Detection Of Biotin Research Articles

Detection of four new single-stranded telomeric DNA binding proteins by means of an optimized protein blotting procedure

The preservation and replication of telomeres are likely to involve multiple protein interactions. We describe a procedure for detecting sequence-specific telomeric DNA binding proteins in crude nuclear extracts. The technique involves electrophoretic transfer of SDS-PAGE fractionated crude nuclear proteins onto PVDF membranes with subsequent incubation in 2% (wt/vol) bovine serum albumin blocking solution. Incubation of the blocked filters with a 5'-biotin-labeled telomeric DNA probe under optimal binding conditions and subsequent biotin detection by means of peroxidase-linked streptavidin complexes reveals sequence-specific protein-telomeric DNA interactions. Using this technique, we identified 13 proteins that specifically bind the single-stranded telomere repeats of (TTAGGG)n, four of which have not been characterized as telomere binding so far.

Oligonucleotides as Hybridization Probes to Localize Phytoplasmas in Host Plants and Insect Vectors

ABSTRACT Protocols have been developed using 20- to 24-mer oligodeoxynucleotides, originally designed as polymerase chain reaction primers, as hybridization probes for the nonradioactive detection of Italian clover phyllody (ICPh) phytoplasma in plant (Chrysanthemum carinatum) and leafhopper (Euscelidius variegatus) tissue. In situ hybridization of paraffin-embedded tissue sections was carried out using oligodeoxynucleotides 5' end-labeled with either Cy5 fluorochrome, biotin, or digoxigenin. The Cy5-labeled oligonucleotide probes that hybridized to phytoplasmas present in plant tissue were visualized by confocal microscopy. The biotin- and digoxigeninlabeled probes were detected in both plant and insect tissue using a chromogenic alkaline phosphatase-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate reaction. An enhancement of a signal was observed in plant tissue when a tyramide signal-amplification procedure was incorporated into the biotin or digoxigenin detection systems. The results obtained using these techniques with the ICPh phytoplasma system showed that they can provide a rapid means of confirming vector status in insects. Due to the potential ability of short, labeled, oligonucleotide probes to specifically distinguish between different phytoplasmas present in multiple infections, this technique should provide a powerful new tool for epidemiological and vector ecology studies.

Electrochemical Detection of Biotin Using an Interaction between Avidin and Biotin Labeled with Ferrocene at a Perfluorosulfonated Ionomer Modified Electrode

The electrochemical behavior of biotin labeled with ferrocene was investigated at a perfluorosulfonated ionomer (Nafion®) film-coated glassy carbon electrode (GCE). The electrode response of the labeled biotin (LB) was improved by about fifty-fold in sensitivity by using a Nafion®-coated GCE, compared with a plain GCE. It was found by a solid-phase avidin–biotin binding assay that the labeling with ferrocene hardly affected the affinity of a biotin moiety in the labeled biotin to avidin. The avidin-biotin interaction was estimated from the change of the electrode response caused by the formation of a complex with avidin and the labeled biotin. Furthermore, the detection of biotin was performed by a competitive reaction between the labeled biotin and unlabeled biotin for the limited binding sites of avidin.

Control of electrocatalytic oxidation of NADH using an interaction between labeled biotin with dopamine and avidin

A new avidin biotin assay was investigated on the basis of electrochemical detection. Dopamine labeled biotin (LB) was used as a bifunctional compound to mediate the oxidation of β-nicotinamide adenine dinucleotide (NADH). The labeled biotin exhibited an effective electrocatalytic activity toward the oxidation of NADH as well as a specific binding with avidin. The labeled biotin’s function as a mediator decreased when it bound with avidin, hence decreasing the electrode response. The detection of avidin and biotin was performed from the suppression of mediated current by the interaction between protein and ligands.

Application of AFLP to Molecular Genetic Analysis in Peach.

AFLP analysis, using FITC (fluorescein isothiocyanate) and biotin detection systems, was applied to molecular genetics to obtain the information on reproducibility, efficiency and frequency of polymorphisms, using the peach as the experimental subject. An almost identical band pattern was obtained from different methods of DNA extraction and between replications. FITC detection with DNA sequencer made AFLP analysis more accurate and efficient and biotin detection simplified the cloning of the target bands. AFLP analysis produced 2.5 polymorphic bands per primer between 'Akame' and Juseitou', which were almost 20 times more than those obtained by RAPD analysis. Thirty eight AFLPs were obtained out of 334 amplified fragments among 8 cultivars by 8 primer combinations, which could discriminate even closely related cultivars. In conclusion, we confirmed that AFLP analysis could provide useful genetic markers to execute the cultivar identification, the parentage, and mapping work in peach.

Detection of endogenous biotin in various tissues: novel functions in the hippocampus and implications for its use in avidin-biotin technology

Significant amounts of endogenous biotin were detected by avidin-peroxidase in fixed rat kidney, liver, and brain. The staining was indistinguishable from the true signals of immunoreactivity and could not be consistently blocked by pretreatment with avidin. The finding that certain neurons in the hippocampus contain more biotin than neurons in other areas of the brain suggests that biotin might have novel functions in the brain other than its well-known role as cofactor of carboxylases. Critical examination of published immunohistochemical localization studies on rat kidney strongly suggests that many false-positive results have been considered as true signals. Interference of endogenous biotin in any study using avidin-biotin technology must be considered if biological tissues are involved. The published data obtained by this method should therefore be reevaluated. Furthermore, appropriate controls, blockers and caution in interpreting results must be exercised, not only in immunohistochemistry but also in any applications of avidin-biotin technology.

Fluorescence detection of biotin using post-column derivatization with OPA in high performance liquid chromatography

A sensitive and selective high-performance liquid chromatographic method with post-column derivatization, using o-phthalaldehyde (OPA) and 3-mercaptopropionic acid (3-MPA), is described for the determination of biotin in pharmaceutical preparations. The use of 3-MPA gives intense fluorescent derivative and improves the stability of biotin fluorophore towards oxidation to the picomole level. The fluorophore was detected at 453 nm (excitation at 342 nm). The calibration graph was linear for 20–200 ng per injection. The detection limit of biotin under these conditions was about 10 ng per injection. The RSDs were 1.9–3.4%. This method could be applied to pharmaceutical preparations without interference of other compounds.

Competitive nonseparation electrochemical enzyme binding/immunoassay (NEEIA) for small molecule detection

A nonseparation electrochemical enzyme binding/immunoassay (NEEIA) for the detection of small molecules via a competitive format is described. The NEEIA concept is based on the use of a microporous gold electrode, which serves as both the working electrode, and solid phase for the immobilization of binding protein/antibody through a chemisorbed layer of thioctic acid. Competitive assays are performed by incubating the small molecule of interest and an alkaline phosphatase (ALP) labeled analyte competitor (conjugate) with the modified electrode. Surface bound conjugate is spatially resolved from unbound conjugate by introducing the substrate ( p-aminophenyl phosphate) through the backside of the microporous gold electrode. The substrate diffuses rapidly through the microporous gold electrode where it first encounters surface bound conjugate. The enzymatically generated product ( p-aminophenol) is subsequently oxidized at the electrode (+190 mV vs. Ag/AgCl). Due to the competitive nature of the assay, the magnitude of the amperometric signal is inversely proportional to the concentration of analyte in the sample. Detection of biotin, digoxin and digitoxin in buffer is demonstrated with detection limits of 1, 0.1 and 10 nM, respectively. In addition, it is shown that digoxin can be measured in undiluted sheep serum with a detection limit of 1 nM, demonstrating that the proposed competitive NEEIA format can be employed for the detection of small molecules directly in complex matrices without any discrete separation or washing steps.

Voltammetric detection of avidin and biotin by biotin labeled with cysteine

An avidin-biotin assay was developed from a voltammetric procedure using biotin labeled with cysteine. Mercury(II) as a marker was used to detect avidin and biotin, because the oxidation wave of mercury decreases when the cysteine part of labeled biotin(LB) complexes with mercury(II).The formation of the mercury(II)-cysteine complex is suppressed when the LB binds to the biotin site of avidin. Accordingly, the concentration of avidin can be estimated from the increasing mercury peak current. Detection of biotin is also carried out by a competitive reaction of biotin and the LB to the binding site on avidin, where the addition of biotin decreases the peak current of mercury. Limits of detection for avidin and biotin were in the 10–9 mol/L range. The length of the spacer between the cysteine and biotin was investigated. It was observed that the strength of binding increased with increasing length of spacer. Size considerations rules out steric influences, so it is suggested that the binding constant depends on hydrophobic interactions in the binding site.

Ultrastructural and biochemical detection of biotin and biotinylated polypeptides in Schistosoma mansoni.

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 micrograms/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite.

Time-resolved fluoroimmunoassay for pituitary adenylate cyclase activating polypeptide 27 (PACAP27) using europium (III) ion chelate labeled streptavidin-biotin complex

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel peptide hormone and has a variety of biological action. In studies of the physiological behaviour of endogenous PACAP, the determination of PACAP levels in biological materials require a highly sensitive and specific method. Therefore, we developed a sensitive time-resolved fluoroimmunoassay (TR-FIA) for PACAP27 which is the biologically important fragment of PACAP. Accordingly, we developed TR-FIA using a biotinylated PACAP27 (b-PACAP27) as a tracer and europium (III) chelate labeled streptavidin-biotinylated bovine serum albumin complex as a detection of biotin on solid phase. A measurable range of PACAP27 was 7.8–1000 pg ml −1 by the proposed TR-FIA. For measurement of biological samples, the samples were purified to eliminate substances which interfered with the TR-FIA. The mean recovery of PACAP27 using commercially reversed phase column was 74.7% ( n = 12). The various tissues, extracts and plasma concentrations of rat could be measured by the proposed TR-FIA.

Detection of transcripts via fluorescencein-situ hybridization and confocal microscopy in whole mounts of roots ofArabidopsis thaliana

We report a modification of a previously published procedure (de Almedia Engler et al., 1994) aimed at the fluorescent detection of RNA-RNA hybrids. Transcripts ofARAth;Cyc1;1 were located in whole mounts of roots ofArabidopsis thaliana afterin-situ hybridization with digoxigenin-and biotin-labeled antisense probes. Digoxigenin- and biotin-labeled hybrids were detected by FISH with an AP-conjugated antibody against digoxigenin or AP-conjugated streptavidin for detection of biotin, with the UV-excitable dye ELF™ as a substrate. Alternatively, biotin-labeled hybrids were detected with HRP-conjugated streptavidin, with fluorescein tyramine as a substrate. Images were recorded via confocal microscopy. We report on the different fixation and reaction conditions needed for obtaining the highest signal-to-noise ratio, as well as on the requirements for mounting media, filter sets, excitation wavelengths, and recording modes used for recording confocal data sets.

29] Fluorophore-linked assays for high-performance liquid chromatography postcolumn reaction detection of biotin and biocytin

Publisher Summary This chapter describes high-performance liquid chromatography (HPLC) postcolumn reaction detection systems in which two types of protein-binding assay principles are used. In a competitive-binding approach, the fluorescent probe 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) competes with the analyte (biotin or biocytin) for the same binding sites on avidin. Two noncompetitive binding approaches are also described, in which the interaction that takes place is between the analyte (biotin or biocytin) and avidin or streptavidin. In these cases (strept)avidin is labeled with fluorescein isothiocyanate (FITC). On binding with the analyte, the fluorescence intensity of (strept)avidin–FITC increases in a manner proportional to the concentration of the analyte. The coupling of the binding assays with HPLC results in a highly selective detection of biotin and its derivatives owing to their selective interaction with (strept)avidin. These types of approaches also lead to enhanced sensitivity because the HPLC detection is fluorescence based rather than the conventional ultraviolet (UV) absorbance-based detection. The chapter also describes an absorbance-based postcolumn reaction detection system. In this system, biotin competes with and displaces the dye 2-(4'-hydroxyphenylazo)benzoic acid (HABA), where the free and bound HABA have different absorbance characteristics.

31] Bioluminescence competitive binding assays for biotin based on photoprotein aequorin

Publisher Summary This chapter describes competitive binding assays for biotin that use the bioluminescent protein aequorin as the label. Heterogeneous and homogeneous competitive binding assays using aequorin bioluminescence have been developed for detection of biotin. In the heterogeneous system, an aequorin–biotin conjugate competes with unlabeled biotin for binding to immobilized avidin, followed by a separation step to remove the unbound conjugate. Bioluminescence assays are best performed on luminometers operated in a photon-counting mode. A variety of such instruments is commercially available. In addition, modern scintillation counters may be used to perform bioluminescence assays. The relative amounts of avidin and biotinylated aequorin (conjugate) influence the response characteristics of the assay. Therefore, the amounts of avidin and biotinylated aequorin must be optimized in order to enhance assay performance. The development of a homogeneous assay for biotin using aequorin is based on the ability of avidin in solution to inhibit the bioluminescence of biotinylated aequorin. To establish the optimal amount of avidin to use in the assay, a binder dilution curve is constructed by varying the amount of avidin in the presence of a fixed amount of biotinylated aequorin, and measuring the degree of signal inhibition.

Determination of the extent of protein biotinylation by fluorescence binding assay.

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.

Applying hollow fibres for separating free and bound label in continuous-flow immunochemical detection

On-line liquid chromatography-immunochemical detection (LC-ICD) provides the possibility to individually monitor cross-reactive compounds overcoming the need of tedious fraction collection. ICD is performed as a post-column reaction detection system and is based on a two-step immunoreaction. In the first step unlabelled antibodies are added to the LC effluent and allowed to react with antigens (analytes) eluting from the LC column. The amount of analytes bound to the antibodies is measured by adding, in a second step, labelled antigen to the reaction mixture. For quantitation, free and bound label need to be separated prior to detection. The present paper describes a hollow fibre module (HFM), which can be used for this purpose. Separation of free and bound label occurs on discrimination by size. Using biotin as a model compound, a detection limit of 30 nmol/1 can be reached employing anti-biotin antibodies and a low-molecular-mass fluorescence label in the LC-ICD system. Additional to low-molecular-mass labels, the HFM allows the use of small enzyme labels. In this context, horseradish peroxidase-labelled biotin was used as a label in combination with antibodies in the immunochemical detection of biotin. This allows future implementation of commercially available enzyme immunoassay kits in continuous-flow immunochemical detection.

The Visualization of Peroxisomal Proteins Containing a C-Terminal Targeting Sequence on Western Blot by Using the Biotinylated PTS1-Receptor

A procedure to visualize proteins containing a C-terminal peroxisomal targeting signal (PTS1) in complex protein mixtures was developed using a bacterially expressed, biotinylated form of the human PTS1-receptor. The binding of this fusion product to purified PTS1-containing proteins that were separated by SDS–PAGE and blotted onto nitrocellulose was detected by means of streptavidin–alkaline phosphatase and shown to be both saturable and specific. When applied to total tissue extracts, in addition to PTS1-containing proteins various endogenous biotinylated proteins were visualized. Therefore, a two-step staining procedure was optimized whereby the endogenous biotinylated proteins were shielded with a blue precipitate, followed by incubation with the biotinylated receptor and detection of the resulting PTS1-receptor/PTS1-protein complexes with a phosphatase reaction coupled to the formation of a red-colored precipitate. This relatively inexpensive, simple, and fast technique enabled us to visualize a variety of PTS1-containing proteins. In addition, the information presented in this study can be used to facilitate the identification and characterization of receptor–ligand interactions in general and to eliminate interference by endogenous biotinylated proteins intrinsic to the streptavidin–biotin detection system.

Electrochemical evaluation of avidin-biotin interaction using N-iodoacetyl- N-biotinylhexylenediamine

The avidin-biotin assay was investigated by an electrochemical procedure based on the chemical reaction between cysteine containing a thiol group and N-iodoacetyl- N-biotinylhexylenediamine (IB). In the presence of avidin and biotin this reaction, whereby the thiol group combines with IB, is controlled because IB has a biotin part; that is, avidin and biotin are detected indirectly by measurement of iodide ion or cysteine. To achieve a high sensitivity of detection, Hg(II) was introduced as a marker that interacts with cysteine and the oxidation peak from Hg(0) to Hg(II) was measured. The sensitivity of detection of avidin was at the level of 10 −9 M. The relative standard deviation at 1 × 10 −8 M avidin was 4.8% ( n = 5). On the other hand, a response curve to detect biotin was obtained by the competitive reaction between IB and biotin for the limited binding sites of avidin. The change in peak current enables the detection of biotin at the level of 10 −9 M. This method has the advantage that it is not necessary to separate free IB from bound IB.

Protein adsorption to titania surfaces

Titanium and its alloys are used widely in the manufacture of orthopaedic and dental implants. Their popularity is encouraged by the excellent biocompatibility of the surface oxide layer and the phenomenon of osseointegration that occurs following surgical implantation into bone. This term describes the fixation of a medical device through the formation of a direct interface with bone tissue. However, the processes that lead to osseointegration are not fully understood and, in particular, the contribution of selective protein adsorption to clinical success is the subject of debate. The aim of this study was, therefore, to investigate the adsorption of serum proteins to titanium oxide (titania) and to non-integrating surfaces as controls using a number of methods. In situ labelling of proteins with biotin and subsequent detection with avidin peroxidase on electroblots and an ELISA method gave the best results. Differences were observed between protein adsorption to the two classes of surfaces, with selective adsorption of vitronectin to the osseointegrating surfaces. It is suggested that this may influence the subsequent behaviour of cells and the process of osseointegration.

Application of immunostaining for muscle specific actin in detection of myoepithelial cells in breast fine‐needle aspirates

Myoepithelial cells play an important role in the interpretation of breast fine-needle aspiration biopsy, since these cells are believed to be a component of a benign process in breast lesions. Myoepithelial cells are usually easy to recognize, however, other cells can morphologically simulate myoepithelial cells and make the distinction difficult. To assess the feasibility of immunostaining as an adjunct to identify myoepithelial cells, we have used immunocytochemistry using monoclonal antibody against muscle specific actin (MSA) in our breast fine-needle aspirates. Herein, we report our experience in immunocytochemical detection of myoepithelial cells using labeled Streptavidin Biotin Detection System on destained Papanicolaou stained smears and cell block preparations obtained from breast aspirates.