Affinity Magnetic Bead Research Articles

Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis

The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.

Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests.

Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/μL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

Precocene II, a Trichothecene Production Inhibitor, Binds to Voltage-Dependent Anion Channel and Increases the Superoxide Level in Mitochondria of Fusarium graminearum.

Precocene II, a constituent of essential oils, shows antijuvenile hormone activity in insects and inhibits trichothecene production in fungi. We investigated the molecular mechanism by which precocene II inhibits trichothecene production in Fusarium graminearum, the main causal agent of Fusarium head blight and trichothecene contamination in grains. Voltage-dependent anion channel (VDAC), a mitochondrial outer membrane protein, was identified as the precocene II-binding protein by an affinity magnetic bead method. Precocene II increased the superoxide level in mitochondria as well as the amount of oxidized mitochondrial proteins. Ascorbic acid, glutathione, and α-tocopherol promoted trichothecene production by the fungus. These antioxidants compensated for the inhibitory activity of precocene II on trichothecene production. These results suggest that the binding of precocene II to VDAC may cause high superoxide levels in mitochondria, which leads to stopping of trichothecene production.

Performance of Filters in Medical Protein Separation System Using Superconducting Magnet

Medical proteins such as monoclonal antibodies or immunoglobulin are important as medicine for cancer and other uses. Today, we can easily sort and analyze medical proteins using various types of commercially available affinity magnetic beads. However, separation systems for these medical proteins have a very low separation rate, and the cost of the product is extremely high. We successfully developed a high gradient magnetic separation system using a cryocooler-cooled low-temperature superconducting magnet and conducted experiments on separating affinity magnetic nanobeads. Our system demonstrated very high separation efficiency and can achieve low costs with a large production rate compared to systems now used in this field. The design of a filter to trap and recover the nanobeads is important to this application. In order to achieve a filter with a high trapping ratio of magnetic beads, the parameters need to be optimized because the ratio depends on the dimensions and arrangement of the filter made of fine magnetic metal fibers. In this study, we investigated the performance of filters in the high gradient magnetic separation system. The test results show 97.8% of the magnetic nanobeads in pure water were captured, and 94.1% of the total beads were collected.

Magnetically Promoted Rapid Immunoreactions Using Functionalized Fluorescent Magnetic Beads: A Proof of Principle

Accurate detection and monitoring of disease-related biomarkers is important in understanding pathophysiology. We devised a rapid immunoreaction system that uses submicrometer polymer-coated fluorescent ferrite (FF) beads containing both ferrites (magnetic iron oxide) and fluorescent europium complexes. FF beads were prepared by encapsulation of hydrophobic europium complexes into the polymer layers of affinity magnetic beads using organic solvent. A sandwich immunoassay using magnetic collection of antibody-coated FF beads to a specific place was performed. Brain natriuretic peptide and prostate-specific antigen were selected as target detection antigens to demonstrate the feasibility of this approach. An immunohistochemical staining using magnetic collection of antibody-coated FF beads onto carcinoma cell samples was also performed. The sandwich immunoassays, taking advantage of the magnetic collection of antibody-coated FF beads, detected target antigens within 5 min of sample addition. Without magnetic collection, the sandwich immunoassay using antibody-coated FF beads required long times, similar to conventional immunoassays. Using the magnetic collection of antibody-coated FF beads, immunohistochemical staining enabled discrimination of carcinoma cells within 20 min. This proof of principle system demonstrates that immunoreactions involving the magnetic collection of antibody-coated FF beads allow acceleration of the antigen-antibody reaction. The simple magnetic collection of antibody-coated FF beads to a specific space enables rapid detection of disease-related biomarkers and identification of carcinoma cells.

Improvement of a High-Gradient Magnetic Separation System for Trapping Immunoglobulin in Serum

Recently, affinity magnetic beads have been widely used in immunomagnetic cell sorting (IMCS) technology. Today, we can easily sort and analyse DNA and antibodies (immunoglobulin) using various types of affinity magnetic beads available in the market. The diameters of the affinity magnetic beads used in immunomagnetic cell sorting are above approximately 1 ¿m because of the low magnetic fields induced by permanent magnets. At present, nanosized affinity magnetic beads are strongly desired to achieve high resolutions. We have been studying and attempting to develop a high-gradient magnetic separation (HGMS) system that employs a superconducting magnet to induce a considerably higher magnetic field than that induced by a permanent magnet. The objective is to trap smaller nanosized affinity magnetic beads using a filter made of fine stainless steel wool. In a previous study, we constructed a prototype of a desktop-type HGMS system using a cryocooler-cooled LTS magnet; we conducted preliminary experiments on trapping nanosized magnetic particles and investigated the magnetic field distribution and magnetic force around a magnetic wire in the filter by means of a numerical simulation. In this study, we succeeded in producing prototype nanobeads covered with the biosurfactant of a high-affinity ligand system for immunoglobulin G and M. Furthermore, we attempted to improve the recovery of nanobeads by adding a resonance circuit to the HGMS system. In practice, the trapped nanobeads attract one another and agglomerate due to their remaining magnetization when the magnetic field is decreased to 0 T. Therefore, the nanobeads and wire are demagnetized in the AC magnetic field by the resonance circuit, making good use of the superconducting magnet of the HGMS system.

<i>In Vitro</i> Selection of Oligonucleotide Acid Aptamers against Pathogenic Vibrio Alginolyticus by SELEX

Detection of pathogenic microorganism is very necessary in the control of infectious disease prevailing in aquiculture animals. However, most of the present techniques can not meet the need of the quick field inspection. Systematic evolution of ligands by exponential enrichment (SELEX) is a new molecular recognition way for generating high affinity oligonucleotide acid aptamers, a new nucleotide acid material, which have been widely used in the detections of proteins, cells and so on. In the present paper, the technology was applied to select the high affinity aptamers against pathogenic microorganism Vibrio alginolyticus, which could be used for the rapid field detection of the microorganism. Based on the designment of the ssDNA library of 76 nucleotide acids with 35-base random region, the SELEX system for the selection of the high affinity aptamers against Vibrio alginolyticus was established. In the SELEX system, asymmetric PCR was proved to be a better amplification method for the ssDNA library than the reported affinity magnetic bead method, and the corresponding parameters of the asymmetric PCR were also studied and optimized. The affinity of the final ssDNA library increased by nearly 200% compared with the original library. Cloning and sequencing of the final ssDNA library showed that there were at least two kinds of ssDNAs with different length in the affinity ssDNA library: one was 76 bases, another was 149 bases. Simulation of the secondary structures showed that the secondary structures of the two fragments were different greatly, suggesting that the two fragments could bind to different sites of V. alginolyticus surface.

Design and Test of Filter of High Gradient Magnetic Separation System for Trapping Immunoglobulin in Serum

Recently, affinity magnetic beads have been widely used in immunomagnetic cell sorting (IMCS) technology. Today, we can easily sort and analyze DNA and antibodies (immunoglobulin) using various types of affinity magnetic beads available in the market. The diameters of these affinity magnetic beads used in immunomagnetic cell sorting are limited to above approximately 1 mum because of the low magnetic fields induced by permanent magnets. Now, nano-sized affinity magnetic beads are strongly desired to achieve high resolutions. We have been studying and attempting to develop a high-gradient magnetic separation (HGMS) system that employs a superconducting magnet to induce a considerably higher magnetic field than that induced by a permanent magnet in order to trap smaller nano-sized affinity magnetic beads by a filter made of fine stainless steel wool. In this study, we constructed a prototype of a desktop-type HGMS system using a cryocooler-cooled LTS magnet and conducted preliminary experiments on trapping the nano-sized magnetic particles. Furthermore, we investigated the magnetic field distribution and magnetic force around a magnetic wire in the filter by means of a numerical simulation.

Standardized Peptidome Profiling of Human Urine by Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Peptidome profiling of human urine is a promising tool to identify novel disease-associated biomarkers; however, a wide range of preanalytical variables influence the results of peptidome analysis. Our aim was to develop a standardized protocol for reproducible urine peptidome profiling by means of magnetic bead (MB) separation followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). MBs with defined surface functionalities (hydrophobic interaction, cation exchange, and metal ion affinity) were used for peptide fractionation of urine. Mass accuracy and imprecision were calculated for 9 characteristic mass signals (M(r), 1000-10,000). Exogenous variables (instrument performance, urine sampling/storage conditions, freezing conditions, and freeze-thaw cycles) and endogenous variables (pH, urine salt and protein concentrations, and blood and bacteria interferences) were investigated with urine samples from 10 male and 10 female volunteers. We detected 427 different mass signals in the urine of healthy donors. Within- and between-day imprecision in relative signal intensities ranged from 1% to 14% and from 4% to 16%, respectively. Weak cation-exchange and metal ion affinity MB preparations required adjustment of the urinary pH to 7. Storage time, storage temperature, the number of freeze-thaw cycles, and bacterial and blood contamination significantly influenced urine peptide patterns. Individual urine peptide patterns differed significantly within and between days. This imprecision was diminished by normalization to a urinary protein content of 3.5 microg. This reliable pretreatment protocol allows standardization of preanalytical modalities and facilitates reproducible peptidome profiling of human urine by means of MB separation in combination with MALDI-TOF MS.

Single‐Step Purification of Recombinant Thermus aquaticus DNA Polymerase Using DNA‐Aptamer Immobilized Novel Affinity Magnetic Beads

A DNA aptamer specific for Thermus aquaticus DNA polymerase (Taq-polymerase) was immobilized on magnetic beads, which were prepared in the presented study. The effect of various parameters including pH, temperaturem and aptamer concentration on the immobilization of 5'-thiol labeled DNA-aptamer onto glutaric dialdhyde activated magnetic beads was evaluated. The binding conditions of Taq-polymerase on the aptamer immobilized magnetic beads were studied using commercial Taq-polymerase to characterize the surface complexation reaction. Efficiency of affinity magnetic beads in the purification of recombinant Taq-polymerase from crude extracts was also evaluated. For this case, the enzyme "recombinant Taq-DNA polymerase" was cloned and expressed using an Amersham E. coli GST-Gene Fusion Expression system. Crude extracts were in contact with affinity magnetic beads for 30 min and were collected by magnetic field application. The purity of the eluted Tag-polymerase from the affinity beads, as determined by HPLC, was 93% with a recovery of 89% in a one-step purification protocol. Apparently, the system was found highly effective as one step for the low-cost purification of Taq-polymerase in bacterial crude extract.