An enzyme-linked immunometric assay for cortisol based on idiotype-anti-idiotype reactions

Cortisol levels in body fluids are useful for monitoring the function of the pituitary-adrenal axis. Here, we established an “enzyme-linked immunometric assay” (a noncompetitive-type ELISA) for cortisol based on idiotype-anti-idiotype reactions. Six different anti-idiotype monoclonal antibodies that recognized the variable regions of a newly established anti-cortisol antibody were generated using hybridoma technology; these were two β-type and four α-type anti-idiotype antibodies, recognizing the paratope and framework regions, respectively. An immunometric assay was established using a combination of a selected α-type and a selected β-type antibody. The analyte (cortisol) was captured by an excess amount of anti-cortisol antibody immobilized on microplates, and the unoccupied paratope was saturated with the β-type antibody. Hapten-occupied anti-cortisol antibody, with less steric hindrance, was then selectively bound by the α-type antibody, labeled with biotin. The amount of biotin residue on the microplates was colorimetrically monitored using a peroxidase-labeled streptavidin. This assay had an approximately threefold higher sensitivity (detection limit: 90 pg = 248 fmol cortisol) than a competitive ELISA using the same anti-cortisol antibody, as well as a practical specificity for providing reasonable determination of normal urinary cortisol levels.     

Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal.

The interactions between Cd(2+) and the C-terminal region of phytochelatin (PC) synthase using recombinant wild-type and mutant PC synthase were studied. We show that site-directed mutagenesis of Cys residues at C(358)C(359)XXXC(363)XXC(366) motif decreases the number of Cd(2+) and other heavy metal ions interacting with the enzyme, and that the motif binds the metals discriminatingly. The optimum binding ratio of PC synthase to Cd(2+) was also determined. The findings indicate that Cys exists as a free SH residue and that it is involved in the regulation of PC enzyme activity by transferring the metals into closer proximity with the catalytic domain. These results are important in understanding heavy metal detoxification mechanisms in higher plants, a step towards phytoremediated-applications.     

Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate: application to enzyme-linked immunosorbent assay for urinary and serum cortisol.

Measurement of cortisol levels in body fluids is important for monitoring pituitary gland and adrenal functions. To develop a specific and standardized enzyme-linked immunosorbent assay (ELISA), a novel monoclonal anti-cortisol antibody has been generated using a reasonably designed haptenic derivative. Spleen cells were prepared from the BALB/c or A/J mouse, which had repeatedly been immunized with a conjugate of 4-(2-carboxyethylthio)cortisol (CET) and bovine serum albumin, to be fused with P3/NS1/1-Ag4-1 myeloma cells. After four fusion experiments, one hybridoma clone secreting a practical antibody has been established. The resulting monoclonal antibody CS#38 (isotype gamma1, kappa) showed an affinity constant (K(a)) for cortisol of 1 x 10(9) M(-1) and provided a practical calibration curve (detection limit, 0.26 ng per assay) in a homologous ELISA system employing horseradish peroxidase-labeled CET as a labeled antigen. Cross-reactivities with related C-21 steroids were acceptably low: 11-deoxycortisol (4.3%), cortisone (4.0%), corticosterone (1.9%), progesterone (1.6%), 17alpha-hydroxyprogesterone (12%), 6beta-hydroxycortisol (8.4%), and tetrahydrocortisol (< 0.1%). Urinary and serum cortisol levels of healthy volunteers were determined by this method after methylene chloride extraction to be 39.0 +/- 17.0 microg/day (n = 7) and 80.8 +/- 38.9 ng/mL (n = 10), respectively, both of which are in the reference range.     

Molecular distortion effect on ff-emission in a Pr(III) complex with 4,7-diphenyl-1,10-phenanthroline.

The electronic and structural behaviour of a Pr(III) complex with 4,7-diphenyl-1,10-phenanthroline, [Pr(bathophen)(2)(NO(3))(3)], is investigated with respect to the effect of configuration changes on the Pr(III) centre. [Pr(bathophen)(2)(NO(3))(3)] luminesces from the excited states of the ligand and the metal ion. The fluorescence, ff-emission ((1)D(2)-->(3)H(4)), and phosphorescence bands appear at 394, 608.2 and 482 nm, respectively, in the solid state. In acetonitrile, the complex also shows multiple emissions. From the time-resolved emission and the lifetime measurements, the excitation energy-transfer in [Pr(bathophen)(2)(NO(3))(3)] is clarified, that is, the upper excited triplet level of the ligand acts as an energy donor, while the (1)D(2) levels of Pr(III) is the acceptor. Additionally, the emission phenomena of the complex can be modified by molecular distortion, particularly by rotation of the phenyl groups in the ligand.     

Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced by zinc finger luciferase

The authors describe a pipette type of biosensor for detecting target genes and using a zinc finger protein fused to luciferase (ZF luciferase). The ZF protein binds to a specific DNA sequence, and the target double-stranded (ds) DNA can be detected by monitoring the enzymatic activity of ZF luciferase. A small avidin-immobilized reaction plate is placed on a plastic pipette tip (referred to as Biologi tip). The dsDNA detection procedures are carried out by using a programmable dispensing robot equipped with a photodetector. These procedures include (a) the aspiration of an analyte to capture the biotinylated target dsDNA (a product of a polymerase chain reaction) on the small reaction plate inside the pipette tip, (b) the introduction of ZF luciferase and luciferin into the pipette tip, and (c) migration of the pipette tip to the detection port to measure bioluminescence on the small reaction plate. The emission originating from luciferase activity is observed on the reaction plate containing immobilized biotin-tagged target dsDNA, whereas plates containing non-target or biotinylated single-stranded DNA only do not yield a signal. The intensity of emission increases proportionally to the concentration of dsDNA, and the detection limit of the target dsDNA is as low as 62 pM. An actual genomic DNA sample from Escherichia coli O157 was successfully detected by this automatic analyzer using the Biologi tip equipped with a reaction plate. This indicates that this system has a large potential for practical applications, including in particular point-of-care analyses in hygiene control, food safety testing, and clinical diagnosis.

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Graphical abstract A pipette-type biosensor was developed to detect target genes using a luciferase-fused zinc finger protein, where a small NeutrAvidin-immobilized reaction plate was placed on the tip, and the biotinylated target double-stranded DNA was detected by monitoring the bound luciferase activity.

     

A new biisoflavonoid from the roots of Erythrina variegata

A new biisoflavonoid, biseryvarin A (1a), together with two known compounds were isolated from the roots of Erythrina variegata. The structure of biseryvarin A was established on the basis of spectroscopic evidence. Biseryvarin A is the first dimeric isoflavonoid possessing isoprenoid groups from the genus Erythrina. Biseryvarin A showed low activity against methicillin-resistant Staphylococcus aureus (MRSA).     

An analysis of Y2O3:Eu thin films for thermographic phosphor applications

A comparative study of the luminescent properties of Y₂O₃:Eu³⁺ phosphor powders and thin films sputtered from targets prepared from combustion synthesized powders is reported. Thin films of (Y_0.96Eu_0.04)₂O₃ were deposited on silicon substrates. Films deposited at 600 °C had both monoclinic and cubic phases of Y₂O₃, which developed to an oriented cubic phase after annealing. Films and powders showed a linear dependence of the intensity of the ⁵D₇→⁷F₂ (611 nm) transition with temperature in the range 26-660 °C with an average rate of change of 1.8×10⁻⁴ °C⁻¹. The rate of change appears to be dependent on the Eu³⁺ concentration. This work shows that these thin films can be used as thermographic phosphors for remote temperature measurements.     

Semiempirical Pm3 Quantum Mechanics Calculations of Carbohydrate Crystals

ABSTRACT

Miniature crystal models of six small carbohydrates were examined using the PM3 semiempirical quantum mechanics method. The minicrystal structures, consisting of 27 sugar residues, were optimized by the combined procedure of partial optimization and reconstruction of the model, while maintaining the original crystal symmetries. All of the minicrystals were successfully optimized without exhibiting a great increase in an energy at any reconstruction step. Some minicrystals showed a prolonged behavior of optimization cycles. A major source of structure change appeared to arise from hydroxyl group rotations wherein the largest movements mostly occurred in an early optimization stage. No significant deformations in geometry of either residues or hydrogen bonds were observed in the final minicrystal structures. The thermodynamic values calculated for the optimized minicrystals were in reasonably good agreement with the literature data. The present study indicated that the PM3 semiempirical method successfully predicted the basic features of intermolecular hydrogen bonding involved in a condensed system.     

Initial step of anthracene‐sensitized photoacid generation from diphenyliodonium hexafluorophosphate in an epoxy matrix studied by steady‐state and laser‐flash photolyses

The anthracene‐sensitized photodecomposition of diphenyliodonium hexafluorophosphate was studied in an epoxy matrix. From steady‐state photolysis, the generation of protons, which are considered to be the actual initiators of the polymerization of epoxides, was confirmed. In addition, 9‐phenylanthracene was detected as a main product from anthracene. From time‐resolved laser‐flash photolysis, a broad absorption band with a peak at about 500 nm was observed that was thought to be due to the precursor of 9‐phenylanthracene. On the basis of these results, we propose electron transfer from anthracene in the excited singlet state to the diphenyliodonium cation as the initial step of photoacid generation. This process is followed by fast chemical reactions, which generate 9‐phenylanthracene and protons, and back electron transfer from the diphenyliodine radical to the anthracene cation radical. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2937–2946, 2001