Antibody immobilization on magnetic particles

Abstract

Magnetic particles (MNPs) offer attractive possibilities in biotechnology. MNPs can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide (Fe3O4) MNPs with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat IgG), bovine serum albumin (BSA) as blocking agent, and a complementary antibody labelled with FITC (anti-goat IgG). The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated MNPs, a 5% (w/v) concentration of BSA in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.

Designing novel nano-immunoassays: antibody orientation versus sensitivity

“…The second method consists on the use of the polysaccharidic region located in the Fc region of the antibody and the use of aminated nanoparticles. To our knowledge, few contributions in this line can be found on literature with magnetic microbeads and still less with magnetic nanoparticles [25][26][27][28][29][30][31][32]. The biofunctionalization of magnetic nanoparticles suffers some limitations when compared to surfaces and microbeads, such as easy aggregation with small changes of pH and/or ionic strength, polymeric shell instability, batch to batch MNPs irreproducibility, or presence of surfactants.…”

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…This is because the orientation and confirmation of antibodies are primarily based on the hydrophobic and polar interactions. [20] Furthermore, upon nanoparticle-antibody association, these charge interactions are being altered and thereby resulting in antibody destabilization. [21] However, there are studies showing functionally active antibody-nanoparticle conjugates developed from random conjugation, which clearly shows that these effects are strongly dependent on antibody species as well.…”

Magnetic dragging of vascular obstructions by means of electrostatic and antibody binding

“…in all three cases, the antibody-bound MPs were washed once and suspended in PBS buffer (1 mL, 2xl0 8 MPs). Particles coated using the above methods are stable for one month at 4 °C [19], [20]. Lastly, 100 ilL of coated MPs were added to the Eppendorf tube containing the clot and the volume was adjusted to 1 mL using PBS buffer.…”