Volatile organic compounds in selected micro-environments.

A program of sampling for volatile organic compounds (VOCs) in ambient air was undertaken in selected locations and micro-environments in Perth, Western Australia to characterise concentrations of target VOCs and to determine the relative strength of the contributing sources to ambient air in different micro-environments in a major Australian city. Twenty-seven locations were sampled and, of the forty-one target compounds, 26 VOCs were detected in the samples collected. The highest concentrations were recorded for benzene, toluene, ethylbenzene, xylenes (BTEX), chloroform and styrene. The maximum 12-h toluene and benzene concentrations observed were from a basement carpark and were 24.7 parts per billion (ppb) and 5.6 ppb, respectively. The maximum xylenes concentration was 29.4 ppb and occurred in a nightclub where styrene was also detected. A factor analysis of the data was undertaken. Two key factors emerge that appear to be associated with petroleum and motor vehicles and environmental tobacco smoke. A third significant occurrence was a high concentration of chloroform that was observed at a sports centre complex with a swimming pool text and was uncorrelated with other compounds in the data set. This study indicates that locations associated with motor vehicles and petrol fuel, tobacco and wood smoke and chlorinated water represent the major risks for personal exposure to VOCs in Perth.

Inherited prion disease with an alanine to valine mutation at codon 117 in the prion protein gene.

A large English family with autosomal dominant segregation of presenile dementia, ataxia and other neuropsychiatric features is described. Diagnoses of demyelinating disease, Alzheimer's disease, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker syndrome have been attributed to particular individuals at different times. An Irish family, likely to be part of the same kindred, is also described, in which diagnoses of multiple sclerosis, dementia, corticobasal degeneration and new variant CJD have been considered in affected individuals. Molecular genetic studies have enabled the classification of this disease at the molecular level as one of the group of inherited prion diseases, with the substitution of valine for alanine at codon 117 of the prion protein gene (PRNP). Only three other kindreds have been described world-wide with this mutation and only limited phenotypic information has been reported. Here we describe the phenotypic spectrum of inherited prion disease (PrPA117V). The diversity of phenotypic expression seen in this kindred emphasizes the logic of molecular classification of the inherited prion diseases rather than classification by specific clinicopathological syndrome. Indeed, inherited prion disease should be excluded by PRNP analysis in any individual presenting with atypical presenile dementia or neuropsychiatric features and ataxia, including suspected cases of new variant CJD.

Measurement of albumin in nasal lavage using the technique of chemiluminescent immunoassay.

A method has been developed that uses chemiluminescent acridinium esters rather than radioactive iodine in an immunoassay for albumin. Albumin is a protein derived from plasma sources found in nasal fluids. As such, it can be used as an important marker of plasma exudation in experimental rhinology. The assay was developed as an alternative to radioimmunoassay for a number of reasons: chemiluminescent assays do not require radioactive materials and the complex safety related aspects of their usage; the assays are easy to perform and do not require expensive equipment; and the assay is capable of the sensitivity required to detect very small changes in albumin concentrations in nasal fluids. The assay measures albumin with a sensitivity of 1.2 ng/ml. The working range of the assay is 1.0-23.7 micrograms/ml with an interassay coefficient of variation < or = 15%. This working range encompasses the range of albumin usually found in nasal lavage samples from normal volunteers.

Employment of a phenoxy-substituted acridinium ester as a long-lived chemiluminescent indicator of glucose oxidase activity and its application in an alkaline phosphatase amplification cascade immunoassay.

This paper describes the employment of a novel phenoxy-substituted acridinium ester (di-ortho-bromophenyl-AE) as a chemiluminescent endpoint indicator for ligand binding assays. The reactivity of this compound is such that it is capable of generating a high-intensity chemiluminescent signal at neutral pH. Under these conditions, when present in excess, it has been used as an indicator of hydrogen peroxide generation by the action of glucose oxidase (GOx, EC 1.1.3.4) on glucose substrate. The resulting chemiluminescent signal is a long-lived glow. The magnitude of the chemiluminescent signal is directly proportional to the quantity of GOx present and has been used to measure GOx with a sensitivity of 1.8 x 10(-16) mol. In addition, this ability to monitor GOx activity has been utilized in an alkaline phosphatase (ALP, EC 3.1.3.1) amplification cascade assay. Here ALP catalyzes the formation of FAD from a prosthetogenic substrate FADP. FAD, a cofactor for a number of oxidase enzymes, then converts inactive apo-GOx to holo-GOx, the activity of which is monitored by the chemiluminescent endpoint and facilitates detection of ALP over the range 10(-15) to 4.1 x 10(-19) mol. The clinical utility of this system has been demonstrated by application to the assay of human thyrotrophin (TSH, sensitivity 0.005 mU/liter).

Water immiscible solvent based immunoassay.

A competitive monoclonal antibody-based immunoassay which quantifies a hydrophobic hapten (Rx) in water immiscible solvents, obviating the need of a pre-extraction step, has been developed. Approximately linear dose response profiles of analyte, over the range 1-20 ugml-1 in the hydrophobic solvents, hexane, toluene and xylene were obtained. UV spectrophotometric analyses of Rx dosed hexane confirm the phenomenon of antibody-mediated transfer of analyte from the organic to the aqueous milieu. Preliminary data on the effect of water immiscible solvents on the immunoreactivity of a monoclonal antibody in free solution are presented. The potential industrial applications of water immiscible solvent based immunoassays are discussed.

Urinary growth hormone excretion as measured by a sensitive immunochemiluminometric assay.

A sensitive immunochemiluminometric assay with a detection limit of 1.1 microU/L was developed for the measurement of urinary growth hormone (UGH). The assay was shown to be specific and precise. There was a good correlation between serum growth hormone (GH) and UGH concentrations in 20 patients with acromegaly and six volunteers following an intravenous injection of recombinant GH. We concluded therefore that UGH measurements appear to provide a satisfactory index of GH secretion. The use of the assay in the investigation of growth disorders was assessed. We studied 11 pre-pubertal children, six of normal stature, and five of short stature, over a 6-month period. Sequential fortnightly measurements of UGH were carried out and height velocity was determined. The children of short stature grew at a slower rate and excreted less GH than the children of normal stature. However, we observed considerable within-individual variability in GH excretion in both groups (CV 22-98%). We therefore recommend that sequential UGH analyses should be carried out and the results interpreted in conjunction with growth measurements. However, further investigations into the renal handling of GH are needed to establish optimum sampling regimes.

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.

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.

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.

Chemiluminescent immunoassay (CLIA) for urinary albumin.

A simple chemiluminescent immunoassay (CLIA) for urinary albumin has been developed based on the use of a chemiluminescent acridinium ester-labelled human albumin and a commercially available antiserum. It includes two incubation steps and a second polyethylene glycol-assisted antibody separation. The sensitivity of detection is 0.016 mg/l, the assay working range is 0.1-5 mg/l, and the inter-assay CVs are less than or equal to 15%. Using 10- and 50-fold sample dilutions in assay buffer, a wide working range (1-250 mg/l) is obtained covering normal and pathological conditions. Timed overnight urine samples (bed rest conditions) were collected on three consecutive days for each patient. Albumin excretion rate (AER) was 4.7 +/- 2.7 micrograms/min (mean +/- SD), range 1-15.9 micrograms/min in 36 healthy subjects (17 male, 19 female, ages 4-56 years), with day-to-day variations of 28.5 +/- 20% (mean +/- SD), range 3.3-76.1%. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the disadvantages of short shelf-life and health and safety hazards associated with radioisotopes. Results compare favourably with those obtained using a commercially available RIA kit.

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.

Development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone.

Abstract We describe the development and validation of a two-site immunochemiluminometric assay for rat growth hormone-releasing hormone (GHRH) based on the affinity purification of polyclonal rabbit antisera to rat GHRH using a human 1-29 GHRH affinity column. Assay sensitivity is 3.2 pg/ml using 100 mul of unextracted sample and the working range for the assay within 15% confidence limits is 64 to 5,000 pg/ml. Rat hypothalamic extract and secreted material demonstrated a single large peak of immunoreactive material coeluting with synthetic rat GHRH on high-performance liquid chromatography with a smaller, earlier peak which probably represents methionine sulphoxide [Met(O)(27)] GHRH. Extracted material diluted in parallel to the standard curve. Incubated rat hypothalami readily released measurable amounts of rat GHRH which responded appropriately to depolarization with 60 mM K(+) in a Ca(2+)-dependent manner.

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.

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.

Effect of surfactants on the intensity of chemiluminescence emission from acridinium ester labelled proteins.

In order to establish optimum conditions for the chemiluminescent (CL) reaction of two acridinium ester labelled proteins (human albumin and rabbit anti-human albumin IgG), we investigated the effects of the following factors known to influence the CL emission: pH, presence of proteins, relative concentrations of components of CL reaction and presence of surfactants. Under optimal conditions of pH and hydrogen peroxide concentration, hexadecyl trimethyl ammonium chloride (CTAC) increased the intensity of the CL reaction of the acridinium ester labelled albumin by 42-fold. Triton X-100, Tween-20, 23 lauryl ether (Brij 35) and sodium dodecyl sulphate (SDS) exerted a much smaller effect. In the case of the acridinium ester labelled antibody, the greatest increase was obtained with Triton X-100 (15-fold) followed by CTAC, Brij 35 and Tween 20 (SDS decreased the emission intensity).

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.

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.

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.

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.