Least detectable concentration and dynamic range of three immunoassay systems using the same antibody

The least detectable concentration (LDC) and dynamic range (DR) of three immunoassay systems are compared using four distinct antibodies (all specific for estradiol but with different affinities) on each system. The systems evaluated include the industry standard, ELISA, and two biosensors, surface plasmon resonance and kinetic exclusion. In all cases, the measurements of inhibition curves (response vs estradiol concentration) were contracted to outside experts (the biosensor manufacturers themselves in the case of the biosensors), each of whom was supplied with the same blind samples. Each biosensor manufacturer also reported an estimate of the equilibrium dissociation constant (Kd) for each of the antibodies. The LDC and DR observed for the kinetic exclusion biosensor are consistent with an interpretation of Kd limited detection while that from the other biosensor and ELISA show limits of detection somewhat above those expected for Kd limited performance. The determined LDC and DR of each biosensor is self-consistent in the sense that none of the inhibition data contradicts theoretical limits associated with the Kd as measured on that system; however, some contradictions are apparent across platforms. The use of multiple antibodies of differing Kd improves confidence that the observed differences in performance are associated with the immunoassay system rather than the particular analyte.     

Calculating optimal modulation periods to maximize the peak capacity in two-dimensional HPLC

Theoretical calculations are presented to optimize modulation period for maximum total peak capacity in comprehensive two-dimensional HPLC (2D-HPLC) taking into account the effect of modulation on the apparent peak capacity of the first-dimension (1D) separation. Results indicate that modulation periods are most favorable when they are adjusted to approximately 2.2-4 times the standard deviation of a 1D peak in order to avoid excessively short run times at the second dimension (2D). Data are presented that effective peak capacities of several thousand in 60 min can be expected for practical 2D-HPLC conditions, utilizing 1D gradient elution followed by 2D isocratic elution, that remain at approximately 50-70% of the theoretical maximum peak capacity. This work suggests that lower modulation frequencies and longer 2D separation times than previously proposed are favorable under realistic chromatographic conditions, alleviating some practical problems associated with 2D-HPLC.     

Hollow fiber optics with improved durability for high-peak-power pulses of Q-switched Nd:YAG lasers

To improve the damage threshold of hollow optical waveguides for transmitting Q-switched Nd:YAG laser pulses, we optimize the metallization processes for the inner coating of fibers. For silver-coated hollow fiber as the base, second, and third Nd:YAG lasers, drying silver films at a moderate temperature and with inert gas flow is found to be effective. By using this drying process, the resistance to high-peak-power optical pulse radiation is drastically improved for fibers fabricated with and without the sensitizing process. The maximum peak power transmitted in the fiber is greater than 20 MW. To improve the energy threshold of aluminum-coated hollow fibers for the fourth and fifth harmonics of Nd:YAG lasers, a thin silver film is added between the aluminum film and the glass substrate to increase adhesion of the aluminum coating. By using this primer layer, the power threshold improves to 3 MW for the fourth harmonics of a Q-switched Nd:YAG laser light.     

Design and evaluation of an antiparallel coupled resonator for chemical sensor applications

In this paper, a new type of quartz crystal resonator in which the electrodes are located on one side has been developed for chemical sensing. The resonator has two electrodes for exciting thickness shear mode (TSM) vibrations on one side of the crystal and a conductive layer on the other side. These electrodes are capacitively coupled with the electric fields in opposite directions, forming an antiparallel coupled resonator (ACR). The resonant characteristics of the ACR were evaluated as a function of gap width between the two electrodes used to excite the TSM. The conductance value was observed to increase with decreasing gap width. We also discovered that the gap should be parallel with the crystallographic x-axis to obtain the highest sensitivity. The frequency response to a viscous loading was almost same as that of a standard quartz crystal microbalance (QCM). The ACR sensor is an attractive alternative to a QCM chemical sensor because it can be easily integrated into packaging and film coatings.     

Noncompetitive detection of low molecular weight peptides by open sandwich immunoassay

Small peptides with less than 1000 in molecular weight are not considered amenable to sandwich immunoassays due to their difficulty of simultaneous recognition by two antibodies. As an alternative, we attempted noncompetitive detection of small peptides by open sandwich enzyme-linked immunosorbent assay (OS-ELISA) utilizing the antigen-induced enhancement of antibody VH/VL interaction. Taking fragments of human osteocalcin (BGP), a major non-collagen peptide produced in bone, as model peptides, OS immunoassay was performed using the cloned VH and VL cDNAs from two anti-BGP monoclonal antibodies either recognizing the N- or C-terminal fragment, respectively. When the clones were used for OS-ELISA with immobilized VL fragment and phage-displayed VH fragment, enhanced VH/VL interaction upon BGP addition was observed. Especially the clone for the C-terminal fragment showed a superior detection limit as well as a wider working range than those of competitive assay. The result was reproduced with purified VH-alkaline phosphatase and MBP-VL fusion proteins, where the latter was directly immobilized onto the microplate wells. The minimum detectable fragment was the hexamer including the C-terminus. This simple approach with a single monoclonal antibody with a short measurement time may prove a useful tool in immunodiagnostics as well as in proteomics research.     

Effect of probe arrangement on reproducibility of images by near-infrared topography evaluated by a virtual head phantom

The effect of the probe arrangement on the reproducibility of topographic images of the concentration changes in oxygenated hemoglobin and deoxygenated hemoglobin is evaluated by a virtual head phantom. A virtual head phantom consists of five types of tissue the 3D structure of which is based on a magnetic resonance imaging scan of an adult head. Localized and broadened brain activation is assumed in a virtual head phantom. The topographic images are obtained from the reflectance detected by the standard probe arrangement and the double-density probe arrangement. The uneven thickness of the superficial layer, which cannot be evaluated by the previous slab model, affects the distribution of measured activation in the topographic image, and this reduces the position reproducibility of near-infrared (NIR) topography with the standard probe arrangement. The overlapping measurements by the double-density probe arrangement can improve the reproducibility of the image obtained by NIR topography.     

Development of integrated whole body counter

Instead of conventional human counter using NaI(Tl) scintillator in scanning bed geometry, integrated whole body counter was developed. This is constituted by 10 detectors that are located just above the objective organs in order to improve the identification of nuclides. Two sets of p-type arrayed planar HPGe detectors composed of two crystals are used for lungs, and two sets of p-type high efficiency coaxial HPGe detectors are used for gastrointestinal tract. Similarly n-type HPGe is used for chest or thyroid or skull and the other p-types are used for the liver and kidney, respectively. An electric cooling system by adiabatic expansion was adapted as cooling apparatus for all detectors with the object of asphyxia prevention and continuous operation. In the efficiency calibration, Japan Atomic Energy Research Institute (JAERI) phantom with newly designed tissue equivalent lungs, which contain 241Am homogeneously distributed, was applied to lung detector, and Bottle Manikin Absorbtion (BOMAB) phantom regulated by American National Standards Institute (ANSI) was also applied to detectors for trunk.     

Effects of photo-induced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine on physical properties of cross-linked polyethylene in artificial hip joints

Osteolysis caused by wear particles from polyethylene in the artificial hip joints is a serious issue. We have used photo-induced radical graft polymerization to graft 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer onto the surface of cross-linked polyethylene (CLPE-g-MPC) in order to reduce friction and wear at the bearing surface of the joint. The physical and mechanical properties of CLPE and CLPE-g-MPC were not significantly different, expect that the friction coefficient of untreated CLPE cups was 0.0075, compared with 0.0009 for CLPE-g-MPC cup, an 88% reduction. After 3.0 × 10⁶ cycles in the hip joint simulator test, we could not observe any wear of CLPE-g-MPC cups. We concluded that the advantage of photo-induced radical graft polymerization technique was that the grafted MPC polymer gave a high lubricity only on the surface and has no effect on the bulk properties of the CLPE substrate.     

Excellent ferroelectricity of thin poly(vinylidene fluoride-trifluoroethylene) copolymer films and low voltage operation of capacitors and diodes

Characteristics of metal-ferroelectrics-metal (MFM) capacitors and meta -ferroelectrics-insulator-semiconductor (MFIS) diodes with poly(vinylidene fluoridetrifluoroethylene) [P(VDF-TrFE)] copolymer films thinner than 100 nm were investigated. The films were prepared by spin cast process and were annealed at 140 degrees C in air for crystallization. The ferroelectric properties are equivalent to those of oxide ferroelectrics, even under low voltage or high frequency operation.     

Site-specific functionalization of proteins by organopalladium reactions

A new carbon-carbon bond has been regioselectively introduced into a target position (position 32 or 174) of the Ras protein by two types of organopalladium reactions (Mizoroki-Heck and Sonogashira reactions). Reaction conditions were screened by using a model peptide, and the stability of the Ras protein under the reaction conditions was examined by using the wild-type Ras protein. Finally, the iF-Ras proteins containing a 4-iodo-L-phenylalanine residue were subjected to organopalladium reactions with vinylated or propargylated biotin. Site-specific biotinylations of the Ras protein were confirmed by Western blot and LC-MS/MS.