GNPs carrying selected gp120 high-mannose oligosaccharides were previously shown to bind 2G12 and to compete with 2G12/gp120 binding while demonstrated by surface plasmon resonance (SPR), NMR, and cellular neutralization experiments [25]

GNPs carrying selected gp120 high-mannose oligosaccharides were previously shown to bind 2G12 and to compete with 2G12/gp120 binding while demonstrated by surface plasmon resonance (SPR), NMR, and cellular neutralization experiments [25]. specific immunoglobulins (IgGs) against carbohydrates. Two nanometer platinum glyconanoparticles bearing oligosaccharide epitopes of HIV or TGFB2 were used as antigens to coating ELISA-plates. A ~3,000-collapse Icariin improved detection of specific IgGs in mice immunized against respect to the well known BSA-glycoconjugate ELISA was accomplished. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human being dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the recognition of biomarkers. Intro The detection of anti-glycan antibodies in serum is definitely of mounting interest for the evaluation of carbohydrate-based vaccines and pathogen illness as well as for the detection of biomarkers in diseases like malignancy. The profiling of human being serum antibodies has shown that a considerable portion of circulating antibodies is definitely directed against carbohydrates [1]. The affinity of anti-carbohydrate antibodies towards their epitopes, demands Icariin a multivalent demonstration of the carbohydrate-ligands and highly sensitive testing methods. Furthermore, the low large quantity of Icariin anti-carbohydrates antibodies in serum during pathological claims and/or early illness hampers their use as biomarkers for quick analysis. The coupling of carbohydrates on a scaffold (carrier) allows the multiple demonstration of these antigens in an enzyme-linked immunosorbent assay (ELISA) [2]. However, while protein covering of ELISA plates is definitely a well-established strategy, equivalent strategies for the direct coating of carbohydrates have been hampered by technical limitations. Early efforts to detect antibodies against bacterial polysaccharides by ELISA showed the difficulty to absorb carbohydrates to the assisting materials. This problem was solved by conjugation of the polysaccharides to positive charged poly-lysine scaffold, which allowed the immobilization of the producing neoglycoconjugate to ELISA plates [3]. Soon afterwards, glycolipids were efficiently used to coating ELISA surfaces for a type 14 polysaccharide, only (TetraPn-GNP) or in combination with the small peptide OVA323-339 of ovalbumin (TetraPnOv-GNP) [19]. Like a control (Number 1C), GNPs bearing only glucose (Glc-GNP) or galactose (Gal-GNP) were also included. The oligosaccharides are conjugated to the same aglycon, a thiol-ending amphiphilic linker Icariin to attach them to the gold surface. A glucose conjugate is definitely incorporated as inner component to modulate the denseness of the antigenic oligosaccharides on the surface [22]. Nunc MaxiSorp plates were selected for the GNP-ELISA, as related altered polystyrene slides were previously used to prepare microarrays of polysaccharides and proteoglycans [23]. GNPs were adsorbed within the MaxiSorp surface because of the high hydrophilicity. Open in a separate window Number 1 Platinum glyconanoparticles used in this work to coating ELISA plates for anti-carbohydrate-antibodies detection.(A) High-mannose type undecasaccharide present within the HIV gp120 surface and GNPs carrying the tetramannoside (TetraMan) or dimannoside (DiMan), partial structures of the viral gp120 high-mannose undecasaccharide, to detect 2G12 antibody. (B) Repeating unit of type 14 capsular polysaccharide and GNPs transporting the tetrasaccharide epitope (TetraPn) of the Pn14PS and the T-helper OVA323-339. (C) GNPs transporting glucose or galactose as control. Detection of anti-HIV monoclonal antibody 2G12 Like a proof of basic principle, we setup a GNP-ELISA for the detection of the anti-HIV human being monoclonal antibody 2G12. The 2G12 antibody is one of the broadly neutralizing antibodies against HIV-1 and binds to a conserved high-mannose cluster on HIV gp120 [24]. GNPs transporting selected gp120 high-mannose oligosaccharides were previously shown to bind 2G12 and to compete with 2G12/gp120 binding as shown by surface plasmon resonance (SPR), NMR, and cellular neutralization experiments [25]. In particular, TetraMan-GNPs were able to bind 2G12 with high avidity (nanomolar range) and inhibit 2G12/gp120 connection in the micromolar range as measured by SPR and NMR. On the contrary, the analogue DiMan-GNPs did not display significant binding to 2G12 actually at higher concentration [25]. For this reason, in the present study, we selected TetraMan-GNP for the detection of 2G12 and DiMan-GNP as control to exclude non-specific.