Sensitive Immunoradiometric Assay Research Articles

The Production and Characterisation of Monoclonal Antibodies Against Human Prolactin and the Development of a Two-Site Immunoradiometric Assay

Monoclonal antibodies against human prolactin (PRL) have been produced and characterised and used to develop a sensitive two-site immunoradiometric assay (IRMA). Nine anti-PRL monoclonal antibodies were assessed for reactivity in immunoblotting experiments with PRL, hPL, hGH and pituitary gland extract. There was no detectable crossreactivity with hPL or hGH. In liquid phase radioimmunoassay (RIA) studies using three of the antibodies there was no detectable crossreaction from hPL or hGH. Five antibodies were positive in immunocytochemical studies using sections of human pituitary gland. Using FPLC purified monoclonal antibodies, a two-site IRMA was developed that could assay PRL over the range 17.5-3500 mIU per litre and was readily adapted to assaying serum samples from patients. The two-site IRMA could be performed within one day without loss of sensitivity and has potential as a rapid and simple method for screening clinical samples.

Normal growth hormone response to the 75 g oral glucose tolerance test measured by immunoradiometric assay.

Estimation of plasma growth hormone (GH) during a 50 g oral glucose tolerance test (OGTT) is widely used in the investigation of suspected acromegaly and in monitoring its response to treatment. A plasma GH of < 2 mUlL measured in terms of International Reference Preparation (I RP) 66/217 by radioimmunoassay (RIA) is considered normal in the majority of UK labora­ tories, although data from the UK External Quality Assessment Scheme reveal that cut-offs as low as I mUj l.; and as high as 10mUIL, are used. Two developments suggest a need for reapprai­ sal of the normal GH response. Firstly, since 1982 the 50 g OGTT has been superseded by a 75 g OGTT in the diagnosis of diabetes mellitus, I and it would seem desirable to standardise the pro­ cedure for both conditions. Secondly, the intro­ duction of sensitive immunoradiometric assays (IRMA) for GH has made it possible now to measure levels that are below the detection limit of most RIA This short study was performed, therefore, to define the normal plasma GH response measured by IRMA during a 75 g OGTT. The normal subjects comprised 16 men and 8 women (mean age 28·6 years, range 21-48) who did not abuse alcohol and had no clinical evidence of pituitary, liver or renal disease. They were fasted overnight and rested in the sitting position for 45 min prior to insertion ofa Venflow catheter in the antecubital fossa at - 30 min. Following two basal samples, at -IS and 0 min, 75 g of glucose in 200 mL water (freshly prepared) was given orally at 09.00 h. Blood was collected at 30 min intervals for ISO min into heparinised tubes on ice, and the plasma assayed for GH using the Boots-Celltech Sucrosep kit (Boots-Celltech Diagnotics Ltd, Slough, Bucks, UK), and glu­ cose. Samples from individual patients were all

A sensitive immunoradiometric assay for the quantification of murine monoclonal antibodies in human serum

The clinical investigation of murine monoclonal antibodies (MoAbs) as potential immunotherapeutic agents necessitates their quantification in human serum. The present paper reports the development of a sensitive, non-competitive “sandwich” immunoradiometric assay specifically optimized for measurement of murine IgG in human serum. Affinity-purified goat anti-mouse IgG antibody covalently bound to acrylic microspheres serves as the solid-phase antibody and 125I-labelled goat anti-mouse IgG antibody functions as tracer. All assay reagents were obtained from commercial sources. The assay is sensitive (capable of detecting 20 ng ml −1 of murine IgG), specific (< 0.0001% cross-reactivity with human IgG), reproducible (intra-assay %RSD < 6.3%), and rapid (a 100-tube assay can routinely be processed in 3 h). The assay has a working concentration range of 20–2000 ng ml −1 and is suitable for measuring MoAbs of any antigenic specificity. The assay was validated for use with human serum and cell culture media by comparison studies with flow cytometric and immunonephelometric methods and by high-performance size-exclusion chromatographic studies. Application of this assay should facilitate further investigation of murine MoAbs as potential immunotherapeutic agents.

Luteinizing hormone secretion patterns in boys at the onset of puberty measured using a highly sensitive immunoradiometric assay.

Pulsatile LH secretion was studied in 3 prepubertal and 11 early pubertal boys by measuring plasma LH concentrations at 10-min intervals from 1200-1800 h and from 2400-0600 h using an immunoradiometric assay with a lower limit of detection of 0.10 IU/L. Plasma testosterone (T) was measured hourly. In the prepubertal boys plasma LH was not detectable during the daytime but at night 20- to 300-min periods of detectable, but low (less than 0.5 IU/L) plasma LH values occurred. A discrete episodic LH pattern was discernible, and the median number of pulses was 2 during the 6-h nocturnal sampling periods. Plasma T was not detectable (less than 1.0 nmol/L). In the pubertal boys most daytime plasma LH values were greater than 0.3 IU/L, with periods of values of 0.1-0.3 IU/L and short periods of undetectable levels as well. At night definite pulses, up to 4.7 IU/L, were found in all boys. The median number of pulses was 4 during the 6-h nocturnal sampling period. Plasma T was detectable at night in 5 of these 11 boys. The results strongly suggest that at the onset of puberty prepubertal boys (G1) have no LH secretion during the day but intermittent gonadotrophic activity during the night. In early puberty LH secretion increases in amplitude as well as frequency to a clear pulsatile pattern during the night, sometimes with pulses during the day as well.

Relationship between the concentration and rate of change of calcium and serum intact parathyroid hormone levels in normal humans.

We employed variable rates of infusion of EDTA or calcium gluconate to examine the relationships between the concentration and rate of change of serum total and plasma ionized calcium and serum immunoreactive intact PTH concentrations in normal subjects. Use of a sensitive immunoradiometric assay specific for PTH-(1-84) made it possible to determine the full range of PTH responses to perturbations in the extracellular calcium concentration. By progressively increasing the rate of administration of calcium gluconate or EDTA during rapid or slow infusions, linear rates of change in the serum calcium concentration were achieved in eight men. The slopes were 2-fold greater during the rapid infusions. Both the calcium infusions decreased serum PTH-(1-84) levels from a mean of 23.2 +/- 2.0 (+/- SE) to 6.4 +/- 1.0 and 5.6 +/- 1.0 ng/L for the rapid and slow infusions, respectively, with no obvious rate dependence. The rapid or slow EDTA infusions increased serum PTH levels to the same maximal extent (95.0 +/- 20.2 and 99.9 +/- 14.5 ng/L, respectively), also with no significant rate dependence. Thus, there was no effect of the rate of change of calcium on the PTH response, which was reflected in similar inverse sigmoidal relationships between PTH and serum total and plasma ionized calcium when the data for the slow and rapid infusions were pooled separately. Similar sigmoidal curves were found in normal women. These data suggest the feasibility of using calcium and EDTA infusions combined with an intact PTH assay to define the relationships between circulating levels of PTH-(1-84) and calcium in states of normal and deranged parathyroid physiology.

Use of Sensitive Immunoradiometric Assay for Thyrotropin in Clinical Practice

Although measurement of thyrotropin (thyroid-stimulating hormone; TSH) by radioimmunoassay was a major advance in the laboratory diagnosis of thyroid failure--replacing the time-consuming TSH stimulation test--it was not sufficiently sensitive to discriminate reliably between euthyroid and hyperthyroid patients. Measurement of the TSH response to thyrotropin releasing hormone (TRH) served this purpose, however. The recent development of TSH assays that are severalfold more sensitive and more specific than conventional radioimmunoassays has allowed distinction of euthyroid from hyperthyroid patients and eliminated the need for the TRH test. Although undetectable levels of TSH, compatible with hyperthyroidism, are occasionally noted in euthyroid patients with severe nonthyroidal illness and during the first trimester of pregnancy, false-positive results are less often recorded for TSH than for free or total thyroid hormone measurements. Measurement of TSH by sensitive immunoradiometric assay is currently the most useful first-line test of thyroid function in patients with suspected thyroid disease and, in addition, has a valuable role in monitoring the dose of thyroxine replacement therapy.

Immunoradiometric assay for basal thyroid-stimulating hormone levels: strategy for the management of thyroxine replacement.

Basal levels of thyroid-stimulating hormone (TSH) as determined by a highly sensitive immunoradiometric assay (IRMA) were evaluated in 30 clinically euthyroid patients receiving levothyroxine (T4) replacement therapy for primary hypothyroidism. In each patient, the serum TSH level had been normal as determined by conventional TSH radioimmunoassay while the patient had been receiving a constant dosage of T4 for at least three months before the study. Correlation of the TSH levels by IRMA with the concurrent concentrations of serum T4 and triiodothyronine (T3) showed that basal TSH levels by IRMA were not predictable from the serum T4 and/or T3 levels. We observed a relatively high frequency of suppression of basal TSH levels (8/30, 27%), a finding compatible with, though not necessarily indicative of, overmedication with T4. In view of the reasonable suspicion that modest but protracted overmedication with T4 may be detrimental and that suppressed TSH levels are commonly observed in conventionally managed patients, and since suppression of TSH levels is now identifiable, it seems prudent to revise the guidelines of T4 replacement therapy accordingly. In addition to maintaining clinical euthyroidism, we propose that titrating the T4 dosage to establish lower normal TSH concentrations without suppression (eg, 0.1 to 3.0 microU/ml) would assure the resolution of frank hypothyroidism, reduce the likelihood of persistent goiter, and minimize the potential for subtle but chronic overmedication.

Intestinal development in the suckling rat: effect of insulin on the maturation of villus and crypt cell functions.

The influence of insulin on the postnatal development of intestinal functions linked to villus cells (sucrase, lactase, maltase and aminopeptidase) and crypt cells (secretory component of immunoglobulins, SC) has been studied in suckling and weanling rats. At 9 days of age, the animals received a daily injection of insulin 12.5 mU g-1 body weight day-1 for 4 days. Compared with saline-treated controls, insulin had no effect on the development of the intestinal mucosal mass parameters determined in the jejunum, ileum and colon. A premature appearance of sucrase was noted in isolated jejunal villus and crypt cells, the level of activity reached by the enzyme being dependent of the amount of insulin injected. By 6 and 12 h after a single injection of the hormone (12.5 mU g-1 body weight), sucrase activity was detected in all the cell fractions along the villus-crypt axis. In villus cells of insulin-treated rats, maltase, lactase and aminopeptidase activities were significantly (P less than 0.001) increased (+201%, +50%, +207%, respectively, vs. controls), whereas the concentration of SC measured by a sensitive immunoradiometric assay was enhanced over the controls by 75% in villus cells, 83% in crypt cells and 172% in the liver. Weanling rats treated from day 10 to day 20 postpartum with 12.5 mU insulin also exhibited a higher intestinal production of SC (+93%, P less than 0.01) than did saline controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum Free Thyrotropin subunit in Congenital Isolated Thyrotropin Deficiency.

In two patients with congenital isolated thyrotropin (TSH) deficiency, serum TSH determined by a sensitive immunoradiometric assay (IRMA) was consistently undetectable. The basal levels of serum free TSH-alpha subunit (TSH-alpha) determined by a specific radioimmunoassay (RIA) were elevated in the hypothyroid state, and decreased to the undectable level during displacement therapy with thyroid hormone. The serum free TSH-alpha significantly increased following intravenous administration of thyrotropin releasing hormone (TRH). Serum free TSH-beta subunit (TSH-beta) was undectable. These findings suggest that TSH deficiency in this disease is not due to absence of thyrotroph in the pituitary gland or deficiency of TSH-alpha, but to abnormalities of the TSH-beta gene.

Optimum replacement dose of thyroid hormone assessed by highly sensitive TSH determination in patients with congenital hypothyroidism.

Serum thyroid hormone concentrations were measured in 100 samples from 25 patients with congenital hypothyroidism who were clinically well while receiving L-T4 therapy. Thyroxine concentrations were significantly higher than those of controls (p less than 0.01), while triiodothyronine was not significantly different. These samples were divided into four groups according to serum thyroid stimulating hormone concentrations as measured by highly sensitive immunoradiometric assay (IRMA-TSH). Serum thyroid hormone concentrations were compared among groups. The replacement dose of L-T4 and serum thyroid hormone in groups with undetectable IRMA-TSH were significantly higher than those in groups with normal or increased IRMA-TSH. These results show that serum thyroxine concentrations increase in most patients with congenital hypothyroidism on L-T4 therapy. Therefore, thyroxine concentrations above normal are not necessarily of clinical significance if IRMA-TSH is detectable. Undetectable IRMA-TSH might indicate the necessity for a reduction in the L-T4 replacement dose in patients with congenital hypothyroidism.

Highly Sensitive Determination of TSH in the Follow-Up of TSH-Suppressive Therapy of Patients with Differentiated Thyroid Cancer

Basal and TRH-stimulated TSH levels were determined in 72 patients with differentiated thyroid cancer on hormonal treatment, using a highly sensitive immunoradiometric assay (IRMAclon, Henning). 43 patients were under treatment with levothyroxine (T4), 29 patients with triiodothyronine (T3). In 33/43 patients (77%) under T4- and in 18/29 patients (62%) under T3-treatment basal TSH levels were below 0.1 mU/l and levels stimulated with 200 micrograms TRH i.v. were below 0.5 mU/l. 3 patients showed a significant response (to above 0.5 mU/l) in the TRH test despite basal values of less than 0.1 mU/l. In 2 patients with elevated basal TSH levels (0.23 and 0.60 mU/l, resp.) in the IRMAclon, total suppression of TSH secretion was suggested by a failure of TSH to rise after TRH. By retesting these samples in an own TSH IRMA, basal and stimulated TSH values were below 0.1 mU/l. In conclusion, basal and TRH-stimulated TSH levels are well correlated in most patients with thyroid cancer under hormonal treatment. However, in some cases (5/72) determination of basal TSH could not clearly define the degree of thyrotropic suppression. Thus, TRH testing is still necessary to establish definitely complete TSH suppression in patients with thyroid carcinoma under suppressive treatment.

Studies on the pituitary and thyroid function in patients with nonthyroid illnesses

Serum thyroid hormone and TSH concentrations were determined in patients with nonthyroidal illnesses (NTI) including 26 patients with chronic renal failure on hemodialysis, 15 patients with end-stage malignancy and 21 normal controls. Serum TSH levels were measured by a highly sensitive immunoradiometric assay Kit (RIA-gnost hTSH) and free-T4 levels were determined not only by both back-titration method and T4 derivative method, but also by magnesium precipitation method using equilibrium dialysis. TSH levels in normal controls ranged from 0.6 to 2.4 microU/ml. In 40 of 41 patients with NTI, serum T3 levels were less than 100 ng/dl, and 37 patients had diminished T3 levels, as compared with those in normal controls. Moreover, serum T4 levels were also diminished in 19 patients with NTI, and T4 levels related significantly to serum TBG levels in all patients with NTI (r = +0.76, p less than 0.01), suggesting that the diminution of T4 levels in patients with NTI can be explained partly by decreased TBG levels. However, it was remarkable that free T4 levels in serum, determined by RIA kits, were decreased in approximately half of the patients with NTI. Free T4 levels by the magnesium precipitation method were still lower in these patients when compared with those in normal controls. Serum TSH levels in patients with NTI showed a wide scattering from below to above the normal range, and the mean value was not significantly different from that in normal controls. No significant relations were found between TSH levels and T4, T3, or free T4 levels in patients with NTI.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of subcutaneous injection of a long-acting analogue of somatostatin (SMS 201–995) on plasma thyroid-stimulating hormone in normal human subjects

SMS 201-995 (SMS), a synthetic analogue of somatostatin (SRIF) has been shown to be effective in the treatment of the hypersecretion of hormones such as in acromegaly. However, little is known about the effects of SMS on the secretion of thyroid-stimulating hormone (TSH) in normal subjects. In this study, plasma TSH was determined with a highly sensitive immunoradiometric assay, in addition to the concentration of SMS in plasma and urine with a radioimmunoassay, following subcutaneous injection of 25, 50, 100 μg of SMS (4 subjects/dose) or a placebo (6 subjects) to normal male subjects, at 0900 h after an overnight fast. The plasma concentrations of SMS were dose-responsive and the peak levels were 1.61±0.09, 4.91±0.30 and 8.52±1.18 ng/ml, which were observed at 30, 15 and 45 min after the injection of 25, 50 and 100 μg of SMS, respectively. Mean plasma disappearance half-time of SMS was estimated to be 110±3 min. Plasma TSH was suppressed in a dose dependent manner and the suppression lasted for at least 8 hours. At 8 hours after the injection of 25, 50 and 100 μg of SMS, the plasma TSH levels were 43.8±19.4, 33.9±9.4 and 24.9±3.2%, respectively, of the basal values. The results suggest that SMS suppresses secretion of TSH from its suggest that SMS suppresses secretion of TSH from the normal thyrotrophs in man and thus also that attention should be paid to possible hypothyroidism during the long-term treatment of patients such as those with acromegaly with this potent analogue of SRIF.

Serum thyrotrophin in patients with destructive pituitary lesions assessed by a sensitive immunoradiometric assay.

Using a sensitive immunoradiometric assay, serum concentrations of thyrotrophin (TSH) were measured both basally and after intravenous thyrotrophin releasing hormone (TRH) in two groups of patients with destructive pituitary lesions. Group A patients, who had more extensive pituitary hormone deficiencies and biochemical evidence of hypothyroidism at the time of study, had significantly lower mean serum TSH concentrations than the group B patients who were biochemically euthyroid. Basal TSH was below the lowest value seen in a control group of normal subjects in 5 of the 8 group A patients. In all patients TSH increased significantly after injection of TRH; the peak values being significantly correlated with the basal. In contrast to recent studies, which have emphasised other mechanisms in the production of secondary hypothyroidism, our data suggest that inadequate production of immunoassayable TSH is a common cause of secondary hypothyroidism in patients with destructive pituitary disease. Little, if any, additional clinical information is to be gained by measurement of TSH after TRH stimulation in this circumstance.

Ultrasensitive assay of thyroid stimulating hormone in patients with acute non-thyroidal illness.

Serum TSH in critically ill euthyroid patients is generally within the normal range when measured with conventional radioimmunoassays. Sensitive immunoradiometric assays allow detection of low levels of serum TSH. We assessed this method in a prospective study of 34 euthyroid patients admitted to our critical care unit. Serum TSH ranged from 0.12 to 3.60 mU/l and was significantly lower for the whole group than in the controls (P less than 0.001), as also were serum total T4 and T3 values (P less than 0.001). However, 21 patients had a serum TSH within the normal range (group 1) and 13 patients (33%) had a serum TSH less than 0.40 mU/l (group 2). The two groups did not differ in age, sex, type and severity of illness, outcome, and serum T4 and T3 levels. However, the magnitude of TSH increase from the baseline value after the i.v. injection of 200 micrograms of TRH, assessed by the 30 min TSH/basal TSH ratio was significantly higher in group 2 (P less than 0.05). These results suggest that a substantial proportion of patients with acute illness have a clearly low serum TSH, unaccounted for by age, sex, type or quantified severity of illness, serum T4 and T3 levels. In addition, the secretory capacity of the pituitary to exogeneous TRH is significantly enhanced in those patients with low basal TSH.

Economy and efficiency in routine thyroid-function testing: use of a sensitive immunoradiometric assay for thyrotropin in a general hospital laboratory.

We measured thyrotropin (TSH) with a sensitive immunoradiometric assay (IRMA) in 2329 consecutive serum samples received for thyroid-function tests from hospital and general practice. Of these, 185 (7.9%) had TSH values less than 0.2 milli-int. unit/L: 33 (1.4%) were hyperthyroid, 20 (0.9%) were being treated for hyperthyroidism, 115 (4.9%) were receiving L-thyroxin, and 17 (0.7%) were clinically euthyroid but had severe non-thyroidal illnesses. In the first 506 serum samples, we also measured free thyroxin, free triiodothyronine (FT3), and total thyroxin. Thyroliberin (thyrotropin-releasing hormone, TRH) tests performed on 84 patients showed that an undetectable initial TSH (usually ascribable to therapy with thyroxin) predicted a flat TRH response. All untreated thyrotoxic patients had undetectable TSH. Experience confirmed that this TSH assay, in conjunction with a supplementary assay of FT3 when the TSH concentration is less than twice the limit of detection, is efficient and economical for routine evaluation of thyroid function in an unselected population.

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.

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.

Improved survey of TSH-suppressive therapy by sensitive determination of TSH in patients with thyroid carcinoma

The aim of hormonal treatment in differentiated thyroid carcinoma is complete inhibition of TSH secretion. As highly sensitive immunoradiometric assays (IRMA) discriminate between suppressed basal TSH (TSHb) levels in hyperthyroid patients and normal values in euthyroid subjects, the question arises whether TSHb levels could possibly substitute TRH-testing in the follow-up of these patients. Therefore we studied the sensitivity of TSHb and TRH-testing by IRMA and conventional RIA to control treatment with levothyroxine and triiodothyronine.

Circadian and pulsatile thyrotropin secretion in euthyroid man under the influence of thyroid hormone and glucocorticoid administration.

The inhibitory action of thyroid hormones (TH) and glucocorticoids on circadian and pulsatile TSH secretion was investigated in groups of five normal men by sampling blood every 10 min for 24 h (start, 1750 h). Serum TSH was measured by a sensitive immunoradiometric assay. Continuous infusion of 50 micrograms T3 or 250 micrograms T4 for 8 h (1900-0300 h) significantly suppressed serum TSH levels (T3, P less than 0.025; T4, P less than 0.05; by paired t test). Administration of 3 g sodium ipodate 7 h before TH infusion did not alter the TSH response to T3, but T4-dependent suppression was abolished. Pulsatile TSH secretion [basally, 5.8 +/- 1.3 (+/- SD) pulses/24 h, as analyzed by the PULSAR program; 6.8 +/- 1.9 by the Cluster program] was not significantly altered by any of the experimental conditions. The additional finding of blunting of the TSH response to TRH after TH alone or ipodate and T3 suggests a predominantly pituitary feedback action of TH exerted via conversion of T4 to T3. In contrast, bolus injections of 4 mg dexamethasone (dex) at 1900 and 2200 h abolished TSH pulses for at least 6 h (PULSAR, 6.6 +/- 1.6 pulses/24 h basally vs. 3.6 +/- 3.0 under dex; Cluster, 7.0 +/- 2.7 pulses/24 h basally vs. 1.6 +/- 1.6 under dex). Dex administration also resulted in a prompt, sustained, and significant suppression of basal TSH (P less than 0.0005). Together with a normal serum TSH response to TRH (in separate experiments 1, 9, and 19 h after dex administration), these data suggest that glucocorticoid feedback occurs at a suprapituitary level.