4-phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.     

An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction

The mechanism of peroxidase-catalysed oxidation of luminol by H₂O₂ was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfuly tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10^?9M for IgG and 10^?11M for T4, the overall time of the assay being 5–15 min.     

Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD‐methoxy‐pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets. Copyright © 2015 John Wiley & Sons, Ltd.     

Chemiluminescence enzyme immunoassay (CLEIA) for the determination of chloramphenicol residues in aquatic tissues.

A competitive indirect chemiluminescence enzyme immunoassay (ic-CLEIA) for chloramphenicol (CAP) residues in shrimp has been developed. After optimization (incubation time, concentration of Tween-20, concentration of PBS and pH), the method gave a limit of detection of 0.01 ng/mL and a detection range of 0.03-23.7 ng/mL, with an ED(50) of 0.47 ng/mL. The method has been validated on spiked shrimp samples in terms of precision (intra- and interassay coefficient variations of less than 10% and 15%, respectively) and accuracy (mean recovery 95-123%). The assay performance is better than the ELISA method which is widely used to detect chloramphenicol and indicates that the CLEIA method can be used to test aquatic samples instead of ELISA.     

Enzyme immunoassay of (24R)-brassinosteroids

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.     

Beta-adrenergic stimulation evokes a rapid, Ca2+-dependent stimulation of endocytosis, hexose and amino acid transport associated with increased Ca2+ fluxes in mouse kidney cortex

The beta-adrenergic agonist 1-isoproterenol evokes an acute (less than 5 min) stimulation of endocytosis, hexose transport and amino acid transport, measured by the temperature-sensitive uptake of HRP, 3H-DG and 14C-AIB, in mouse kidney cortex slices. This stimulation is concentration dependent and is maximal at 10(-8)-10(-7) M isoproterenol. Peroxidase cytochemistry showed that the hormonal increase in HRP uptake is confined to proximal tubules. The rapid membrane response is abolished in a calcium-free medium and by the beta-adrenergic antagonist propranolol, indicating Ca2+- and beta-adrenoreceptor-dependence. Isoproterenol (1 microM) rapidly (less than 30 sec) stimulates the influx and efflux of 45Ca in cortex slices. Isoproterenol also decreased mitochondrial 45Ca and increased soluble 45Ca. These results indicate that beta-adrenergic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular (mitochondrial) calcium. An increase in cytosolic Ca2+ concentration appears to be the regulatory signal for these membrane transport processes.     

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

A rapid and sensitive 384‐well microtitre format chemiluminescent enzyme immunoassay for 19‐nortestosterone

We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19‐nortestosterone (19‐NT) in bovine urine. Anti‐19‐NT polyclonal antibodies were raised in rabbits using a 19‐NT‐hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384‐well black polystyrene microtitre plates and HRP‐labelled 19‐NT activity was measured using an efficient chemiluminescent substrate (SuperSignal® ELISA Femto) after 3 min incubation. Emitted light was recorded using a conventional, photomultiplier‐tube‐based microtitre plate reader or a sensitive back‐illuminated, cooled CCD camera. The developed method fulfils all the requirements of precision (intra‐ and inter‐assay CV < 10%) and accuracy (mean recovery 94–112%), with a detection limit of 0.03 ppb (1.1 × 10^?9 mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP‐labelled tracer (thus lowering the limit of detection with respect to colorimetry) and reduces analysis time. The 384‐well microtitre plate cuts the sample/reagent volume (20 µL), a five‐fold reduction with respect to the conventional 96‐well microtitre plate. The developed method is suitable for high‐throughput screening of 19‐NT in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays. Copyright © 2003 John Wiley & Sons, Ltd.     

Antimicrobial lignans derived from the roots of Streblus asper

Four new lignans, (7′R,8′S)-4,4'-Dimethoxy-strebluslignanol (1), 3'-Hydroxy-isostrebluslignaldehyde (2), 3,3'-Methylene-bis(4-hydroxybenzaldehyde) (3), and 4-Methoxy-isomagnaldehyde (4), and six known lignans (5–10), were isolated from the roots of Streblus asper. The structures of these molecules were elucidated through various spectroscopic methods of analysis, including 1D and 2D NMR. The stereochemistry at the chiral centres was determined using the CD spectrum and from coupling constant and optical rotation data. Compounds 1–6 showed good antimicrobial activity against Saccharomyces cerevisiae (ATCC 9763), Bacillus subtilis (ATCC 6633), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 11775), and Staphylococcus aureus (ATCC 25923), with MIC values ranging from 0.0150 to 0.0940 μM.     

Isolation and Structure of Cotylenin E

²-Casein was modified by trypsin, chymosin or phosphoprotein phosphatase. Each modified ²-casein was fortified to the test-milk which was pretreated at 10°C by immobilized thermolysin to curdle non-enzymatically at 35°C. Fortification with chymosin-modified ²-casein, devoid of the hydrophobic moiety near the C-terminus, increased the curd tension as did native ²-casein. The test-milk fortified with trypsin-modified ²-casein, which has been deleted some part of the hydrophilic region near the N-terminus, had similar curd tension to unfortified test-milk. The test-milk fortified with dephosphorylated ²-casein, formed a softer curd than that with native ²-casein. Trypsin-modified ²-casein or dephosphorylated ²-casein retained its associative ability, but chymosin-modified ²-casein revealed negligible ability to associate.

These results led to the conclusion that the phosphoryl group on the hydrophilic moiety of ²-casein contributes to the increase in curd tension, while the hydrophobic moiety is responsible for the association of ²-casein.     

Inhibition of horseradish peroxidase byN-ethylamide ofo-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence ofN-ethylamide ofo-sulfobenzoylacetic acid (EASBA) at pH 5.0–7.5. In the oxidation ofo-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases bothK _m andV _m in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.