An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.     

Cytometric bead array (CBA) for the measurement of cytokines in urine and plasma of patients undergoing renal rejection

Renal rejection is associated with an active immune response regulated by cytokines and in which immunocompetent cells are involved. Previous studies have measured high levels of cytokines in the urine and plasma in various renal dysfunction states. However, some methods used to measured cytokines hinder their use as a diagnostic tool in renal rejection. In this report, cytokine levels were determined in the plasma and urine of kidney transplant patients, with renal rejection and without it, using a cytometric bead array (CBA) technique. Concentrations of six human cytokines (IL-2, IL-4, IL-5, IL-10, TNF-alpha and INF-gamma) were established. Results show that patients who develop renal rejection presented high levels of IL-10 and IFN-gamma cytokines in plasma and urine compared to patients without renal rejection. The CBA technique displayed greater sensitivity in the determination of cytokines in urine than the conventional ELISA technique. Finally, when standard cytokines in plasma and in urine were compared, it was observed that, in plasma, levels of IL-4, IL-5, IL-10, TNF-alpha and IFN-gamma were detected, whereas in urine the levels detected were of IL-4, IL-5, IL-10 and IFN-gamma. These results indicate that the CBA assay is a sensitive method to measure cytokines in urine. In kidney transplant patients undergoing acute renal rejection, the presence of cytokines in urine reflects renal damage and could be a useful method in the diagnosis of renal rejection.     

Pre- and postoperative accommodative convergence/accommodation (AC/A) ratio: preliminary study of computer-assisted analyses

A group of 15 patients who underwent bilateral medial rectus muscle recession for esodeviation were analyzed for factors most closely correlated with the outcome. The data evaluated included age of onset of strabismus, refractive error, year of surgery, age of surgery, sex, preoperative esodeviation, and clinical assessment of preoperative heterophoria. Linear regression analysis was used to evaluate the relationships of these parameters. Computer-assisted logistic regression analysis and discriminant analysis were used to asses predictive variables for a postoperative increase in the AC/A ratio.     

Feasibility of using ultra-high field (7 T) MRI for clinical surgical targeting

The advantages of ultra-high magnetic field (7 Tesla) MRI for basic science research and neuroscience applications have proven invaluable. Structural and functional MR images of the human brain acquired at 7 T exhibit rich information content with potential utility for clinical applications. However, (1) substantial increases in susceptibility artifacts, and (2) geometrical distortions at 7 T would be detrimental for stereotactic surgeries such as deep brain stimulation (DBS), which typically use 1.5 T images for surgical planning. Here, we explore whether these issues can be addressed, making feasible the use of 7 T MRI to guide surgical planning. Twelve patients with Parkinson's disease, candidates for DBS, were scanned on a standard clinical 1.5 T MRI and a 7 T MRI scanner. Qualitative and quantitative assessments of global and regional distortion were evaluated based on anatomical landmarks and transformation matrix values. Our analyses show that distances between identical landmarks on 1.5 T vs. 7 T, in the mid-brain region, were less than one voxel, indicating a successful co-registration between the 1.5 T and 7 T images under these specific imaging parameter sets. On regional analysis, the central part of the brain showed minimal distortion, while inferior and frontal areas exhibited larger distortion due to proximity to air-filled cavities. We conclude that 7 T MR images of the central brain regions have comparable distortions to that observed on a 1.5 T MRI, and that clinical applications targeting structures such as the STN, are feasible with information-rich 7 T imaging.     

An immunoassay for the measurement of (1-->3)-beta-D-glucans in the indoor environment

An inhibition enzyme immunoassay was developed for quantitation of (1-->3)-beta-D-glucans in the indoor environment. Immunospecific rabbit antibodies were produced by immunization with bovine serum albuminconjugated laminarin.The laminarin calibration curve ranged from 40 to 3000 ng/ml.Another (1-->3)-beta-D-glucan (curdlan) showed a similar inhibition curve, but was less reactive on a weight basis. Pustulan, presumed to be (1-->3)-beta-D-glucan, also showed immunoreactivity in the assay. Control experiments indicated that this was due to (1-->3)-beta-D-glucan structures. Other non-(1-->3)-beta-D-glucan polysaccharides did not react. (1-->3)-beta-Dglucan was detectable in dust from a variety of occupational and environmental settings. We conclude that the new assay offers a useful method for indoor (1-->3)-beta-Dglucan exposure assessment.     

Quantitative ballistocardiography (Q-BCG) for measurement of cardiovascular dynamics

In the seventies of the past century ballistocardiography had been thought to be obsolete in cardiology for impossibility of objective calibration. In the present work the quantitative ballistocardiography (Q-BCG) for measurement of systolic force (F) and minute cardiac force (MF) in sitting subject was described. The new principle of piezoelectric transducer enabled to register the force caused by the heart and blood movement, which was not measured before. The calibration proved that the action of the force on the transducer was expressed quantitatively without the amplitude-, time-, and phase deformation. The close relationship of skeletal muscle force and F was proved. The F and MF changed under different physiological conditions (age, partial pressure of oxygen, body weight, skeletal muscle force). It was shown that the systolic force (F) and minute cardiac force (MF) are the physiological parameters neurohumorally regulated similarly as the heart rate or systolic volume.     

An evaluation of three methods for the measurement of diamine oxidase (DAO) activity in amniotic fluid.

The activity of diamine oxidase (DAO) in amniotic fluid was measured by three different methods. The first two, one colorimetric and the other fluorometric, depended on the coupled peroxidase assay. The other was a radiochemical procedure. The latter proved to be a reliable method, free from interference and linear over a wide range of time and substrate concentration. The coupled peroxidase assay was shown to be affected by a variety of factors. It was not linear with time, owing to a lag period that varied from sample to sample. The hydrogen peroxide-peroxidase reaction was inhibited by high substrate concentrations and by undialyzed amniotic fluid.     

Steroid isotopic standards for gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS)

Carbon isotope ratio (CIR) analysis of urinary steroids using gas chromatography-combustion isotope ratio mass spectrometry (GCC-IRMS) is a recognized test to detect illicit doping with synthetic testosterone. There are currently no universally used steroid isotopic standards (SIS). We adapted a protocol to prepare isotopically uniform steroids for use as a calibrant in GCC-IRMS that can be analyzed under the same conditions as used for steroids extracted from urine. Two separate SIS containing a mixture of steroids were created and coded CU/USADA 33-1 and CU/USADA 34-1, containing acetates and native steroids, respectively. CU/USADA 33-1 contains 5α-androstan-3β-ol acetate (5α-A-AC), 5α-androstan-3α-ol-17-one acetate (androsterone acetate, A-AC), 5β-androstan-3α-ol-11, 17-dione acetate (11-ketoetiocholanolone acetate, 11k-AC) and 5α-cholestane (Cne). CU/USADA 34-1 contains 5β-androstan-3α-ol-17-one (etiocholanolone, E), 5α-androstan-3α-ol-17-one (androsterone, A), and 5β-pregnane-3α, 20α-diol (5βP). Each mixture was prepared and dispensed into a set of about 100 ampoules using a protocol carefully designed to minimize isotopic fractionation and contamination. A natural gas reference material, NIST RM 8559, traceable to the international standard Vienna PeeDee Belemnite (VPDB) was used to calibrate the SIS. Absolute δ¹³C_VPDB and Δδ¹³C_VPDB values from randomly selected ampoules from both SIS indicate uniformity of steroid isotopic composition within measurement reproducibility, SD(δ¹³C) < 0.2‰. This procedure for creation of isotopic steroid mixtures results in consistent standards with isotope ratios traceable to the relevant international reference material.     

Minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR): quantitative measures for selectivity ratios obtained by screening assays

Development of inhibitor compounds selective against undesirable targets is critical in drug discovery. Selectivity ratios for candidate compounds are evaluated by dividing potencies from two assays assessing the off-target and target. Because all potency measurements have underlying uncertainty, understanding error propagation is essential to interpreting selectivity data. Assay noise introduces ambiguity in the statistical significance of selectivity ratios, particularly at low replicate numbers when compounds are often prioritized for subsequent testing. The ability to differentiate potency results for any pair of compounds in one assay is evaluated using a metric called minimum significant ratio (MSR). Potency results of one compound tested in a pair of assays can be differentiated by the minimum significant selectivity ratio (MSSR). To differentiate selectivity ratios for any pair of compounds, we extend this concept by proposing two new parameters called the minimum significant ratio of selectivity ratios (MSRSR) and confidence in ratio of selectivity ratios (CRSR). Importantly, these tools can be used after a single selectivity measurement. We describe these methods and illustrate their usefulness using structure-activity relationship data from a Janus kinase inhibitor project, in which these tools informed a cogent retesting strategy and enabled rapid and objective decision making.     

Labeless AC impedimetric antibody-based sensors with pgml(-1) sensitivities for point-of-care biomedical applications

This paper describes the development and characterisation of labeless immunosensors for (a) the cardiac drug digoxin and (b) bovine serum albumin (BSA). Commercial screen-printed carbon electrodes were used as the basis for the sensors. Two methods were used to immobilise antibodies at the electrode surface. Aniline was electropolymerised onto these electrodes to form a thin planar film of conductive polyaniline; the polyaniline film was then utilised as a substrate to immobilise biotinylated anti-digoxin using a classical avidin-biotin affinity approach. As an alternative approach, poly(1,2-diaminobenzene) was electrodeposited onto the carbon electrodes and this modified surface was then sonochemically ablated to form an array of micropores. A second electropolymerisation step was then used to co-deposit conductive polyaniline along with antibodies for BSA within these pores to produce a microarray of polyaniline protrusions with diameters of several mum, containing entrapped anti-BSA. The resulting antibody grafted electrodes were interrogated using an AC impedance protocol before and following exposure to digoxin or BSA solutions, along with control samples containing a non-specific IgG antibody. The impedance characteristics of both types of electrode were changed by increasing concentrations of antigen up to a saturation level. Calibration curves were obtained by subtraction of the non-specific response from the specific response, thereby eliminating the effects of non-specific adsorption of antigen. Both the use of microelectrode arrays and affinity binding protocols showed large enhancements in sensitivity over planar electrodes containing entrapped antibodies and gave similar sensitivities to our other published work using affinity-based planar electrodes. Detection limits were in the order of 0.1ngml(-1) for digoxin and 1.5ngml(-1) for BSA.     

A simple Na(2)SO(3) feeding method for K(L)a measurement in large-scale fermentors

A simple and convenient method for measuring K(L)a in large-scale fermentors was proposed. This method was based on the measurement of the dissolved oxygen concentration under steady state conditions established by an equivalency of the sulfite ion feed and chemical oxidation rates. This method had the following advantages: It was a steady state method, and so it was not necessary to consider the response lag of a dissolved oxygen probe and the response lag due to gas phase mixing in fermentors. The oxygen content of the effluent gas in this measuring system was nearly the same as that of the sparged air. Therefore, it was possible to use the oxygen partial pressure of the sparged air for the calculation of the driving force of oxygen transfer. The detailed information on the kinetics of sulfite oxidation was not necessary, because the dissolved oxygen concentration in steady state was not influenced by sulfite oxidation rates. The K(L)a measurement was finished in as short a period as 150 s, even in a fermentor with a volume of 10 m(3). Since the amount of Na(2)SO(4) accumulation in the test fermentors was very small because of the quick measurement, the K(L)a values obtained by this method were applicable to the electrolyte-free system. Furthermore, we could discharge the used liquid from the fermentors into a drain without any pretreatment due to the low salt concentration.     

An Italian program of external quality control for chromogranin A (CgA) assay: state of the art of CgA measurement

Chromogranin A (CgA) is a secretory protein produced by many neuroendocrine cells. Circulating levels of CgA have been found to be elevated in a variety of neuroendocrine tumors and may facilitate the diagnosis and management of patients with functioning as well as non-functioning forms. However, up to now the analytical methods used for assaying intact CgA and CgA-derived peptides in the circulation of patients have not been monitored in Italy by an external quality control program. Within the framework of a Ministry of Health project an external quality control program was developed to investigate the state of the art of CgA determination in Italy and to monitor the performance of laboratories carrying out this assay. This paper deals primarily with the former of these aspects. Every laboratory received the study protocol together with a questionnaire to be returned before receipt of the samples to be assayed. Serum and plasma samples obtained from a pool of routine specimens were prepared at three different concentrations of CgA, aliquoted, frozen at -80 degrees C and mailed in dry ice to the participating laboratories. Of the 43 laboratories, 21 used IRMA, 21 used ELISA and one used RIA. There was a wide range in the time of kit utilization and the number of samples assayed per year, which indicated that the participating group was heterogeneous with regard to their experience in the determination of CgA. Most laboratories routinely used serum and plasma for IRMA and ELISA, respectively, and different data fitting approaches were employed. Further analyses will investigate the possible influence of these preanalytical factors on laboratory performance.     

A role for fractalkine and its receptor (CX3CR1) in cardiac allograft rejection

The hallmark of acute allograft rejection is infiltration of the inflamed graft by circulating leukocytes. We studied the role of fractalkine (FKN) and its receptor, CX(3)CR1, in allograft rejection. FKN expression was negligible in nonrejecting cardiac isografts but was significantly enhanced in rejecting allografts. At early time points, FKN expression was particularly prominent on vascular tissues and endothelium. As rejection progressed, FKN expression was further increased, with prominent anti-FKN staining seen around vessels and on cardiac myocytes. To determine the capacity of FKN on endothelial cells to promote leukocyte adhesion, we performed adhesion assays with PBMC and monolayers of TNF-alpha-activated murine endothelial cells under low-shear conditions. Treatment with either anti-FKN or anti-CX(3)CR1-blocking Ab significantly inhibited PBMC binding, indicating that a large proportion of leukocyte binding to murine endothelium occurs via the FKN and CX(3)CR1 adhesion receptors. To determine the functional significance of FKN in rejection, we treated cardiac allograft recipients with daily injections of anti-CX(3)CR1 Ab. Treatment with the anti-CX(3)CR1 Ab significantly prolonged allograft survival from 7 +/- 1 to 49 +/- 30 days (p < 0.0008). These studies identify a critical role for FKN in the pathogenesis of acute rejection and suggest that FKN may be a useful therapeutic target in rejection.     

A radioimmunoassay for the measurement of aminopeptidase (microsomal) in human serum

A radioimmunoassay for the measurement of aminopeptidase (microsomal) (AP) in human serum was developed by using antiserum to human kidney AP. AP purified from kidney and AP present in normal serum and in serum from a patient with obstructive jaundice gave parallel logit-log transformation lines, suggesting immunological identity. The mean concentration of AP in normal serum (n = 104) was 1.33 +/- 0.30 (mean +/- SD) micrograms/ml. Men had significantly higher serum AP levels (1.41 +/- 0.30 micrograms/ml) (p less than 0.005) than women (1.24 +/- 0.28 micrograms/ml). Serum AP levels of patients with hepatoma (2.26 +/- 0.87 micrograms/ml) and cancer of the pancreas or the biliary tract (2.90 +/- 0.67 micrograms/ml) were significantly higher (p less than 0.005) than those of normal subjects. Patients with acute and chronic hepatitis (2.06 +/- 0.66 micrograms/ml) also had significantly higher serum AP levels (p less than 0.005) than normal subjects. In pregnant women, however, the increase in AP activity without the increase in AP concentration showed that the increased AP activity was due to an enzyme other than AP. The enzyme levels and activities in normal serum as well as in patients' sera were significantly correlated (normal, r = 0.77; patients, r = 0.95). Based on the specific activity of AP purified from human plasma, the enzyme activity splitting L-alanyl-beta-naphthylamide is due almost completely to AP in normal subjects and in patients with hepatobiliary diseases.     

Evaluation of isotope ratio (IR) mass spectrometry for the study of drug metabolism

Isotope ratio (IR) mass spectrometry was evaluated for the study of drug metabolism and balance using 13C, 15N2-labelled antipyrine (AP) as a test drug. Rats were given 40 mg kg-1 (13C,15N2)AP intraperitoneally. Breath, urine, faeces and blood were collected. Except for breath, samples were combusted in sealed quartz tubes. The resulting CO2 and N2 were analysed for excess 13C and 15N, relative to pre-dose samples, by IR mass spectrometry. In addition, blood levels of AP and cumulative excretion of urinary AP metabolites were determined by gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) and high-performance liquid chromatography (HPLC) respectively. Excess 13C and 15N levels in blood were comparable with observed levels of AP, and urinary recoveries of 13C (42%) were in good agreement with those calculated from HPLC data (45%). N-Demethylation, one of the important pathways of AP metabolism, was most rapidly determined by excess 13CO2 excretion in breath (8%). The IR mass spectral analysis complemented gas chromatographic/mass spectrum and HPLC analyses, and was less complex.