A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.     

The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients

A method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7-10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti-human IgG followed by an enzyme-labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.     

Homogeneous enzymatic fluorescence immunoassay of serum IgG by continuous flow-injection analysis

A new technique for automated homogeneous immunoassay has been developed and applied to the determination of serum IgG. An enzyme label, horseradish peroxidase (HRP), conjugated to the antibody (anti-human IgG) was inhibited on immunochemical association. The inhibition of activity was monitored as a decrease in the laser-induced fluorescence of dichlorofluorescein, produced by the HRP-catalysed oxidation of leuco-diacetyldichlorofluorescein by hydrogen peroxide. The entire procedure was performed by flow-injection analysis at a rate of 60 samples per hour. Serum IgG concentrations from 1.4 to 25 mg/ml could be determined after a 1:700 dilution, with a within-run precision of ±9.8%.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

Crystal structure and excimer fluorescence of dibenzoylmethanatoboron difluoride

Fluorescence properties of dilute and concentrated solutions of dibenzoylmethanatoboron difluoride and its crystals were studied. Excimers are formed in concentrated solutions and crystals of dibenzoylmethanatoboron difluoride. The crystal structure of dibenzoylmethanatoboron difluoride was determined by X-ray diffraction analysis. It was found that the molecules of dibenzoylmethanatoboron difluoride are coplanar with an interplanar distance of 3.6 .     

Rapid and simple method for the identification of apolipoprotein E isomorphic phenotypes from whole serum.

For the identification of apolipoprotein E isomorphic phenotypes, fresh or thawed serum was analyzed without prior delipidation or other pretreatment. Using 5% polyacrylamide gels with a 40 mm interelectrode distance, the isoforms were separated by isoelectric focusing in immobilized pH gradients ranging from pH 5 to 6.5, and transferred onto polyvinylidene difluoride membranes by contact blotting for 1 h. The apolipoprotein E isoforms were identified following immunostaining. The electrophoresis required less than 2 h and the entire procedure could be completed within 6 h.     

Availability and development of an enzyme immunomicrosensor based on an ISFET for human immunoglobulins

A critical evaluation of the potentiometric response of an enzyme immuno-ISFET sensor has demonstrated that it is an effective, simple sensor for human immunoglobulin (IgG). The sensor was constructed using an immobilized human IgG membrane and an ISFET. The assay procedure involves the competitive immunochemical reaction of ureuse-labelled anti-human IgG with human IgG in samples and membrane-bound IgG and the electrochemical determination of membrane-bound urease activity. A linear relationship was obtained between the initial rate of response and the logarithm of IgG concentration from 0.1 to 2.0 mg ml^?1.     

Preparation of an aliphatic-substituted hypervalent iodine compound, tetracoordinate 1,2-iodoxetane 1-oxide, and its application to oxidation

A tetracoordinate 1,2-iodoxetane was prepared by the fluorination of a tricoordinate 1,2-iodoxetane with xenon difluoride followed by hydrolysis. The tetracoordinate 1,2-iodoxetane oxidized alcohols and a sulfide to the corresponding aldehydes and ketones, and a sulfoxide, respectively, in good to moderate yields under mild conditions.     

Improved Primary Structure Determination for Peptides and Proteins by Electroblotting in Combination with Sequencing

Abstract

A rapid and simple method of determining the primary structure of large synthetic peptides and natural proteins, using electroblotting on polyvinylidene difluoride membranes followed by fluorescence labelling and gas-phase sequencing, is described.     

The synthesis of naphthothiopyranopyranones and naphthothiopyranopyranthiones

The synthesis of fused tetracyclic naphthothiopyranopyranones from dihydronaphthothiopyranones I or II has been studied. Compounds I or II have been cyclised in good yield to the corresponding dioxaborin difluoride complexes III, IV, XIII and XIV by treatment with acetic or propionic anhydride and boron trifluoride etherate. These complexes and the Vilsmeier reagent reacted to produce fused tetracyclic 3-substituted naphthothiopyranopyranones V, VI, XV or XVI . The reaction of dioxaborin difluoride complexes III or IV with dimethylthioformamide (DMTF) afforded dimethylaminovinyldioxaborin difluoride complexes IX or X . Treatment of IX or X with hydrochloric acid solution gave naphthothiopyranopyranones VII or VIII . The reaction of VII, VIII, XV or XVI with DMTF/acetic anhydride yielded new products, which was identified as naphthothiopyranopyranthions XI, XII, XVII or XVIII .     

Ipso-amidation of arylboronic acids: Xenon difluoride-nitriles as efficient reagent systems

The xenon difluoride-mediated, ipso-amidation of boronic acids has been achieved for the first time under mild conditions. This method provides a simple, one-pot procedure for the direct synthesis of a series of anilides from the corresponding arylboronic acids and alkyl/aryl nitriles. Arylboronic acids bearing electron donating groups gave anilides in high yields, while moderate yields were observed for those bearing electron withdrawing groups. A plausible mechanism involving the formation of an aryl radical cation through single electron transfer by xenon difluoride, followed by the nucleophilic addition of the nitrile, is proposed.     

Rearrangement of phenylethenes on reaction with iodine-xenon difluoride

Phenyl-substituted ethenes react with iodine and xenon difluoride to provide difluorinated products. Iodofluoro intermediates react with xenon difluoride to produce transient iodine difluoride species that lose IF and F(-) and produce carbocations.     

PMMA/PVDF共混对PVDF的β相构型影响的研究

采用溶液法制备了不同含量的聚甲基丙烯酸甲酯/聚偏二氟乙烯(PMMA/PVDF)共混薄膜,利用傅立叶变换红外光谱(FTIR)、X射线衍射谱(XRD)、和差热分析法(DSC)对共混薄膜的结晶行为进行了分析。结果表明,共混物中PMMA的含量对PVDF的β相构型有明显影响:PMMA/PVDF=30/70共混物中β相含量最高。为提高PVDF薄膜的铁电性能提供了新的研究方法。     

Synthesis of chromones

A general procedure for the preparation of chromones from the boron difluoride complex of o-hydroxyaryl methyl ketones is described. One of the best methods used for the preparation of chromones and benzochromones is the treatment of o-hydroxyaryl methyl ketones with the dimethylacetal of dimethylformamide followed by treatment with sulfuric acid (1). We previously had shown (2) that benzochromone 4 was prepared in reasonable yield by the following reaction sequence.     

Genetic studies of human apolipoproteins: XIV. A simple agarose isoelectric focusing gel method for apolipoprotein E phenotyping

A new and simplified procedure is described for apolipoprotein E (APO E) phenotyping from native plasma or serum samples. Diluted or dialyzed samples are separated on agarose isoelectric focusing gels followed by protein blotting on nitrocellulose membranes. APO E banding patterns are localized immunologically using polyclonal goat anti-APO E antiserum as the primary antibody and rabbit anti-goat IgG conjugated with alkaline phosphatase as the secondary antibody. The method was used in parallel with our previously described polyacrylamide gel system to screen 110 unrelated and healthy US whites. Both gel systems gave identical APO E phenotypes, and allele frequencies were comparable with reported US white values. This simplified method can be used on a large number of population and clinical samples with minimum cost and effort.     

Structural analysis of recombinant proteins prepared by semi-dry electroblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Proteins that were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were electroblotted onto polyvinylidene difluoride membranes in procedures to prepare homogeneous recombinant proteins for direct N-terminal sequence analysis. A semi-dry blotting procedure was employed to immobilize protein bands on the membranes for subsequent sequence analysis. This method has been used routinely to evaluate the quality of recombinant proteins, which are present in crude cell extracts produced by different expression systems or under different expression conditions. N-Terminal processing, amino acid misincorporation, as well as the inefficient secretion of recombinant proteins can be detected by direct N-terminal sequence analysis of the purified electroblotted samples. Consequently, time-consuming chromatographic procedures can be eliminated. These procedures are also especially valuable for determining degradation sites of a purified recombinant protein, as well as evaluating multiple gene products expressed by isolated cluster genes.     

A highly sensitive method for quantification of myosin light chain phosphorylation by capillary isoelectric focusing with laser-induced fluorescence detection

Activation of myosin II by phosphorylation of the 20 kDa regulatory light chains (LC20) has been implicated in numerous contractile and motile events, e.g., smooth muscle contraction, cytokinesis, and cell migration. The ability to analyze LC20 phosphorylation in minute samples is critical to determine the importance of LC20 phosphorylation in diverse physiological processes. We have developed a method for the separation and quantification of unphosphorylated, monophosphorylated, and diphosphorylated LC20 with a detection limit of 1 pg (50 amol). LC20 is initially isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to a polyvinlyidene difluoride (PVDF) membrane. The region of the membrane containing the LC20 band (identified by electrophoresis of purified LC20 in a neighboring lane) is cut out and fluorescently labeled with Alexa Fluor 488 C5 maleimide. The labeled LC20 is eluted from the membrane with detergent and subjected to capillary isoelectric focusing (CIEF) to separate unphosphorylated, mono-, and diphosphorylated LC20, which are detected and quantified by laser-induced fluorescence (LIF). A linear relationship between log(peak area) and log(LC20 amount) is observed over the range of 50 amol-150 fmol. Quantification of LC20 phosphorylation by CIEF with LIF detection was compared with three commonly used methods with much lower levels of sensitivity: urea/glycerol-PAGE with Western blotting, phosphorylation by [gamma-32P]ATP with Cerenkov counting, and phosphorylation by [gamma-32P]ATP followed by SDS-PAGE, autoradiography, and scanning densitometry. All four methods gave very similar quantitative results, the major difference being that the new method exhibits 3000-fold enhanced sensitivity. This method is therefore applicable to quantitative analysis of phosphorylation of minute quantities of LC20.     

Boron Difluoride β-Diketonates: IV. Reaction of p-Alkyl-substituted Benzoylacetonates with N-Bromosuccinimide

Bromination of p-alkyl-substituted boron difluoride and chromium(III) benzoylacetonates with N-bromosuccinimide was studied. It was found that that the boron difluoride complexes are selectively brominated by the alkyl radical, whereas chromium toluoylacetonate, by the central carbon atom of the chelate ring.     

Crystal Structure and Luminescence of Boron Difluoride Acetylacetonate

The structure of boron difluoride acetylacetonate was studied by X-ray diffraction. A pucker of the chelate ring along the line connecting the boron and -carbon atom was detected. The effects of geometric and electronic factors on the fluorescence intensity of boron difluoride -diketonates were discussed.