Antigens magnetic beads

FIGURE 14.5 Multiprotein electrical detection protocol based on different inorganic colloid nanocrystal tracers, (a) Introduction of antibody-modified magnetic beads (b) binding of the antigens to the antibodies on the magnetic beads (c) capture of the nanocrystal-labeled secondary antibodies (d) dissolution of nanocrystals and electrochemical stripping detection (reproduced from [29] with permission). 

Figure 3.24 Schematic representation of the analytical protocol (A) Capture of the ALP-loaded CNT tags to streptavidin-modified magnetic beads by a sandwich DNA hybridization (a) or Ab-Ag-Ab interaction (b). (B) Enzymatic reaction. (C) Electrochemical detection of the product of the enzymatic reaction at the CNT-modified glassy carbon electrode MB, Magnetic beads P, DNA probe 1 T, DNA target P2, DNA probe 2 Abl, first antibody Ag, antigen Ab2, secondary...

Ossendorp, F. A., Bruning, P. F, van den Brink, J. A. M., and de Boer, M (1989) Efficient selection of high affinity B-cell hybidomas using antigen-coated magnetic beads. J Immunol Methods 120, 191—200. 

The detection limit (DL) of the electrochemical immunosensor based on magnetic beads has been estimated to be equal to 8 x 10-3 ng/mL. This depends on the affinity of antibodies for antigen and is defined as the lowest analyte concentration which can be distinguished and is calculated by evaluation of the mean of the blank solution (containing the tracer only) response minus two times the standard deviations [35]. 

Fig. 38.2. Particle-based electrochemical immunoassay protocol. (A) Introduction of antibody-modified magnetic beads to magnet/carbon paste electrochemical transducer surface (B) binding of the IgG antigen to the antibodies on the magnetic beads (C) capture of the gold nanoparticle labelled secondary antibodies (D) electrochemical stripping detection of AuNPs. Reprinted with permission from Ref. [72].

Heterogeneous immunoassay has also been conducted with the antibody immobilized on beads. For instance, mouse IgG (50-100 ng/mL) was detected by ELISA in a glass chip. First, mouse IgG (antigen) was captured by magnetic beads coated with sheep anti-mouse antibody (1.02 x 107 beads/mL). Then the secondary antibody, which was rat anti-mouse conjugated with alkaline phosphatase (0.7 pg/mL), was delivered. Thereafter the substrate, PAPP, was added. It was enzymatically converted to p-aminophenol (PAP), which was electrochem-ically detected by the on-chip interdigital microelectrodes [1016]. 

Heterogeneous immunoassay has also been conducted without the use of an enzyme label. For instance, electrochemical immunoassay of mouse IgG (antigen) was carried out in glass chip. The chip contained magnetic beads coated with the sheep anti-mouse antibody. After flowing in the secondary antibody (rat antimouse conjugated with PAPP), electrochemical oxidative detection of PAP was achieved (i.e., PAP was oxidized to p-quinoneimine) [1016]. 

Step 1. Antigen-coated beads are placed, with plastic tipped forceps, onto the magnetic terminals of the 96-place transfer device (as in Fig. 6) all beads are dropped simultaneously into wells of a Microtiter plate containing either serial serum dilutions or a screening dilution of many differ-... 

Fig. 6. Placing of antigen-coated beads onto iron terminals of device A, preparatory to dropping the beads (simultaneously) into a Microtiter plate whose wells contain diluted human sera. Dropping of beads into the wells is accomplished by simply removing the magnet (device B ) from the iron terminals of device A. ...

Figure 11. Modes of association of antigen/antibody complexes and other biomolecules with magnetic beads.

Non-magnetic beads of a variety of matrices, ready for coupling by various chemistries to oligos, antibodies, antigens, or what-have-you, are available for column or suspension applications from Polymer Laboratories (www.polymerlabs.com). 

The simplest way of fraction separation, however, is when one of the immunoreactants (i.e., either the antigen or the antibody) is immobilized on a solid phase, i.e., immunoreagent modified solid-phase separations, such as plastic tubes, microtitre plate, latex-, glass- or magnetic beads, dipsticks or nitrocellulose membranes [121]. The efl ciency of separation is determined by the nature of the solid surface. Unspecific binding of the label to the solid phase can occur, especially when the latter has hydrophobic properties [96]. The adsorption of the antibody to the solid surface causes its partial inactivation, which can explain some unexpected effects when comparing the same antibody used in liquid and solid-phase systems (see also Section 9.3.4.4). 

Selection is based upon the functional properties of the evolved protein (phenotype). Binding to antigen immobilized in the wells of a microtiter plate or on the surfaces of magnetic beads are frequently used approaches. Flow cytometry sorting systems, some of which can sort up to 108 cells/h, represent a promising new class of selection technique. 

A nonisotopic ELISA method in which serum specimens are added to microtiter wells coated with human Tg is also available. In this method, antibody binding is assessed using a peroxidase-conjugated anti-IgG/o-phenylenedi-amine system. An automated two-step fluorescent enzyme immunoassay has been reported. In this assay, Tg is immobilized on magnetic beads, and anti-human IgG mouse monoclonal antibody is labeled with alkafine phosphatase 4-methyiumbelliferyl phosphate is used as the substrate. IRMA and ELISA both have similar detection limits (approximately 3 to 5 U/mL). A considerably more sensitive radioassay has been reported in which diluted serum is incubated with T-labeled Tg to allow formation of antigen-antibody complexes these complexes are then precipitated by adding solid-phase protein A. Its detection limit is reported to be approximately 0.2 U/mL. 

Immunoenrichment can be applied at the peptide level, particularly in the stable isotope standards and capture by anti-peptide antibodies (SISCAPA) method (140). Target peptides together with their stable isotope-labeled counterparts (used as quantitation references) are enriched by antibodies that are developed against antigens with almost the same sequences. The antibodies are often covalently attached to magnetic beads for sample cleanup. This technique boasts an improved limit of quantitation and increased inter-laboratory reproducibility (141). 

The international reference method to detect and recover E. coli 0157 (ISO EN 16654, Fig. 1) inclndes a step of Immuno-Magnetic Separation (IMS), which requires the nse of magnetic beads coated with polyclonal antibodies against 0157 antigen (Dynabeads anti E. coli 0157, Dynal Ltd, Norway). The cell bead complexes are recovered from the medinm by the application of a magnetic field causing the complexes to be concentrated in the tnbe, and enabling the bulk medium to be discarded. The concentrated cells are next plated onto CT-SMAC for strain recovery and confirmation. Chapman et al. (1994) fonnd that IMS was 100-fold more sensitive for detection of E. coli 0157 H7 than direct culture on isolation media. 

Matsunaga T, Maeda Y, Yoshino T et al (2007) FuUy automated immunoassay for detection of prostate-specific antigen using nano-magnetic beads and micro-polystyrene bead composites, Beads on Beads . Anal Chim Acta 597 331-339... 

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