The early detection of Legionella in water reservoirs, and the prevention of their often fatal diseases, requires the development of rapid and reliable detection processes. A method for the magnetic separation (MS) of Legionella pneumophila by superparamagnetic iron oxide nanoparticles is developed, which represents the basis for future bacteria detection kits. The focus lies on the separation process and the simplicity of using magnetic nanomaterials. Iron oxide nanoparticles are functionalized with epoxy groups and Legionella-specific antibodies are immobilized. The resulting complexes are characterized with infrared spectroscopy and tested for the specific separation and enrichment of the selected microorganisms. The cell-particle complexes can be isolated in a magnetic field and detected with conventional methods such as fluorescence detection. A nonspecific enrichment of bacteria is also possible by using bare iron oxide nanoparticles (BIONs), which we used as a reference to the nanoparticles with immobilized antibodies. Furthermore, the immunomagnetic separation can be applied for the detection of multiple other microorganisms and thus might pave the way for simpler bacterial diagnosis.

The demand for purified antibodies is ever-rising. This study presents a nanoparticle-based material for efficient magnetic separation of immunoglobulin G (IgG) with high binding capacity. The characteristics include: (i) Cost-effective bare iron oxide nanoparticles are used as the solid phase on which optimized protein A-based ligands are directly immobilized. An additional chemical modification or activation is not needed. (ii) Oriented immobilization of the ligands is promoted using a C-terminal peptide tag, containing amino acids with an affinity for iron oxide. (iii) The immobilized ligand consists of eight polymerized B-domains of Protein A. This affinity adsorbent for antibody capture allows a recovery of up to 418 mg IgG per gram of particle, which exceeds the state of the art of magnetic nanoparticles as well as microparticles. Particles with a lower ligand density show a higher percentage recovery of IgG, which allows for a cost-effective design of the adsorbent. Furthermore, the selectivity of the immobilized ligand is shown by means of purification of rabbit polyclonal IgG using rabbit serum.

Monoclonal antibodies are key molecules in medicine and pharmaceuticals. A potentially crucial drawback for faster advances in research here is their high price due to the extremely expensive antibody purification process, particularly the affinity capture step. Affinity chromatography materials have to demonstrate the high binding capacity and recovery efficiency as well as superior chemical and mechanical stability. Low-cost materials and robust, faster processes would reduce costs and enhance industrial immunoglobulin purification. Therefore, exploring the use of alternative materials is necessary. In this context, we conduct the first comparison of the performance of magnetic nanoparticles with commercially available chromatography resins and magnetic microparticles with regard to immobilizing Protein G ligands and recovering immunoglobulin G (IgG). Simultaneously, we demonstrate the suitability of bare as well as silica-coated and epoxy-functionalized magnetite nanoparticles for this purpose. All materials applied have a similar specific surface area but differ in the nature of their matrix and surface accessibility. The nanoparticles are present as micrometer agglomerates in solution. The highest Protein G density can be observed on the nanoparticles. IgG adsorbs as a multilayer on all materials investigated. However, the recovery of IgG after washing indicates a remaining monolayer, which points to the specificity of the IgG binding to the immobilized Protein G. One important finding is the impact of the ligand-binding stoichiometry (Protein G surface coverage) on IgG recovery, reusability, and the ability to withstand long-term sanitization. Differences in the materials’ performances are attributed to mass transfer limitations and steric hindrance. These results demonstrate that nanoparticles represent a promising material for the economical and efficient immobilization of proteins and the affinity purification of antibodies, promoting innovation in downstream processing.

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