Streptavidin Magnetic Particles Research Articles

Serum Level of Biotin Rather than the Daily Dosage Is the Main Determinant of Interference on Thyroid Function Assays

Introduction: Several case reports stress that high-dose biotin causes incorrect laboratory results. However, the extent of this interference in children is not systematically studied. Aim: To assess factors associated with biotin interference on thyroid function tests in subjects with biotinidase deficiency. Method: The study included 44 children who were treated with oral biotin (Group 1, median dose: 10 mg/day [25–75p; 10–10], age: 1.83 years [1.04–2.90]) and 30 healthy subjects (Group 2, age: 1.05 years [0.37–3.37]). Thyroid function tests were performed with two different assays, and streptavidin-coated particles were used in order to remove biotin from serum samples of cases with biotin interference. Results: The measurements were first performed with Beckman Coulter. In Group 1, remarkably high levels of fT3 and fT4 were found in 26 (59.1%) and 25 (56.8%) patients, respectively. Thyroid hormone functions were all normal in Group 2. Significantly higher serum biotin levels were detected in interference-positive children (p < 0.001). The serum biotin levels in Group 1 showed a strong positive correlation with fT3 (r = 0.867, p < 0.001) and fT4 levels (r = 0.905, p < 0.001). A serum biotin level of 80.35 µg/L was found to be the best cut-off value for predicting interference (sensitivity: 96.2% and specificity: 94.4%). When analyzed with Siemens Advia Centaur XP, all thyroid function tests were normal in both groups except in one patient (2.27%) with slightly elevated fT3 level in Group 1. Repeated tests with Beckman Coulter after neutralization of biotin with streptavidin magnetic particles in serum samples of the interference-positive cases revealed normal thyroid hormone levels. Conclusion: Interference is an important problem in thyroid function tests in nearly 60% of all children receiving biotin treatment for biotinidase deficiency. Serum levels of biotin rather than the dosage are the main determinant of interference, which can be eliminated by choosing appropriate laboratory methods.

Controlling Ligand Surface Density on Streptavidin-Magnetic Particles by a Simple, Rapid, and Reliable Chemiluminescent Test.

The use of functionalized magnetic particles is increasing because they simplify the analytical process and yield promising results in a wide range of applications. Particularly, streptavidin-coated magnetic beads offer the possibility of rapid and very efficient grafting of biomolecules. Unfortunately, current methods to monitor and compute this grafting process are cumbersome and scarce. We describe herein a simple, rapid, and reliable chemiluminescent assay we have developed to check the grafting rate of functionalized magnetic beads. The power of the assay also relies on its ability to predict the amount of ligands required to obtain a precise grafting rate. In addition, results were correlated with a more general parameter in material functionalization characterization like surface ligand density. Finally, the assay was validated for a wide variety of biotinylated biomolecule sizes, ranging from small molecules (around 200 Da) to antibodies (around 150 kDa). This approach will allow a precise quantification and prediction of the functionalization of magnetic particles that is of enormous importance for quality control in many applications.

Interferon gamma transcript detection on T cells based on magnetic actuation and multiplex double-tagging electrochemical genosensing

Interferon-γ is a proinflammatory cytokine, and its production is related with effective host defense against intracellular pathogens. Therefore, the level of interferon-γ is considered a good biomarker for intracellular infections. It is also useful for the assessment, treatment progression and follow-up of non-communicable diseases, including cancer and autoimmune disorders, among others. This work addresses the development of a novel interferon-γ release assay (IGRA) to evaluate the expression of interferon-γ transcripts as biomarker produced by isolated T cells, as a main advantage. The method sequentially combined three different types of magnetic separation, including the immunomagnetic separation of the T cells performed on antiCD3 modified magnetic particles, the retrotranscription and multiplex double-tagging PCR on polydT-modified magnetic particles and, finally, the electrochemical genosensing on streptavidin magnetic particles as a support. This approach is able to quantify the levels of cellular interferon-γ produced by as low as 150 T cells with outstanding analytical features. The detection of interferon-γ transcripts is performed from only 100 μL of whole blood which can be potentially obtained by fingerprick, demonstrating a further clear advantage to be considered as a promising strategy for the quantification of this important biomarker in several clinical applications.

Wheatstone bridge-giant magnetoresistance (GMR) sensors based on Co/Cu multilayers for bio-detection applications

A Wheatstone bridge-giant magnetoresistance (GMR) sensor was successfully developed for a potential biomaterial detection. In order to achieve this, a giant magnetoresistive [Co(1.5nm/Cu(1.0nm)]20 multilayer structures have been fabricated by DC magnetron sputtering method, showing a magnetoresistance (MR) of 2.7%. The X-Ray diffraction (XRD) patterns showed that Co/Cu film multilayer has a high degree of crystallinity with a single peak corresponding to face-centered cubic (111) structure at 2θ = 44.1°. Co/Cu multilayers exhibit a soft magnetic behavior with the saturation magnetization (Ms) of 1489 emu/cc and the coercivity (Hc) of 11.2 Oe. The magnetite Fe3O4 nanoparticles used as a bimolecular labels (nanotags) were synthesized via co-precipitation method, exhibiting a soft magnetic behavior with Ms of 77.16 emu/g and Hc of 49 Oe. XRD patterns and transmission electron microscopy (TEM) images showed that Fe3O4 was well crystallized and it grew in their inverse spinel structure with an average size of around 10 nm. The GMR sensor design was used to detect a biomolecules of streptavidin magnetic particles with concentration 10, 20, 30, and 40 μl/ml and α-amylase enzyme with consentration 10, 20, 30, and 40 μl/ml captured using polyethylene glycol (PEG)/Fe3O4 nanoparticles. Various applied magnetic fields of 0-650 Gauss have been performed using electromagnetic with the various currents of 0-5 A. Here, the final value of the output voltage signals for the streptavidin magnetic particles concentration is 1.2 mV (10 μl/ml). The output voltage changes with the increase of concentration. It was reported that the output voltage signal of the Wheatstone bridge exhibits log-linear function in real time measurement of the concentration of streptavidin magnetic particles and α-amylase enzyme respectively, making the sensor suitable for use as a biomolecule concentration detector. Thus, the combination of Co/Cu multilayer, Wheatstone bridge, magnetite and PEG polymer has potential application to be used in bio-detection applications where ultra-small bio-labels are needed.

Microplate magnetic chemiluminescence immunoassay for detecting urinary survivin in bladder cancer.

Survivin is a tumor marker for bladder cancer; however the role of urinary survivin levels has not been fully elucidated due to the limitations of current detection methods. Based on two survivin-specific monoclonal antibodies (McAbs) already confirmed through enzyme linked immunosorbent assays, the present study aimed to establish a microplate magnetic chemiluminescence immunoassay (CLIA) for the detection of urinary survivin levels and evaluate its application for the diagnosis of patients with bladder cancer. Horseradish peroxidase and biotin conjugates were used to label two different anti-survivin McAbs, respectively. The labeled antibodies combined with survivin to form a sandwiched immune complex. The streptavidin magnetic particles (MPs) served as the solid phase and the separator. The relevant parameters involved in the immunoassay, including the immunoassay reagents used and the physicochemical parameters were optimized. Then, urine samples from 130 patients with bladder cancer and 113 healthy controls were detected, and analyzed using the established method. The method was linear to 1,000 ng/ml survivin with a detection limit of 0.83 ng/ml. The intra- and inter-assay coefficients of variation were <8, and <11%, respectively. The concentration of diluted survivin and the dilution ratios gave a linear correlation of 0.9989. The results demonstrated that the urinary survivin levels in patients with bladder cancer were significantly higher (P<0.001) compared with that in healthy controls. At a survivin concentration of 2.0884 ng/ml, the sensitivity and specificity were 86.9 and 61.9%, respectively. Furthermore, the urinary survivin levels were positively correlated with metastatic stage, histological stage and recurrence (P<0.01). In conclusion, the present study preliminarily proposed a microplate magnetic CLIA for survivin detection and further evaluated the value of urinary survivin as a diagnostic marker for bladder cancer.

Glycotechnology for Decontamination of Biological Agents: A Model Study Using Ricin and Biotin-Tagged Synthetic Glycopolymers

Two types of biotin-tagged glycopolymers carrying lactose or glucose in clusters along the polyacrylamide backbone were prepared and subjected to decontamination analyses with the plant toxin ricin. A buffer solution containing the toxin was treated with one glycopolymer followed by streptavidin-magnetic particles. Supernatant solutions were analyzed with surface plasmon resonance and capillary electrophoresis, and revealed that the lactose glycopolymer "captured" this toxin more effectively than the glucose polymer. Free toxin was not detectable in the supernatant after treatment with the glycopolymer and magnetic particles; >99% decontamination was achieved for this potentially fatal biological toxin.

Isolation and identification of proteins binding to the major breakpoint region(mbr) of bcl2 gene

Objective We have previously found that mbr is a regulatory element of the bcl2 gene. The objective of this study is to isolate and identify the proteins binding to the 37 mbr in the 3′ -end of the mbr. Methods Streptavidin magnetic particles were ligated to concatameric oligonucleotides of 37 mbr and incubated with the nuclear extracts of Jurkat cells. The DNA-binding proteins were eluted and then resolved by SDS-PAGE. After silver staining, the protein bands were excised and subjected to MALDI-TOF MS. Results Several protein bands were detected after the isolation with magnetic particles, and Splicing factor, proline- and glutamine-rich(SFPQ), Poly(ADP-ribose) polymerase 1(PARP), and promyelocytic leukemia protein(PML) were identified by MALDI-TOF MS. Conclusion Several proteins were isolated and identified from the 37 mbr-protein complex. Results of this study establish a foundation for further study of the mechanisms by which mbr executes its regulatory function.

Differential Control of the CCAAT/Enhancer-binding Protein β (C/EBPβ) Products Liver-enriched Transcriptional Activating Protein (LAP) and Liver-enriched Transcriptional Inhibitory Protein (LIP) and the Regulation of Gene Expression during the Response to Endoplasmic Reticulum Stress

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a stress response program that protects cells early in the response and can lead to apoptosis during prolonged stress. The basic leucine zipper transcription factor, CCAAT/enhancer-binding protein beta (C/EBPbeta), is one of the genes with increased expression during ER stress. Translation of the C/EBPbeta mRNA from different initiation codons leads to the synthesis of two transcriptional activators (LAP-1 and -2) and a transcriptional repressor (LIP). The LIP/LAP ratio is a critical factor in C/EBPbeta-mediated gene transcription. It is shown here that the LIP/LAP ratio decreased by 5-fold during the early phase of ER stress and increased by 20-fold during the late phase, mostly because of changes in LIP levels. The early decrease in LIP required degradation via the proteasome pathway and phosphorylation of the translation initiation factor, eIF2alpha. The increased LIP levels during the late phase were due to increased synthesis and increased stability of the protein. It is proposed that regulation of synthesis and degradation rates during ER stress controls the LIP/LAP ratio. The importance of C/EBPbeta in the ER-stress response program was demonstrated using C/EBPbeta-deficient mouse embryonic fibroblasts. It is shown that C/EBPbeta attenuates expression of pro-survival ATF4 target genes in late ER stress and enhances expression of cell death-associated genes downstream of CHOP. The inhibitory effect of LIP on ATF4-induced transcription was demonstrated for the cat-1 amino acid transporter gene. We conclude that regulation of LIP/LAP ratios during ER stress is a novel mechanism for modulating the cellular stress response.

Stoichiometry and Localization of the Stator Subunits E and Gin Thermus thermophilus H+-ATPase/Synthase

Proton-translocating ATPases are central to biological energy conversion. Although eukaryotes contain specialized F-ATPases for ATP synthesis and V-ATPases for proton pumping, eubacteria and archaea typically contain only one enzyme for both tasks. Although many eubacteria contain ATPases of the F-type, some eubacteria and all known archaea contain ATPases of the A-type. A-ATPases are closely related to V-ATPases but simpler in design. Although the nucleotide-binding and transmembrane rotor subunits share sequence homology between A-, V-, and F-ATPases, the peripheral stalk is strikingly different in sequence, composition, and stoichiometry. We have analyzed the peripheral stalk of Thermus thermophilus A-ATPase by using phage display-derived single-domain antibody fragments in combination with electron microscopy and tandem mass spectrometry. Our data provide the first direct evidence for the existence of two peripheral stalks in the A-ATPase, each one composed of heterodimers of subunits E and G arranged symmetrically around the soluble A(1) domain. To our knowledge, this is the first description of phage display-derived antibody selection against a multi-subunit membrane protein used for purification and single particle analysis by electron microscopy. It is also the first instance of the derivation of subunit stoichiometry by tandem mass spectrometry to an intact membrane protein complex. Both approaches could be applicable to the structural analysis of other membrane protein complexes.

Magnetic Particles as Labels in Bioassays: Interactions between a Biotinylated Gold Substrate and Streptavidin Magnetic Particles

Magnetic particles (MPs) have been attracting much interest as a labeling material for advanced biological and medical applications, such as biomagnetic separation, drug delivery, magnetic resonance imaging, and hyperthermia. In most of these applications, the MPs have been designed to specifically interact with a target, such as cells or proteins, moving freely in a solution. However, for surface-based applications, such as magnetic biosensing, these MPs must bind specifically with a target that is immobilized onto a planar substrate. Consequently, new interaction phenomena, which influence the binding of the MPs to the substrate, have to be taken into account. To achieve adequate binding characteristics and to optimize the MPs toward substrate labeling, these physicochemical interactions should be properly identified. In this paper, the interactions between 16 commercially available streptavidin MPs and a biotinylated gold substrate were monitored in real time by surface plasmon resonance technology and the particle surface coverage was calculated by optical microscopy. On the basis of the type of interactions, the MPs studied in this paper could be classified into three different cases: (I) MPs that bind to the biotinylated substrate via the specific streptavidin−biotin interactions, without showing any nonspecific interactions; (II) MPs that do not bind to the substrate; and (III) MPs that bind to the biotinylated substrate via nonspecific interactions rather than via specific streptavidin−biotin interactions. The three cases were understood by determining the surface charges of both the particle and the substrate in ζ potential measurements. It was found that binding of MPs to the substrate was strongly dependent on the amount and the sign of the charges on both surfaces. The strong influence of electrostatic interactions was validated by simulating the total interaction force between a streptavidin MP and a biotinylated substrate by use of the Derjaguin−Landau−Verwey−Overbeek (DLVO) theory, while the gravitational force and the streptavidin−biotin force were accounted for. Finally, we conclude that apart from a well-controlled streptavidin coating, the surface charge of the particle and the substrate plays a pivotal role in the construction of MP assays on surfaces.

Preparation and application of streptavidin magnetic particles

Two kinds of streptavidin magnetic particles, namely streptavidin GoldMag particles and streptavidin amino terminal particles were prepared by the methods of physical adsorption and covalent interaction respectively. The streptavidin coated on magnetic particle surface, crucial to many applications, was greatly influenced by the choice of the different buffer. Compared with Dynalbeads®M-270 streptavidin, the binding capacity for biotin of different streptavidin magnetic particles was determined by enzyme inhibition method, and the coupling capacity and activity of biotinylated oligonucleotide on their surface were also analyzed. The results indicated that the streptavidin GoldMag particle prepared by physical adsorption was stable in STE (NaCl-Tris-EDTA) buffer that was frequently used in nucleic acid hybridization and detection. The streptavidin amino terminal particles prepared by covalent interaction could be used both in STE buffer and PBS (phosphate buffered saline) buffer. The biotin binding capacity for 1 mg of streptavidin GoldMag particles and streptavidin amino terminal particles was 4950 and 5115 pmol respectively. The capacity of biotinylated oligonucleotide (24 bp) coupled on 1 mg of GoldMag and amino terminal magnetic particles was 2839 and 2978 pmol separately. These data were about 6–7 times higher than those of Dynabeads®M-270 streptavidin. The hybridization results with FITC-labeled complementary probe on magnetic particle surface demonstrated that the oligonucleotide coupled on streptavidin magnetic particles had high biological activity.

Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box) within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

Novel Target Sequences for Pax-6 in the Brain-specific Activating Regions of the Rat Aldolase C Gene

Upstream activating sequences of the rat aldolase C gene are shown here to confer brain-specific expression in transgenic mice. In addition to binding sites described previously for the brain-expressed POU proteins Brn-1 and Brn-2 (Skala, H., Porteu, A., Thomas, M., Szajnert, M. F., Okazawa, H., Kahn, A., and Phan-Dinh-Tuy, F. (1998) J. Biol. Chem. 273, 31806-31814), we have identified two novel DNA elements critical for an interaction with a brain-specific, high affinity DNA-binding protein. Characterization of this binding protein showed it to be sensitive to thiol oxidation and stable to heat at 100 degrees C. This protein was purified on the basis of its thermostability and its selective adsorption to streptavidin magnetic particles via a biotinylated multimer of its target DNA binding site. Liquid chromatography coupled to tandem mass spectrometry analysis, binding competition with consensus oligonucleotides, and antibody supershift assays led to its identification as the homeodomain paired protein Pax-6. This result suggests that the brain-specific aldolase C gene could constitute a new target for the transcription factor Pax-6, which is implicated increasingly in neurogenesis.

Methods to determine biotin-binding capacity of streptavidin-coated magnetic particles

Two assays to determine the biotin-binding capacity of streptavidin magnetic particles are described and compared. The two assays are based on the use of biotinylated alkaline phosphatase and biotinylated fluorescein, respectively. Also, an assay for bound protein is presented. When the biotin-binding methods are combined with the protein assay, the specific activity can be determined. The fluorescent version is used to compare the streptavidin magnetic particles from several manufacturers.

Organelle isolation by magnetic immunoabsorption.

Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, choroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins for their ability to immune-isolate chloroplasts.

Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography.

The vast majority of probes used in fluorescence in situ hybridization (FISH) contain repetitive DNA. This DNA is usually competed out of a hybridization reaction by the addition of an unlabeled blocking agent, Cot-1 DNA. We have successfully removed repetitive DNA from two complex FISH probe sets: a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) single human chromosome library and genomic DNA. The procedure involved hybridizing in solution a DOP-PCR-amplifiable probe set with a 50-fold excess of biotin-labeled Cot-1 DNA, and capturing the Cot-1 DNA-containing hybrids using streptavidin magnetic particles, followed by purification and reamplification of the unbound fraction. Probes were checked for depletion of repeats by hybridization to chromosomes without Cot-1 DNA. Results showed hybridization patterns comparable to those achieved with untreated probes hybridized with Cot-1 DNA.

Preservation of cytotoxic function during multi-cycle immunomagnetic cell separations of human NK cells using a new type of magnetic bead

The isolation from human peripheral blood lymphocytes of natural killer (NK) cell populations by a novel magnetic cell sorting (MACS) procedure yielded large amounts of viable cells with active cytotoxic function Non-adherent B cell-depleted lymphocytes were sequentially labelled with specific monoclonal antibodies, biotin-conjugated second antibody, FITC-conjugated streptavidin and biotin-conjugated magnetic particles (diameter 50–150 nm). In the magnetic field of a permanent magnet, positively labelled cells were retained on columns with a ferromagnetic matrix. When OKT3 was used for the depletion, 96–99% of the T cells were removed. The resulting non-labelled NK cell population contained 78–89% Leu11b + and 87–96% Leu19 + cells. Magnetic retention of NK cells mediated by anti-Leu19 yielded about 81% and retention mediated by anti-Leu11b about 80% of total cells as determined by positive fluorescence. The resulting labelled and unlabelled cell subpopulations maintained their full NK activity as determined in 4 h cytotoxicity assays against human K562 tumor cells. The viability of non-labelled cells was fully preserved, whereas that of labelled cells slowly decreased with increasing numbers of preparative cycles. Furthermore, the ability of the isolated NK cells to show enhancement of their NK cytotoxicity after preincubation with IL-2 was maintained. The cytotoxic function of NK cells was also preserved when two or more MACS cycles using the same or different antibodies were carried out. The saving of time and the physiological condition of the isolated cells offer valuable advantages over FACS procedures.