Dynex: multiplex ELISA technology

Conventional enzyme-linked immunosorbent assay (ELISA) is a gold standard for screening antibodies and testing for protein or antigen presence. A significant limitation of this assay resides in the fact that only one analyte can be assessed per microplate well. Here, we describe and investigate a new technology consisting of an automated ELISA system in which up to 10 analytes can be measured within one single well, thus improving productivity, accuracy, and repeatability by reducing the amount of human labor required. Another strength of the platform is that a user can load any necessary sets/subsets of beads to perform required assays, with improved flexibility compared to manufactured-loaded arrays for multiplex analysis. We also demonstrate that this system can be used to determine the pathogenicity (i.e., presence of Shiga toxins) and serotype (i.e., Escherichia coli O157) of E. coli isolates.     

High-temperature sulfidation of Fe-Nb alloys

The sulfidation behavior of Fe-Nb alloys containing up to 30 w/o Nb was studied over the range of 600–900°C in 0.01 aim. S₂ vapor. All alloys were two-phase, consisting of an Fe-rich solid solution and Fe₂Nb, and followed the parabolic rate law at all temperatures. Scales consisted of two layers-an outer layer of FeS and an inner, complex layer which contained some FeS, FeNb₂S₄ (possibly some FeNb₃S₆), NbS₂, and intermetallic particles which were either completely or only partially sulfidized. Platinum markers were located always at the interface between the two layers, which corresponded to the original metal surface. Activation energies were 18±3 kcal/mol in close agreement with the 19.8 reported for pure iron. The sulfidation rate decreased markedly with increasing Nb content of the alloys. The decrease is attributed to increasing amounts of Fe₂Nb with increasing Nb, the net effect being that the diffusion path for outward iron diffusion through the inner layer is reduced as the Nb content increases. An analysis of the structure of NbS₂ reveals that it is easily intercalated with Fe between loosely bonded layers of S-Nb-S. The S-Nb-S layers are covalently bonded which results in very low diffusivities of either S or Nb in pure NbS₂. Although intercalated Fe tends to change the Van der Waal's type bonding between layers to more ionic or covalent, Fe diffuses readily between the layers in NbS₂. Intercalation of Fe also increases the concentration of sulfur defects in NbS₂, which in turn increases the diffusivity of sulfur. Nb was observed to be immobile. Thus, it is thought that either outward iron diffusion or inward sulfur diffusion in the inner layer is the rate-controlling step, in spite of the close agreement of activation energies with that of the sulfidation of pure iron.     

Indentation Creep in Single-Crystal Cubic Zirconia at Room Temperature

Indentation creep of yttria- and calcia-fully-stabilized single-crystal zirconia has been observed at room temperature. The Knoop hardnesses decreased by 15% and 12% of their conventional short-time values, respectively, for indentation times of 100000 s.     

Positional analysis of isovaleroyl triglycerides using proton magnetic resonance with Eu(fod)3 and Pr(fod)3 shift reagents: I. Model compounds

The proton magnetic resonance spectra of isomeric triglycerides of isovaleric and palmitic acids in the presence of the downfield and upfield chemical shift reagents Eu(fod)₃ and Pr(fod)₃ were studied. The resonance profiles of the γ-protons on the isovaleroyl chains are distinctive for each of the four possible triglyceride isomers at low shift reagent/triglyceride ratios. With either reagent, the well defined γ-methyl isovalerate doublets can be used to identify 2-isovaleroyl and 1,3-isovaleroyl structures and to analyze isomeric mixtures. This technique will be useful for positional analysis of natural cetacean triglycerides containing isovaleric acid.     

Development of an operational fitness test for the Royal Air Force

Since 2002, the Royal Air Force (RAF) has been working towards developing role-related physical tests for use as an operational fitness test (OFT). The purpose of this study was to establish reliability of the OFT (comprising four tests), investigate gym-based tests as predictors of performance and establish performance standards. Fifty-eight RAF personnel performed the OFT on three occasions. A separate cohort carried out fitness and anthropometric tests before performing the OFT, by way of establishing performance predictors. Documented evidence and views of an expert panel were used to determine OFT standards. Reliability ranged from moderate to good for three tests, with one test (Dig) showing poor reliability. The 95% limits of agreement for the prediction models ranged from good to poor (6.7-34.2%). The prediction models were not sufficiently accurate to estimate confidently OFT performance, but could be used as a guide to quantify likely outcome and training needs.     

Physical demands of firefighter search and rescue in ambient environmental conditions

This study investigated the physiological responses and limitations to a simulated search and rescue scenario in a high-rise building under ambient conditions. Sixteen firefighters performed the scenario under four conditions: standard duration breathing apparatus (SDBA) and 45 mm hose; extended duration breathing apparatus (EDBA) and 45 mm hose; SDBA and 70 mm hose; EDBA and 70 mm hose. Core temperature, skin temperature and heart rate were monitored. In four of 32 trials the casualty was rescued; the remainder of the trials were terminated for safety, high core temperature or shortage of air. Final core temperature and heart rate were higher in the EDBA (39.1 degrees C; 72% heart rate reserve (HRR)) than SDBA conditions (38.6 degrees C; 67%HRR). No differences were observed between hose sizes. The scenario proved too onerous to complete successfully in the majority of cases. Replacing SDBA with EDBA eliminates air supply as a limiting factor, but brings with it challenges of managing thermal strain.     

Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity

We developed a novel bivalent antibody fragment, the linear(L-) F(ab')₂, comprising tandem repeats of a heavy chain fragmentV_H–C_H1–V_H–C_H1 cosecreted with a light chain.Functional humanized L-F(ab')₂ directed against p185^HER2 wassecreted from Escherichia coli at high titer (100 mg/l) andpurified to homogeneity. The L-F(ab')₂ binds two equivalentsof antigen with an apparent affinity (K_d = 0.46 nM) that iswithin 3-fold of the corresponding thioether-linked F(ab')₂fragment The N-terminal site binds antigen with an affinity(K_d = 1.2 nM) that is 4-fold greater than that for the C-terminalsite, as shown by the comparison of L-F(ab')₂ variants containinga single functional binding site. L-F(ab')₂ has greater antiproliferativeactivity than the thioether-linked F(ab')₂ against the p185^HER2-overexpressingtumor cell line BT474. Linear and thioether-linked F(ab')₂ havevery similar pharmacokinetic properties in normal mice, andtheir serum permanence times are respectively 7- and 8-foldlonger than the corresponding Fab fragment L-F(ab')₂ offersa facile route to bivalent antibody fragments that are potentiallysuitable for clinical applications, and that may have improvedbiological activity compared with thioether-linked F(ab')₂ fragments.