1 |
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Immunochemical detection of sulfur mustard-adducts with DNA and proteins: Exploratory research on adducts with proteins
We have developed two modes of a standard operating procedure (SOP) for immunochemical detection of sulfur mustard adducts to DNA in human blood and skin. In the shortened mode data could be generated within 9 h after in vitro exposure of human blood to > 1 μM sulfur mustard. The sensitive mode allows detection of exposure to 50 nM sulfur mustard. The in vivo adduct level in blood in hairless guinea pigs remained constant during the first two weeks after administration of sulfur mustard (0.5 LD50, i.v.), whereas during the following two weeks it decreased to background level. The DNA adduct level in blood of a marmoset was initially in the same range but was decreased to marginal values at day 1 and 7. The DNA adduct level in skin after in vivo skin exposure of hairless guinea pigs had decreased 2- to 3-fold at 2 to 3 days after exposure; after 2 weeks the adduct level was only marginal but still detectable. The SOP could be set up and generate data within one day in an institute elsewhere (MRICD). Monoclonal antibodies have been obtained for adducts of sulfur mustard to hemoglobin and keratin (exposed to 50 μM sulfur mustard). Antibodies raised against adducts of sulfur mustard to keratin clearly showed binding to the horny layer of human skin exposed to a solution of 100 μM sulfur mustard or to saturated vapor of sulfur mustard for 1 min. This opens the way for development of a detection kit that can be applied directly to skin of personnel who are supposedly contaminated by sulfur mustard.
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[Abstract]
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2 |
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Evaluation of the specificity of antigen assays for plasminogen activator inhibitor 1 : Comparison of two new commercial kits
t-PA depleted citrated plasma was used to prepare standards of different molecular forms of plasminogen activator inhibitor 1 (PAI-1). These standards were used to evaluate the specificity of two new PAI-1 antigen assays: the TintElize PAI-1 antigen assay (cat. no. 210221) and the Innotest PAI-1. Differences in specificity were apparent: the Innotest PAI-1 was found to approach a 'grand total assay'.
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[Abstract]
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3 |
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Development of a Fast and Simple Method for Determination of β-Agonists in Urine by Extraction on Empore Membranes and Detection by a Test Strip Immunoassay
A rapid screening method for β-agonists in cattle urine was developed that offers speed and protocol simplicity and that could be performed on the spot. The method consists of an extraction on Empore membranes with strong cation exchange properties and a test strip immunoassay. The Empore extraction provides a fast and simple cleanup of the urines and a concentration step with recoveries ranging from 50-80% for the β-agonists of the aniline type and around 10% for salbutamol and terbutaline. A test strip immunoassay is performed on the diluted extract. With the use of an antiperoxidase antibody, an internal negative control was included, to which all color reductions are compared. Although a portable colorimeter increased the sensitivity of the test, a visual interpretation was sufficient to give a correct yes/no answer. The whole test takes about 30 min and has a visual detection limit for clenbuterol of 1 ng/mL of urine. The test also shows good sensitivity for salbutamol, despite the low recovery with the Empore extraction.
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[Abstract]
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4 |
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Biomonitoring of exposure to chemical warfare agents
An overview is presented of the major methods that are presently available for biomonitoring of exposure to chemical warfare agents, i.e., nerve agents and sulfur mustard. These methods can be applied for a variety of purposes such as diagnosis and dosimetry of exposure of casualties, verification of nonadherence to the Chemical Weapon Convention, health surveillance, assessment of low level exposures (Gulf War Syndrome) and last but not least for forensic purposes in case of terrorist attacks with these agents. This paper will focus on methods that are based on the analysis of long-lived protein adducts of CW agents which are detectable weeks or even months after exposure. Examples of real exposure incidents will be described. © 2006 Springer.
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[Abstract]
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5 |
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Determination of fibrin(ogen) degradation products (FDP) in plasma, using a monoclonal antibody
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6 |
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A bio-immuno assay to determine free tissue-type plasminogen activator (t-PA) in Stabilyte® plasma
This article describes a two-step bio-immuno assay (BIA), which determines active (free) tissue-type plasminogen activator (t-PA) in (acidified) plasma. Plasma samples, diluted in buffer of pH 6.0, are added to the wells of a microtiter plate containing an immobilised monoclonal antibody which binds t-PA without affecting t-PA activity. After an overnight incubation at 4°C, bound t-PA activity is determined by incubating the wells at 37°C with plasminogen, CNBr-digested fibrinogen and D-Val-Leu-Lys-pNA, followed by measurement(s) of the absorbance (A) at 405 nm at timed intervals. The ΔA/t2 or, alternatively, the AA after 4 hours (endpoint determination) give an accurate value for the active t-PA concentration. The assay has a lower detection limit of 3 pg t-PA/ml and does not discriminate between various forms of active t-PA. The intra-assay coefficients at 0.2 and 0.05 ng t-PA/ml were 5.5% and 4.3%, respectively. Active t-PA values in samples (N = 66) determined with this assay and with the t-PA BIA from Chromogenix correlated relatively well (r = 0.78). Within a subset of these samples (N = 18), a negative correlation with both t-PA/PAI-1 (r = 0.79) and PAI-1 antigen (r = 0.78) values was observed. Surprisingly, no correlation was found with values for t-PA antigen (r = 0.21).
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[Abstract]
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7 |
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Soluble fibrin as a molecular marker for a pre-thrombotic state: A mini-review
Increased plasma levels of soluble fibrin are considered as molecular markers of an impending thrombotic event. Several methods to assess soluble fibrin have been in existence for many years. Most of those methods are nonspecific, semiquantitative or too laborious to be used in clinical practice. More recent methods are quantitative and relatively rapid. Some of those methods may, however, not be fully specific; in other cases clinical experience is lacking. Chemicals/CAS: fibrin, 9001-31-4; Biological Markers; Fibrin Fibrinogen Degradation Products; Fibrin, 9001-31-4
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[Abstract]
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8 |
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Rapid detection of fungal α-amylase in the work environment with a lateral flow immunoassay
article |
2006
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Author: |
Bogdanovic, J.
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Koets, M.
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Sander, I.
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Wouters, I.
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Meijster, T.
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Heederik, D.
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Amerongen, A. van
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Doekes, G.
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Keywords: |
Food and Chemical Risk Analysis · Allergen exposure · fungal α-amylase · lateral flow immunoassay · rapid detection of allergens · allergen · amylase · fungal enzyme · airborne particle · ambient air · article · dough · dust exposure · enzyme assay · fluor · human · immunoassay · lateral flow immunoassay · occupational allergy · occupational hazard · priority journal · sensitivity analysis · work environment · workplace · Air Microbiology · Air Pollutants, Occupational · alpha-Amylase · Antigens, Fungal · Dust · Fungi · Immunoenzyme Techniques · Occupational Diseases · Workplace
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Background: Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with direct on-site demonstration of a bioallergen exposure hazard. Objective: In a field study, we evaluated a recently developed LFIA for fungal α-amylase, an important bakery allergen. Methods: Airborne and surface dust (wipe) samples and samples from flours and baking additives used at the workplace were collected in 5 industrial bakeries and tested in the LFIA for fungal amylase. For comparison, amylase was measured in sample eluates with the reference EIA method. Results: Sensitivity of the LFIA was 1 to 10 ng/mL, and of EIA, ∼25 pg/mL. In LFIA, most flour samples, 84% of wipe samples, 26% of personal airborne dust, and none of the 26 ambient air dust samples produced a visible reaction. Wipe samples from dough-making areas and flour samples gave the strongest reactions. All extracts with >5 ng allergen per milliliter showed a positive LFIA reaction. Conclusion: The LFIA for fungal amylase is an easy and rapid method to demonstrate the allergen directly at the worksite in less than 10 to 20 minutes. Similar LFIA methods may be used for other occupational allergens in other work environments. Clinical implications: Lateral flow immunoassays for occupational allergens may be of great value in occupational hygiene surveys to demonstrate directly to workers and supervisors the hazards of work-related bioallergen exposure. © 2006 American Academy of Allergy, Asthma and Immunology. Chemicals / CAS: amylase, 9000-90-2, 9000-92-4, 9001-19-8; Air Pollutants, Occupational; alpha-Amylase, EC 3.2.1.1; Antigens, Fungal; Dust
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[Abstract]
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9 |
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The radial immunodiffusion assay for plasma Histidine-rich Glycoprotein (HRG) based on a polyclonal antibody, shows a different specificity towards the two variants of a common amino acid polymorphism
Recently, two molecular weight forms of Histidine-rich Glycoprotein have been described which explain 59% of the variation in plasma HRG levels. Here, we demonstrate that this can partly be ascribed to a difference in specificity of the immuno assay for HRG towards the two alleles of the polymorphism.
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[Abstract]
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10 |
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Allergenicity attributes of different peanut market types
article |
2016
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Author: |
Koppelman, S.J.
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Jayasena, S.
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Luykx, D.
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Schepens, E.
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Apostolovic, D.
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Jong, G.A.H. de
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Isleib, T.G.
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Nordlee, J.
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Baumert, J.
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Taylor, S.L.
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Cheng, H.
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Maleki, S.
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Keywords: |
Biology · Allergen · Arachis hypogaea · IgE · Immunoassay · Peanut · Food and Nutrition · Healthy Living · Life · FI - Functional Ingredients · ELSS - Earth, Life and Social Sciences
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Four different market classes of peanut (Runner, Virginia Spanish, and Valencia) are commonly consumed in Western countries, but for some consumers peanuts are a main cause of food-induced anaphylaxis. Limited information is available on the comparative allergenicity of these distinct market classes. The aim of this study was to compare allergenicity attributes of different peanut cultivars.The protein content and protein profiles were highly comparable for all tested cultivars. All cultivar samples contained the major allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as assessed by SDS-PAGE and RP-HPLC, although some minor differences in major allergen content were found between samples. All samples were reactive in commercial ELISAs for detection and quantification of peanut protein. IgE-binding potency differed between samples with a maximum factor of 2, indicating a highly comparable allergenicity.Based on our observations, we conclude that peanuts from the main market types consumed in Western countries are highly comparable in their allergenicity attributes, indicating that safety considerations with regard to peanut allergy are not dependent on the peanut cultivar in question. © 2016 Elsevier Ltd.
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[Abstract]
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11 |
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The contact-system dependent plasminogen activator from human plasma: Identification and characterization
Apart from tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), a third PA appears to occur in human plasma. Its activity is initiated when appropriate triggers of the contact system are added, and the activation depends on the presence of factor XII and prekallikrein in plasma. The activity of this, so-called, contact-system dependent PA accounts for 30% of the PA activity in the dextran sulphate euglobulin fraction of plasma and was shown not to be an intrinsic property of one of the contact-system components, nor could it be inhibited by inhibitory antibodies against t-PA or u-PA. We have succeeded in identifying this third PA in dextran sulphate euglobulin fractions of human plasma. Its smallest unit (SDS-PAGE) is an inactive 110 kDa single-chain polypeptide which upon activation of the contact system is converted to a cleaved, disulphide-bridged molecule with PA activity. The native form, presumably, is an oligomer, since the apparent M(r) on gelchromatography is 600,000. The IEP is 4.8, much lower than that of t-PA and u-PA. Although the active 110 kDa polypeptide cannot be inhibited by anti-u-PA, it yet comprises at 37 kDa piece with some u-PA related antigenic determinants. However, these determinants are in a latent or cryptic form, only detectable after denaturation by SDS. The 110 kDa polypeptide is evidently not a dimer of 55 kDa u-PA or a complex of u-PA with an inhibitor. It is probably a PA derived from a gene quite distinct from that of t-PA or u-PA, but sharing some homology with u-PA. The physiological role of this contact-system dependent PA remains to be established. Chemicals/CAS: plasminogen activator, 9039-53-6; Antibodies; Enzyme Precursors; Kallikreins, EC 3.4.21.-; Plasminogen Activators, EC 3.4.21.-; Prekallikrein, 9055-02-1; Urinary Plasminogen Activator, EC 3.4.21.73
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[Abstract]
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12 |
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Effect of a common reference plasma on the inter-laboratory variation of the measurement of total and free protein S: A collaborative study of the Dutch Working Group on Haemostasis Laboratory Diagnosis
The comparability of test results for protein S between laboratories is hampered by a high inter-laboratory variability. The effect of the use and type of common reference plasma on the inter-laboratory variability of the total and free protein S measurement was evaluated. The results of 10 plasma samples measured against a centrally distributed frozen plasma and a centrally distributed lyophilized plasma were compared with those of various local reference plasmas regularly used by the 11 participating laboratories. The mean intra-assay coefficient of variation for total protein S in each laboratory varied from 3.8 to 12.8% (mean intra-assay CV of all laboratories: 7.4 ± 2.3%); for free protein S this range was 3.1 to 13.3% (mean intra-assay CV of all laboratories: 6.6 ± 2.7%). We confirmed the high inter-laboratory coefficient of variation (mean±SD) with the several local reference plasmas for both total protein S (13.4±5.6%; n=10) and free protein S (17.1±7.5%; n= 11). For total protein S, the inter-laboratory CV was reduced to 11.5±4.8% (p=0.005) by using a common frozen reference plasma, while it was increased to 16.8±3.4% (p=0.022) using a common lyophilized reference plasma. For free protein S, these values decreased only statistically significantly for the common lyophilized reference plasma, to 15.1±6.0% (p= 0.008). For free protein S, the dilution factor used was identified as a factor influencing the inter-laboratory variability. This study shows that, for both types of protein S measurements, using one frozen reference plasma shows a slight decrease in inter-laboratory variability, while a common lyophilized plasma shows inconsistent results. It is concluded that further investigation is necessary to examine other sources of variability to increase the comparability of laboratory results for both total and free protein S. Chemicals/CAS: Antibodies; Protein S; Reagent Kits, Diagnostic
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[Abstract]
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13 |
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Identification of determinants of the between-operator variation of the total protein S antigen assay: A collaborative study of the Dutch Working Group on Haemostasis Laboratory Diagnosis
In a previous study, a between-operator variability (CVOBETWEEN) of 9.6% and 15.0% was observed for total protein S antigen assays in 11 laboratories using a frozen or lyophilized reference plasma, respectively, and the need to standardize the use of lyophilized reference plasma was identified. The aim of the present study was to identify further determinants of this CVOBETWEEN in order to improve between-laboratory comparisons of test results for one method for protein S antigen assay. Two protocols were carried out: the first again involving local execution but using a joint standardized and detailed prescription of the technical performance in each laboratory; the second using a central session for all operators with the same prescription but with joint reagent and equipment. In the present study, improved handling of lyophilized reference plasma was included and resulted in comparable CVOBETWEEN of 10.9% and 9.6% for the use of frozen and lyophilized reference plasma for the local test performance. An improvement was found in the CVOBETWEEN in the central session compared with the standardized local performance, showing lower values for the central performance of 8.5 and 6.6% for frozen and lyophilized reference plasma, respectively. Further analysis of the difference between the local and central test performance identified the use of different curve fit options of data evaluation software as a significant source of this difference. Interestingly, the within-operator variability in the central performance was around 2% lower (5.9 and 6.0% for frozen and lyophilized plasma, respectively) than that in the local performance (8.1 and 8.0% for frozen and lyophilized plasma, respectively). Although the reduction is not statistically significant, it suggests an effect of reduction of the workload and simplification of procedures for individual operators on the within-operator variability. In this study, in which 11 operators/laboratories participated, the lowest variability between operators and within laboratories was obtained in the central test performance, which is suggested to be the lowest attainable variability for the measurement of total protein S antigen. The practical factors involved in local performance that require attention to reach similar levels of variability are mainly liquid handling, curve-fit procedures and simplicity of practical procedures. Chemicals/CAS: Antibodies; Antigens; Protein S
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[Abstract]
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14 |
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Use of biosensors for rapid drug residue analysis without sample deconjugation or clean-up: A possible way forward
The drug salbutamol (SBL) is a β-agonist that may be used illegally as an animal growth promoter. SBL is also a good example of a drug which is excreted in the form of glucuronides and sulfates. Such metabolites cause complexities in analysing for the presence of drug residues. In the majority of cases a process of deconjugation and sample clean-up is required prior to analysis. This is both time consuming and causes some loss of accuracy. In this study, the urine of calves treated with SBL orally for 3 d was collected during and after medication. Samples were assayed before and after hydrolysis by two different methods, radioimmunoassay (RIA) and a newly developed biosensor immunoassay (BIA). Some samples were also analysed by GC-MS. The results clearly showed that both screening assays (RIA and BIA) found high concentrations of SBL residues throughout the study. This was especially true in the BIA method. It was also demonstrated that urine sample analysis without the need for deconjugation or clean-up could be achieved. Results obtained by GC-MS tended to be an order of magnitude lower than the corresponding screening test results. This work showed that biosensor based veterinary drug residue testing procedures can be developed which can generate results in real time without the need for time consuming sample preparation.
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[Abstract]
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15 |
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Biochemistry and measurement of fibrinogen
Fibrinogen is a large heterogeneous family of closely related molecules consisting of three pairs of non-identical polypeptide chains: two Aα-, two Bβ- and two γ-chains, held together by disulphide bridges. The heterogeneity of fibrinogen is due to heterogeneities in all three chains. Four main types of assay are used to determine fibrinogen:clotting rate (Clauss), clottable protein, precipitation and immunological assays. Heterogenities may differ from person to person and may affect the apparent fibrinogen concentrations in different assays. A further complicating factor was, until recently, the lack of an international fibrinogen standard. The ratio of Clauss:enzyme immunoassay (EIA) for high + low molecular weight fibrinogen decreases during therapy for acute myocardial infarction and increases again after thrombolytic therapy to above normal values. Furthermore, high molecular weight fibrinogen tends to clot more easily than low molecular weight fibrinogen. This suggests that high molecular weight fibrinogen might be associated with increased thrombolytic risk. Fibrinogen assessed by a functional assay (Clauss) alone is strongly associated with ischaemic heart disease. Although not proven, it is conceivable that a fibrinogen with a Clauss:EIA ratio of > 1 has an even stronger association in epidemiological studies. Chemicals/CAS: disulfide, 16734-12-6; fibrinogen, 9001-32-5; Fibrinogen, 9001-32-5
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[Abstract]
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16 |
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Cost-effectiveness of 4 neonatal screening strategies for cystic fibrosis
OBJECTIVES. The purpose of this work was to assess the costs of 4 neonatal screening strategies for cystic fibrosis in relation to health effects. In each strategy, the first test was the measurement of serum concentration of immunoreactive trypsin. The second step consisted of either a second immunoreactive trypsin test (strategy 1) or a multiple mutation analysis (strategy 2). In strategies 3 and 4, a third step was added to strategy 2: a second immunoreactive trypsin test (strategy 3) or an extended mutation analysis of the cystic fibrosis gene, that is, a denaturing gradient gel electrophoresis analysis (strategy 4). METHODS. We conducted an economic-modeling exercise in the Netherlands based on published data and expert opinions. Subjects were a hypothetical cohort of 200 000 neonates, the approximate number of children born annually in the Netherlands, and we assessed the costs and number of life-years gained as a result of neonatal screening for cystic fibrosis. The costs and effects of changes in reproductive decisions because of neonatal screening were also assessed. RESULTS. Immunoreactive trypsin + immunoreactive trypsin had the most favorable cost-effectiveness ratio of €24 800 per life-year gained. Immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis achieved more health effects than immunoreactive trypsin + DNA + immunoreactive trypsin at lower cost. The incremental costs per life-year gained of the immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis strategy compared with the immunoreactive trypsin + immunoreactive trypsin strategy were €130 700, whereas the incremental costs of the immunoreactive trypsin + DNA strategy compared with the immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis strategy were €2 154 300. When changes in reproductive decisions as a result of neonatal screening are also taken into account, neonatal screening for cystic fibrosis may lead to financial savings of approximately €1.8 million annually, depending on the screening strategy used. CONCLUSIONS. Cystic fibrosis screening for neonates is a good economic option, and positive health effects can also be expected. Immunoreactive trypsin + immunoreactive trypsin and immunoreactive trypsin + DNA + denaturing gradient gel electrophoresis are the most cost-effective strategies. Copyright © 2006 by the American Academy of Pediatrics. Chemicals / CAS: DNA, 9007-49-2; trypsin, 9002-07-7; Trypsin, EC 3.4.21.4
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[Abstract]
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17 |
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Evaluation of a bioimmuno assay for t-PA activity and its relation to PAI-1 activity and antigen levels
article |
1998
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Author: |
Rosén, S.
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Wejkum, L.
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Billing-Claeson, S.
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Ghosh, R.
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Grdic, K.
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Chmielewska, J.
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Meijer, P.
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Kluft, C.
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Tengborn, L.
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Conkie, J.
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Walker, I.
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Keywords: |
Health · Alteplase · Monoclonal antibody · Plasminogen activator inhibitor 1 · Polyclonal antibody · Tissue plasminogen activator · Adult · Blood clot lysis · Clinical article · Controlled study · Female · Fibrinolysis · Human · Immunoassay · Male · Reproducibility · Thromboembolism · Thrombogenesis · Thrombosis
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A chromogenic bioimmunoassay method for determination of t-PA activities has been evaluated on plasmas from healthy individuals and from thromboembolic patients. The assay consists of an initial binding and washing step, whereby plasma t-PA is bound to a specific monoclonal t-PA antibody, which is coated on to microplate wells, followed by a chromogenic plasmin generation step, using the chromogenic plasmin substrate S-2403. Through collection of blood at pH 6 and through maintaining this pH during the binding step, in vitro inhibition of t-PA by PAI-1 is prevented and the resulting t-PA activity reflects the condition in plasma at the time of blood sampling. Through parallel incubation of plasma at pH 8, an estimation of the PAI-1 activity is obtained from the ratio of t-PA activities at pH 6 and 8. A median t-PA activity of 0.46 IU/mL (range 0.05-1.3, n = 69) was obtained for healthy individuals, whereas analysis of plasma from thromboembolic patients resulted in a median value of 0.31 IU/mL (range 0.04-1.5, n = 107). These results were in striking contrast to those obtained from 40 individuals from each group with parallel blood sampling in neutral citrate and incubation at pH 8, where the corresponding activities were 0.03 IU/mL (range 0-0.5) and 0.014 IU/mL (range 0-0.13) respectively, illustrating the severe losses of t- PA activity obtained in vitro unless precautions to avoid inhibition by PAI- 1 are undertaken. Furthermore, the results Showed an inverse correlation of the t-PA activity with PAI-1 activity and antigen levels (r = 0.74-0.91). Elevated concentrations of t-PA antigen in combination with elevated levels of PAI-1 consistently resulted in low t-PA activities. PAI-1 antigen concentrations above 70 ng/mL were always connected with t-PA activities below 0.2 IU/mL and with BIA t-PA ratios below 0.4. Prevention of t-PA inhibition by PAI-1 at pH 8 through addition of the monoclonal PAI-1 antibody MAI-12 resulted in equal t-PA activities as obtained from incubation at pH 6. Altogether, the results suggest that the BIA t-PA assay performed on plasma samples collected in acid medium provides a convenient tool for determination of basal steady state t-PA activities.
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[Abstract]
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18 |
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A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids : Saliva from patients with Sjögren's syndrome contain increased latent and active gelatinase-B levels
Here we describe a new principle for accessing the activity of the different members of the human matrix-metalloproteinases (MMPs) by a colorimetric assay. Using protein engineering, a modified pro-urokinase was made in which the activation sequence, normally recognized by plasmin (ProArgPheLys ↓ IlelleGlyGly), was replaced by a sequence that is specifically recognized by MMPs (ArgProLeuGly ↓ IleIleGlyGly). The active urokinase resulting from the activation of this modified pro-urokinase by MMPs can be measured directly using a chromogenic peptide substrate for urokinase. The assay has been made specific for MMP-9 using an MMP-9 specific monoclonal antibody. Using this antibody MMP-9 is captured from biological fluids or tissue culture media, and MMP-activity of both active and latent MMP-9 can be analysed. We determined the gelatinase-B (MMP-9) activity present in saliva from patients with Sjögren's syndrome. Using a general gelatinase assay with radioactively-labeled gelatinated collagen it was observed that gelatinase activity was slightly, though not significantly, increased in patients: general gelatinase activity in patients versus healthy controls: 17.0 ± 4.9 vs 12.2 ± 2.5 x 104 cpm/ml (p > 0.05, and 44.0 ( 4.0 vs 36.1 ± 1.9 x 104 cpm/ml (p > 0.05), for active and latent gelatinase, respectively. However, using the immunocapture activity assay (using modified urokinase) specifically MMP-9 activity was measured, which was significantly increased in saliva from patients compared to healthy controls: MMP-9 (already active): patients 8.9 ± 2.5 U/mg, controls 1.0 ± 0.5 U/mg (p = 0.002); latent plus active MMP-9: patients 53.1 ± 9.8 U/mg, controls 16.5 ± 2.6 U/mg (p = 0.01). This assay, measuring MMP-9 activity using modified pro-urokinase as a substrate can easily be adapted for the specific detection of the various members of the MMP-family or other difficult to measure proteases, in a format that can be used for high throughput screening of compounds or samples.
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[Abstract]
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19 |
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A sensitive bioimmunoassay for thrombin-cleaved two-chain urokinase-type plasminogen activator in human body fluids
Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a two-chain form (tcu-PA/T), which is virtually inactive in plasminogen activator assays. Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA) for the assessment of tcu-PA/T in human body fluids. In this BIA, urokinase antigen was immuno-immobilized in microtiter plates and treated with cathepsin C, a specific activator of tcu-PA/T, after which plasminogen activator activity was measured. The occurrence of tcu-PA/T was examined in the plasma of 27 healthy individuals and of 17 sepsis patients, and in the synovial fluid of 16 rheumatoid arthritis patients. In addition, the concentration of urokinase antigen and scu-PA were measured in all three groups. In the plasma of the healthy individuals no measurable amounts of tcu-PA/T could be found (< detection limit of 0.2 ng/ml). In the plasma of almost all sepsis patients tcu-PA/T could be detected (median value 0.4 ng/ml). The amount of tcu-PA/T was 12% of the amount of scu-PA and accounted for about 9% of urokinase antigen. In the synovial fluid of all rheumatoid arthritis patients tcu-PA/T could be measured (median value 5.4 ng/ml) at a concentration which was twofold higher than the concentration found for scu-PA. In this group tcu-PA/T contributed to about 47% of the urokinase antigen. From these data we conclude that inactivation of scu-PA by thrombin can take place in vivo under pathological conditions which involve the production of large amounts of thrombin. This way thrombin may regulate fibrinolysis and extracellular proteolysis. The BIA for tcu-PA/T can be of use for further research on the physiological role of tcu-PA/T. Chemicals/CAS: Thrombin, EC 3.4.21.5; Urinary Plasminogen Activator, EC 3.4.21.73
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[Abstract]
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20 |
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Assessment of nerve agent exposure: existiting and emerging methods
The perceived threat of the use of nerve agents by terrorists against civilian targets implies the need for methods for point-of-care (POC) diagnosis. This review presents an overview of methods that are currently available for the assessment of exposure to nerve agents. Since these methods are mostly MS based, they require complex and expensive equipment and well-trained personnel and, consequently, they are not very suitable for rapid POC diagnosis. However, new technologies are emerging that allow, among others, immunochemical detection of acetylcholinesterase inhibited by nerve agents. Also, lab-on-a-chip methodologies are under development. It is anticipated that MS methods will be suitable for POC diagnosis within a few years, due to the miniaturization of equipment and the emergence of methodologies that enable mass spectrometric analysis with little sample pretreatment and that are potentially fieldable, such as direct analysis in real time and desorption electrospray ionization MS.
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[Abstract]
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