 0.05 V .0 4 0 0.5 1.0 IS H (miHP-Int.un(tS/L) Fig. 1. Standardcurvefor the present assay for human TSH (A); lower concentrations of TSH are shown on a larger scale ( ISH concentrationsof 0 to1.0 mlIll4nt unit/I are the results(mean ± SD)offive replicateassays; concentrationsof morethan 1.0 mill-mt. unit/I are the average ofduplicate assays The sensitivity of an immunoassay is affected by many factors. Among the methods used to increase the sensitivity are the introduction of high-affinity antibodies, delayed addition of labeled antigen, prolongation of incubation, utilization of immunometric assays rather than competitive immunoassays, and application of sensitively detectable tracer. Delayed addition of labeled TSH and prolonged incubation have been used to increase the sensitivity of inimunoassay for TSH (10-12) but these modifications are not suitable for use in routine tests. Very recently, Weeks et al. (13) reported a highly sensitive immunochemiluminometric assay involving monoclonal antibody and a sensitive chemiluminescent tracer. More recently, a sensitive radioinununoassay of TSH was developed by using a combination of immunometric assay and monoclonal antibodies (14,15). Immunoenzymoassay of TSH has long been used (16-18), but one problem in this assay is sensitivity. Recently, Imagawa et al. (19) developed a sensitive immunoenzymoassay for TSH in which affinity-purified polyclonal antihuman TSH IgG conjugated with /3--galactosidase was used. However, this method has not been used widely as a routine method. All these reported methods may give false values for TSH in patients with extremely high concentrations of human choriogonadotropin, such as those in early pregnancy. CLINICALCHEMISTRY,Vol. 31, No.4, 1985 635 Table 1. Thyroid Hormone T4 Values In Serum from Five TI Fr.. 14 nmol/L MT3U,% 283 7.33 24.8 286 289 170 5.74 2.97 32.4 26.6 28.6 3.85 277 5.31 Range in normalpregnancy 90-194 2.16-3.85 Serumsamples were obtained at nine to 27.4 22-29 13 Patientswith Graves’ Fr.. T3 This work was supported by a research grant from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare; by a Grant-in-Aid for Scientific Research 56570261 (to NA) from the Ministry of Education, Science, and Culture of Japan; and by a grant from the Clinical Pathology Research Foundation of Japan. References 1. Hershman JM. Utility of the radioiminunoassay of serum trophin in man. Ann Intern Med 74, 481-490 (1971). CLINICAL CHEMISTRY, Vol. 31, No.4, 1985 thyro- Disease TSH, mIIlI-lnt.unltsfL RIA EIA Indsx Indsx 18.3 28.0 407 417 2.5 1.7 <0.2 20.1 12.7 237 247 1.4 2.7 <0.2 19.9 326 2.9 <0.2 7.5-11.5 110-188 weeks of pregnancy.MT3U,macroaggregatedalbumin 13 uptake. The sensitivity of our iinmunoenzymometric assay was 0.2 milli-int. unit/L, five- to 10-fold the sensitivity of conventional competitive radioimmunoassays. This high sensitivity might be the result of use of a combination of monoclonal antibodies specific to the beta subunit of TSH and polyclonal antibodies and to the lowering of the nonspecific binding background. The reproducibility of the procedure was also satisfactory for routine use, and patients with Graves’ disease could be clearly distinguished from normal subjects. Basal TSH in serum of patients with thyrotoxicosis, measured with a highly sensitive radioassay and a chemiluminescent assay, have been reported as <0.2 milli-int. unitJL (14, 15) and <0.05 milli-int. unitlL (13), respectively. The reason why one of our thyrotoxic patients had detectable TSH (0.9 milli-int. unitJL) is not known. Because thyroid disease is common in women of childbearing age, it is often necessary to examine thyroid function in patients who are pregnant. During pregnancy the concentrations of thyroxin-binding globulin, T4, and T3 in serum are increased; the free-T4 or free-T4 index should thus be measured to evaluate thyroid function (20). However, normal values for free T4 differ markedly, depending on the RIA method used (21). Furthermore, Graves’ disease is frequently aggravated in early pregnancy (22), making especially difficult the accurate evaluation of thyroid ftmction, particularly in early pregnancy, where the choriogonadotropin concentration is often increased to as much as 200 000 int. unitsfL (20). As shown in Table 1, in all of our pregnant thyrotoxic patients with Graves’ disease, serum H was falsely detectable by the conventional RIA, but was <0.2 milli-int. unitfL by the modified ixnmunoenzymoassay. This discrepancy possibly is ascribable to cross reactivity of choriogonadotropin in the conventional RIA. For assay of low concentrations of pituitary peptide hormones, monoclonal antibodies specific to the beta subunit of the peptide should be used. During pregnancy, the thyroliberin test is not permitted for thyroid evaluation, and there is still much controversy about the “normal” concentration of free T4 in sera of pregnant subjects. Therefore, a highly sensitive assay for TSH should certainly be useful in these cases. Measurement of basal TSH is the first choice as a thyroid-function test if a highly sensitive assay is available, as suggested by Allen and Watson (14) and Alexander et al. (15). 636 Pregnant <8.0 <0.2 <0.2 0.5 -2.4 2. Mayberry WE, Gharib H, Bilstad JM, Sizemore OW. Radioimmunoassay for human thyrotrophin. Ann Intern Med 74, 471-480 (1971). 3. Nelson JC, Johnson DE, Odell WD. Serum TSH levels and the thyroidal response to TSH stimulation in patients with thyroid disease. Ann Intern Med 76, 47-52 (1972). 4. Hershman JM. Clinical application of thyrotropin-releasing hormone. N Engi J Med 290, 886-890 (1974). 5. Jackson IMD. Thyrotropin-releaning hormone. N Engi J Med 306, 145-155 (1982). 6. Kit instruction leaflet, Enzymun-Test5 TSH. Boehringer Mannheim Immunodiagnostics, Mannheim, F.R.G. 7. Amino N, Hagan SR, Yamada N, Refetofi’ S. Measurement of circulating thyroid microsomal antibodies by the tanned red cell hemagglutination technique: Its usefulness in the diagnosis of autoimmune thyroid disease. Clin Endocrinol 5, 115-125 (1976). 8. Amino N, Yabu Y, Miki T, et al. Serum ratio of triiodothyronine to thyroxine, and thyroxine-binding globulin and calcitonin concentrations in Graves’ disease and destruction-induced thyrotoxicosis. J Clin Endocrinol Metab 53, 113-116 (1981). 9. Clark F, Horn DB. Assessment of thyroid function by the combined use of the serum protein-bound iodine and resin uptake of ‘311-triiodothyronine. J Clin Endocrinol Metab 25, 39-45 (1965). 10. Hall R, Amos J, Ormston B,J. Radioimmunoassay of human serum thyrotrophin. Br Med J i, 582-585 (1971). 11. Pekary AE, Hershman HM, Parlow AF. A sensitive and precise radioimmunoassay for human thyroid-stimulation hormone. J Clin Endccrinol Metal, 41, 676-684 (1976). 12. Wide L, Dahlberg PA. Quality requirements of basal s-TEH assays in predictingan s-TSH response to TEN. Scand J Clin Lab Invest 40, 101-110 (1980). 13. Weeks I, Sturgess M, Siddle K, et al. A high sensitivity immunochemiluminometric assay for human thyrotrophin. Clin Endocrinol 20, 489-495 (1984). 14. Allen KR, Watson D. Thyrotropin as the initial screening test for thyroid disease. Clin Chem 30, 502-503 (1984). 15. Alexander WD, Kerr DJ, Ferguson MM. First-line test of thyroid function. Lancet ii, 647 (1984). Letter. 16. Miyai K, Ishibashi K, Kumahara Y. Enzyme-linked immunoassay of thyrotropin. Clin Chim Acta 67, 263-268 (1976). 17. Kate N, Naruse H, Irie M, Tsuji A. Fluorophotometric enzyme immunoassay of thyroid-stimulating hormone. Anal Biochem 96, 419-425 (1979). 18. Miyai K, Ishibashi K, Kawashima M. Two-site inununoenzymometric assay for thyrotropin in dried blood samples on fflter paper. Clin Chem 27, 1421-1423 (1981). 19. Imagawa M, Ishikawa E, Yoshitake S, et al. A sensitive and specific sandwich enzyme immunoassay for human thyroid-stimulating hormone. Clin Chim Ada 126, 227-236 (1982). 20. Amino N, Yamada T, Mitsuma T, et al. Increase in plasma thyrotropin-releasing hormone in normal human pregnancy. J Clin Endocrinol Metab 53, 1288-1290 (1981). 21. Amino N, Nishi K, Nakatani K, et al. Effect of albumin concentration on the assay of serum free thyroxin by equilibrium radioimmunoassay with labeled thyroxin analog (Amerlex5 Free T4). Clin Chem 29, 321-325 (1983). 22. Amino N, Tanizawa 0, Mon H, et al. Aggravation of thyrotoxicoals in early pregnancy and after delivery in Graves’ disease. J Clin Endocrinol Metab 55, 108-112 (1982).