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Biomedical subjects

I Weeks

Publications and source records attributed to I Weeks.

At least 19 recordsLinked to original sources
PubMed1992

Immunochemiluminometric assays (ICMA) specific for growth hormone releasing hormone 1-44 NH2 and 1-40 OH.

We describe specific two-site immunochemiluminometric assays able to directly measure human growth hormone-releasing hormone 1-44 NH2 and 1-40 OH concentrations in unextracted plasma. A common N-terminal antibody was purified from polyclonal rabbit antisera to growth hormone-releasing hormone 1-44 NH2 on a growth hormone-releasing hormone 1-29 NH2 linked affinity column and labelled with chemiluminescent acridinium ester. C-terminal specific monoclonal antibodies to growth hormone-releasing hormone 1-44 NH2 and 1-40 OH were raised in Balb/C mice and used as solid phase antibodies. Assay of fasting specimens from normal individuals gave medians (and ranges) of 23 pg/ml (2-200) and 30 pg/ml (3-134) for growth hormone-releasing hormone 1-44 NH2 and 1-40 OH, respectively. Samples from a series of acromegalics showed that most have values in the normal range though median values were higher, 56 pg/ml for growth hormone-releasing hormone 1-44 NH2 (P < 0.001) and 52 pg/ml for 1-40 OH (P < 0.001). Using these assays it will be possible for the first time to directly study the physiology and pathophysiology of these two peptides.

Acromegaly↗
PubMed1992

Preparation of a chemiluminescent imidoester for the non-radioactive labelling of proteins.

A chemiluminescent aryl acridinium ester was synthesized which possesses an imidate ester group capable of reacting with proteins under mild conditions. The compound can be detected at levels as low as 5.2 x 10(-19) mol using commercially available luminometers and can therefore be used to produce high specific activity labelled antibodies for use in immunochemiluminometric assays. The imidate ester compares favourably with a previously reported N-succinimidyl ester in terms of its labelling properties but is easier to synthesize, requiring one less step. The compound was used to label affinity purified to synthesize, requiring one less step. The compound was used to label affinity purified sheep antibodies to human parathyroid hormone to demonstrate its utility in a two-site immunochemiluminometric assay for the measurement of intact parathyroid hormone.

Acridines↗
PubMed1989

A rapid and sensitive method for estimating low concentrations of albumin in human urine.

A chemiluminescence immunoassay has been developed for the measurement of albumin concentrations in human urine, as an indicator of diabetic nephropathy. The assay involved competition between analyte albumin and an acridinium ester labelled albumin tracer for binding to a rabbit (anti-human albumin) antibody. Immune complexes were separated using sheep (anti-rabbit immunoglobulin G) antibodies coupled to paramagnetic particles. The total incubation time was ninety minutes at room temperature followed by sedimentation and washing of the solid-phase using a magnetic rack. Chemiluminescence emission was quantified rapidly (2 s) using a commercially available luminometer. The assay was sufficiently sensitive (10 ng/ml) for the detection of microalbuminuria with the advantages of rapidity and use of stable reagents. The assay correlated well with both RIA and rate nephelometry.

Acridines↗
PubMed1989

Magic Lite design and development.

Chemiluminescence immunoassays have now achieved a recognized place in the diagnostic laboratory. The advantages of this non-isotopic technology derive from the use of acridinium esters which can be used to label antigens and antibodies to high specific activities, as well as from optimized immunochemistry. The availability of simple, reliable instrumentation for chemiluminescence measurement together with a range of assay kits offers a logical alternative to traditional radioimmunoassay.

Humans↗
PubMed1988

Comparison of the performance and clinical utility of a carboxy-terminal assay and an intact assay for parathyroid hormone.

A comparison of the performance of a two-site immunochemiluminometric assay for intact parathyroid hormone with that of an in-house radioimmunoassay for carboxy terminal parathyroid hormone has been performed on samples from unselected patients being investigated for hypercalcaemia. The intact parathyroid hormone assay was found to be a simple and robust technique with a broad working assay range (CV less than 10% between 1.8-212 pmol/l) and a detection limit of 0.2 pmol/l. Clinically it is superior to the carboxy terminal assay in its ability to distinguish between patients with hyperparathyroidism from those with other causes of hypercalcaemia especially in the presence of impaired renal function.

Adult↗
PubMed1988

A sensitive chemiluminescence based immunoassay for antibody to staphylococcal peptidoglycan.

A sensitive chemiluminescence based immunoassay is described for measuring antibody to staphylococcal peptidoglycan in blood and dialysates from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Peptidoglycan was isolated from a strain of S. epidermidis obtained from the dialysate of a CAPD patient with peritonitis and after sonication used to coat polystyrene beads. The coated beads were incubated with standard or sample and bound IgG was detected by the addition of affinity-purified goat anti-human IgG labelled with acridinium ester. After a wash stage 0.1 M nitric acid containing 0.1% hydrogen peroxide was added to the beads. Subsequently the chemiluminescence produced following the addition of 0.3 M sodium hydroxide was measured over a 2 s time interval with an automatic luminescence analyser. Using this technique the optimum dilution of serum for detecting antibodies to peptidoglycan was found to be 1/800 and for overnight effluent from CAPD patients the dilution was 1/8. Initial values of serum and dialysate antibody levels from 34 subjects are presented. This method has the advantage that it will detect concentrations of anti-peptidoglycan which are less than 1% of those in sera, the reagents remain stable for long periods and large numbers of samples can be processed on the same day.

Antibodies, Bacterial↗
PubMed1988

A new and rapid immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein was purified to apparent homogeneity from human urine by repeated precipitation with 0.58 mol/l NaCl and gel permeation chromatography under dissociating conditions on Bio-Gel A1.5M. The protein was found to consist of a single polypeptide chain of Mr 100,000 under non-reducing conditions and Mr 75,000 under reducing conditions. Antibodies to Tamm-Horsfall glycoprotein were raised in rabbits and subsequently purified by affinity chromatography using the glycoprotein linked to Sepharose 4B. The specificity of these antibodies was confirmed by Western blotting and by indirect immunofluorescence staining of human kidney tissue. The purified antibodies were labelled with 4-(2-succinimidyloxycarbonylethyl)phenyl-10-methyl-9-acridinium carboxylate fluorosulphonate, an acridinium ester, to a specific activity of 6 X 10(5) photon counts/ng of protein, and used to establish a two-site immunochemiluminometric assay for the measurement of Tamm-Horsfall glycoprotein in serum and urine. The bound and the free fractions were separated by a second antibody to Tamm-Horsfall glycoprotein linked to paramagnetic particles. The bound antibodies were quantified by chemiluminescence. The assay had a sensitivity of detection of 2 ng/ml and a working range, as determined by inter-assay precision profiles, of 30-500 ng/ml. The range in serum samples from volunteers with normal renal function (n = 92) was 74-520 ng/ml and the mean 24-h excretion rate in healthy subjects (n = 32) was 70 +/- 26 mg.

Animals↗
PubMed1987

Circulating TSH levels measured with an immunochemiluminometric assay in patients taking drugs interfering with biochemical thyroid status.

Serum TSH was measured using a high sensitivity immunochemiluminometric assay (ICMA) in patients receiving anticonvulsant drugs, heparin or non-steroidal anti-inflammatory agents (NSAIs) and the results compared with those from groups of age- and sex-matched controls. The TSH results have also been compared with those of estimates of free thyroid hormone levels using Amerlex analogue method reagents. All patients were clinically euthyroid and TSH concentrations were in the normal, euthyroid range and did not differ significantly in any group studied. In contrast, Amerlex free T4 levels were significantly reduced in the patients treated with phenytoin (P less than 0.001), carbamazepine (P less than 0.01), sodium valproate (P less than 0.01) and heparin (P less than 0.001). Patients treated with NSAI agents showed no significant change in free T4 levels. Amerlex free T3 levels were also significantly reduced in the patients treated with phenytoin (P less than 0.005), carbamazepine (P less than 0.005) and heparin (P less than 0.001) but not in those treated with sodium valproate or NSAIs. These data support the view that measurement of circulating TSH with an assay of sufficient precision in the relevant range provides a reliable way of assessing thyroid status and could be used to exclude hyperthyroidism in patients taking the medications investigated in this study.

Adolescent↗
PubMed1987

An immunochemiluminometric assay for serum free thyroxine.

An immunochemiluminometric assay has been developed for the measurement of free T4 concentrations in serum. The assay uses chemiluminescent acridinium ester labelled monoclonal antibodies which react with free T4 in the sample. A T4-rabbit immunoglobulin G conjugate competes for antibody binding sites, immune-complexes containing this being isolated using an anti-immunoglobulin G antibody coupled to paramagnetic particles. Associated chemiluminescence intensity is thus dependent on the free T4 concentration. The assay distinguishes patients with primary thyroid disease from euthyroid subjects and is unaffected by abnormal binding proteins which compromise the diagnostic accuracy of radiolabelled analogue immunoassays. the test yields results which accurately reflect the clinical thyroid status of euthyroid patients with a variety of acute and chronic non-thyroid illnesses. This is again in marked contrast to the aberrant results seen using certain radiolabelled analogue procedures.

Adolescent↗
PubMed1987

Circulating intact parathyroid hormone measured by a two-site immunochemiluminometric assay.

A direct immunoassay for circulating intact human PTH (hPTH) is described. The method relies on the formation of an immune complex of labeled antiamino-terminal PTH antibody, intact hPTH, and solid phase antimidregion PTH antibody. A chemiluminescent aryl acridinium ester is used as label. Serum samples (100 microL) are incubated with labeled antibody, and subsequently the bound fraction is separated by the addition of solid phase antibody. The bound luminescence is quantitated in an automatic luminometer. Luminescence intensity is directly proportional to the amount of intact PTH present in the sample. Only intact PTH was found to react in this system; there was no significant interference from PTH fragments. The assay detection limit of 0.8 pmol/L hPTH-(1-84) allowed detection of intact PTH in the serum of all normal subjects tested. A clear distinction was found between hypercalcemic individuals subsequently proven to have primary hyperparathyroidism and those with malignancies. The assay offers several advantages over previously described PTH immunoassays with regard to specificity, rapidity, and reagent stability. It, thus, provides a valuable means of investigating parathyroid physiology and clinical disorders of extracellular calcium metabolism.

Adult↗
PubMed1987

Studies in patients with hyperparathyroidism using a new two-site immunochemiluminometric assay for circulating intact (1-84) parathyroid hormone.

A recently developed chemiluminescent immunoassay for 1-84 intact parathyroid hormone (PTH) demonstrated increased specificity by virtue of two-site antibody binding and increased sensitivity by use of a chemiluminescent technique. Basal PTH levels were measured in three groups of subjects: (1) normal (n = 82), (2) hyperparathyroidism (n = 31), and (3) patients with hypercalcemia of malignancy (n = 16). There was good discrimination between normal (1.2 to 9.4 pmol/L) and hyperparathyroid subjects (9.2 to 53.4 pmol/L). In persons with hypercalcemia of malignancy all PTH levels were within the normal range (0.8 to 5.2 pmol/L) or suppressed. PTH release was stimulated by the intramuscular injection of 100 IU salmon calcitonin in 6 normal controls, 10 patients with primary hyperparathyroidism due to adenoma, and 5 with four-gland hyperplasia. There was no significant rise in PTH concentration and out of the normal range in the control subjects, but the adenoma patients demonstrated a mean rise of 24.4%, 26%, and 33%, and hyperplasia patients, a mean rise of 37%, 47%, and 37% over basal levels at 120, 180, and 240 minutes. The mean absolute rise in PTH concentration was 13.4 +/- 7.7 pmol/gm of parathyroid tissue in the adenomas and 27.2 +/- 9.5 pmol/gm of parathyroid tissue in the hyperplastic glands; this difference was significant (p less than 0.05). Serial blood samples from a central vein were taken at surgery for hyperparathyroidism, and the rate of decay of the intact hormone was studied in 9 patients after removal of the parathyroid tissue. This decay was rapid with a half-life of 300 seconds. We conclude that this new specific and sensitive intact PTH assay will provide a valuable means of investigating dynamic aspects of parathyroid physiology.

Adult↗
PubMed1986

Measurement of human growth hormone (hGH) using a rapid immunochemiluminometric assay.

A two-site immunochemiluminometric assay for human growth hormone has been developed based on the use of chemiluminescent acridinium ester labelled monoclonal antibodies and a magnetisable particle - polyclonal antibody solid-phase. The assay has an incubation time of 1 h at room temperature and rapid separation and quantitation stages. The sensitivity of detection is 0.12 mU/l and the working range is 0.88 to greater than 100 mU/l for inter-assay CVs less than or equal to 15%. The assay is convenient both technically and clinically since the wide range of growth hormone levels seen in growth hormone deficiency and acromegaly can be quantified accurately in a single test without the need for repeated sample dilution.

Acromegaly↗
PubMed1986

The circadian variation of thyrotrophin in patients with primary thyroidal disease.

We have used a highly sensitive immunochemiluminometric assay (ICMA) for human TSH to study the effect of thyroid status on the circadian variation in TSH levels. Three subjects with Graves' disease, three with toxic multinodular goitre and three with euthyroid multinodular goitre were sampled every hour for 24 h. The results obtained were compared to those from five euthyroid control subjects. It was found that some patients with hyperthyroidism and suppressed basal TSH levels exhibited a 24 h secretory pattern similar to that seen in normal subjects with peak TSH levels occurring at night. In addition two subjects with a euthyroid multinodular goitre demonstrated levels of TSH below the normal range despite being clinically and biochemically euthyroid. TSH suppression in these subjects is probably related to some degree of thyroid autonomy and possible development of hyperthyroidism in the future.

Adult↗
PubMed1986

Chemiluminescent labeled-antibody assay for thyroxin in serum, with magnetic separation of the solid-phase.

A chemiluminescent labeled-antibody immunoassay has been developed for measurement of total thyroxin (T4) in serum. Monoclonal antibodies to T4 labeled with a chemiluminescent acridinium ester are used. Serum samples are incubated with the labeled antibodies and a thyroxin-rabbit immunoglobulin conjugate, then reacted with magnetizable particles containing sheep anti-rabbit immunoglobulin. The total reaction time is 40 min. The chemiluminescence intensity of the solid-phase immune complexes is inversely proportional to the concentration of T4 in the sample. The sensitivity of the assay is 1 nmol/L, and the working range of 20-190 nmol/L is characterized by CVs less than or equal to 10%.

Animals↗
PubMed1985

Measurement of C9 concentrations using an immunochemiluminometric assay.

A 2-site immunochemiluminometric assay is described for the measurement of complement component C9 concentrations in biological fluids as an aid in the diagnosis and management of immune-based diseases. The assay utilises 2 monoclonal antibodies one of which is labelled with a chemiluminescent acridinium ester and one of which is covalently coupled to reprecipitated aminoaryl cellulose. The incubation time is 1 h with simultaneous reagent addition. The working range of the assay is 10-2500 micrograms/1 at CVs less than or equal to 10% and the results are in excellent agreement with those of an immunoradiometric assay. The assay exhibits superior performance to other immunochemical and immunoassay techniques and has the advantage of using stable, non-radioactive reagents.

Antibodies, Monoclonal↗