When the concentrations of 2 or more substances are measured separately, their molar ratios are subject to the additive imprecisions of the different assays. We hypothesized that the cumulative error ...for concentration ratios of peptides containing a common sequence might be minimized by measuring the peptides simultaneously with a "trefoil-type" immunoassay.
As a model of this approach, we developed a dual-label time-resolved fluorescence immunoassay (TRFIA) to simultaneously measure proinsulin, C-peptide, and the proinsulin-C-peptide ratio (PI/C). A monoclonal antibody captures all C-peptide-containing molecules, and 2 differently labeled antibodies distinguish between proinsulin-like molecules and true C-peptide.
The trefoil-type TRFIA was capable of measuring plasma C-peptide and proinsulin simultaneously without mutual interference at limits of quantification of 48 and 8125 pmol/L, and 2.1 and 197 pmol/L, respectively. Within-laboratory imprecision values for the trefoil-type TRFIA ranged between 8.4% and 12% for the hormone concentrations. Unlike the hormone results obtained with separate assays, imprecision did not increase when PI/C was calculated from trefoil assay results (P < 0.05). Peptide concentrations were highly correlated with results obtained in individual comparison assays (r(2) > or = 0.965; P < 0.0001). The total error for PI/C obtained with the trefoil-type TRFIA remained < or = 25% over a broader C-peptide range than with separate hormone assays (79-7200 pmol/L vs 590-4300 pmol/L C-peptide). Preliminary data indicate little or no interference by heterophile antibodies.
The developed trefoil-type TRFIA is a reliable method for simultaneous measurement of proinsulin, C-peptide, and PI/C and provides proof of principle for the development of other trefoil-type multiple-label immunoassays.
The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence ...immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to >
100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death
in vitro as well as
in vivo.
Groundwater‐surface water (GW‐SW) interaction in numerical groundwater flow models is generally simulated using a Cauchy boundary condition, which relates the flow between the surface water and the ...groundwater to the product of the head difference between the node and the surface water level, and a coefficient, often referred to as the “conductance.” Previous studies have shown that in models with a low grid resolution, the resistance to GW‐SW interaction below the surface water bed should often be accounted for in the parameterization of the conductance, in addition to the resistance across the surface water bed. Three conductance expressions that take this resistance into account were investigated: two that were presented by Mehl and Hill (2010) and the one that was presented by De Lange (1999). Their accuracy in low‐resolution models regarding salt and water fluxes to a dense drainage network in a confined aquifer system was determined. For a wide range of hydrogeological conditions, the influence of (1) variable groundwater density; (2) vertical grid discretization; and (3) simulation of both ditches and tile drains in a single model cell was investigated. The results indicate that the conductance expression of De Lange (1999) should be used in similar hydrogeological conditions as considered in this paper, as it is better taking into account the resistance to flow below the surface water bed. For the cases that were considered, the influence of variable groundwater density and vertical grid discretization on the accuracy of the conductance expression of De Lange (1999) is small.
We present a time-resolved fluorescence immunoassay (TR–FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to ...different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25
μl could be analyzed in less than 2
days with a measuring range between 5 and 1250
pg (0.2–50
μg/L or 34.4–8600
pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40
μg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (
P
<
0.0001,
r
2
=
0.99). The TR–FIA method had a similar detection limit (0.16
μg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.
In low lying deltaic areas in temperate climates, groundwater can be brackish to saline at shallow depth, even with a yearly rainfall excess. For primary production in horticulture, agriculture, and ...terrestrial nature areas, the fresh water availability may be restricted to so-called fresh water lenses: relatively thin pockets of fresh groundwater floating on top of saline groundwater. The persistence of such fresh water lenses, as well as the quantity and quality of surface water is expected to be under pressure due to climate change, as summer droughts may intensify in North-West Europe. Better understanding through modelling of these fresh water resources may help anticipate the impact of salinity on primary production. We use a simple model to determine in which circumstances fresh water lenses may disappear during summer droughts, as that could give rise to enhanced root zone salinity. With a more involved combination of expert judgement and numerical simulations, it is possible to give an appraisal of the hazard that fresh water lenses disappear for the Dutch coastal regions. For such situations, we derive an analytical tool for anticipating the resulting salinization of the root zone, which agrees well with numerical simulations. The provided tools give a basis to quantify which lenses are in hazard of disappearing periodically, as well as an impression in which coastal areas this hazard is largest. Accordingly, these results and the followed procedure may assist water management decisions and prioritization strategies leading to a secure/robust fresh water supply on a national to regional scale.
In this randomized, controlled trial involving critically ill patients not receiving early parenteral nutrition, tight glucose control did not affect the length of time that ICU care was needed or ...mortality.
This multicenter, phase 2, placebo-controlled trial involved the use of a humanized antibody — ChAglyCD3 — directed against CD3 in the treatment of new-onset type 1 diabetes mellitus. Patients ...received placebo or ChAglyCD3 for 6 consecutive days and were then followed for 18 months. The insulin dose increased in patients treated with placebo but not in those treated with ChAglyCD3, and residual beta-cell function appeared to be relatively well preserved with ChAglyCD3. This approach may offer a new strategy for the preservation of residual pancreatic function in persons with newly diagnosed type 1 diabetes.
A humanized antibody — ChAglyCD3 — was directed against CD3 in the treatment of new-onset type 1 diabetes mellitus. This approach may offer a new strategy for the preservation of residual pancreatic function in persons with newly diagnosed type 1 diabetes.
The T-cell–mediated autoimmune origin of type 1 diabetes mellitus has prompted efforts to prevent the progression of disease by targeting T lymphocytes.
1
This approach was first tested with the use of cyclosporine.
2
–
4
However, the benefit was lost after the withdrawal of cyclosporine, implying a need for the indefinite administration of this agent, with the attendant risks of chronic immunosuppression. Studies in nonobese diabetic mice indicated that short-term treatment with monoclonal antibodies against CD3 induced long-term remission of established diabetes
5
,
6
through the induction of immune tolerance that involved transforming growth factor β–dependent regulatory T cells.
7
The translation to use . . .
The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive ...function tests such as the proinsulin:C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release.
Between-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5-39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20-57 months (interquartile range).
TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96-0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5-7.1 vs 6.7-9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001) with first-phase C-peptide release during clamp (also with second phase release, only available for age 12-39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release.
The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
To compare cholesteatoma care internationally and to evaluate outcomes, ear surgeons must use the same terminology. However, a clear universal definition on how to describe the extension, ...destruction and accompanying morbidity caused by the cholesteatoma is lacking. The practical applicability by means of interrater agreement is assessed for the STAMCO and the ChOLE classification.
Methods
A total of 134 adult patients derived from the nationwide multicentre study in the Netherlands, entitled Dutch Cholesteatoma Data (DCD) were included. Retrospective analysis of 134 surgical reports according to the STAMCO and ChOLE classification for localisation/extension of the cholesteatoma, complication status and ossicular chain status. Both the percentage agreement and the interrater agreement were determined for each item of the classifications and interrater agreement was compared between the classifications as a whole.
Results
Differences in interrater agreement were found for both the localisation/extension of the cholesteatoma and ossicular chain status. STAMCO classification derived from the surgical report scored better on the localisation/extension of the cholesteatoma, whereas the ChOLE classification derived from the surgical report scored better on the status of the ossicular chain. In both classifications, complication status had a low agreement level but was also poorly registered in the surgical reports.
Conclusion
Both STAMCO and ChOLE will be beneficial in uniform registration of cholesteatoma pathology in practice. Modifications proposed for both classifications may make them even more practical applicable in the future. A common denominator obtained from these two classifications may be incorporated in a standardised surgical report to facilitate evaluation which make outcomes transferable towards both classifications.
We devised a diagnostic approach based on screening plasma for an Aspergillus antigen with use of a sandwich enzyme-linked immunosorbent assay (ELISA), thoracic computed tomographic scanning, and ...radionuclide imaging for managing patients at risk for invasive aspergillosis. We used a decision analytic model to compare this alternative strategy with the conventional strategy, which relies only on the presence of clinical symptoms, persistent fever, and chest roentgenographic findings. Use of the alternative strategy reduced the number of patients who would receive antifungal treatment empirically, but this strategy was more expensive. The specificity of the sandwich ELISA had a significant impact on cost, but the sensitivity did not. A 13% prevalence of infection resulted in equal costs for both strategies. As much as 43.3% of the patients treated empirically could be given liposomal amphotericin B (L-AmB) before the conventional strategy became the most expensive. The costs of the alternative strategy were less than those of the conventional strategy when >5.3% of all patients, irrespective of strategy, were treated with L-AmB.
PDF
