Antibody purification: ammonium sulfate fractionation or gel filtration.
ABSTRACT Antibodies can be purified by a variety of methods based on their unique physical and chemical properties such as size, solubility, charge, hydrophobicity and binding affinity. This chapter focuses on ammonium sulfate precipitation as a convenient first step in antibody purification in that, it allows the concentration of the starting material and the precipitation of the desired protein. The principle of ammonium sulfate precipitation lies in "salting out" proteins from the solution. The proteins are prevented to form hydrogen bonds with water and the salt facilitates their interaction with each other forming aggregates that afterward precipitate out of solution. Gel filtration or size- exclusion chromatography is also discussed in this chapter. Gel filtration is based on the relative size of protein molecules and it is of great value to separate IgMs, exchange buffers and/or desalt solutions. The columns designed to separate the proteins are composed of porous beads and the proteins will flow through the packed column inside and around the beads, depending on its size.
- SourceAvailable from: Tim D Sparwasser[show abstract] [hide abstract]
ABSTRACT: FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.Blood 04/2012; 119(24):5898-908. · 9.06 Impact Factor
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ABSTRACT: Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood.The Journal of clinical investigation 11/2013; · 15.39 Impact Factor