A BAMA with C.1086 gp140 K160N-labeled magnetic beads (MagPlex; Bio-Rad) was used as previously described (72 (link)) to measure concentrations of antigen-specific IgG in secretions and IgA in both secretions and serum depleted of IgG. Briefly, beads reacted with dilutions of the standard (73 (link)) and specimens at 1,100 rpm at 4°C overnight were washed and developed with biotinylated anti-monkey IgG or -monkey IgA (Rockland) followed by phycoerythrin-labeled Neutralite-avidin (SouthernBiotech). The construction of standard curves and interpolation of antibody concentrations were done using Bioplex Manager software after measurement of fluorescence with a Bioplex 200 instrument (Bio-Rad). Concentrations of gp120-specific IgG or IgA in secretions were divided by the total IgG or IgA measured in the sample by an ELISA (74 (link)) to obtain the specific activity (nanograms of IgG or IgA antibody per microgram of total IgG or IgA).
Magplex
Manufactured by Bio-Rad
The MagPlex is a magnetic bead-based assay technology developed by Bio-Rad. It enables the simultaneous detection and quantification of multiple analytes in a single sample. The MagPlex system utilizes color-coded magnetic beads to capture and identify specific target molecules, allowing for efficient and high-throughput analysis.
Automatically generated - may contain errors
Lab products found in correlation
Phycoerythrin labeled neutralite avidin, by Southern Biotech (1 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Pcr clean up extraction kit, by Macherey-Nagel (1 mentions)
Magna pure external lysis buffer, by Roche (1 mentions)
Streptavidin r phycoerythrin sa pe, by Thermo Fisher Scientific (1 mentions)
Bio plex amine coupling kit, by Bio-Rad (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
4 protocols using magplex
1
Quantifying Antigen-Specific Antibodies
2
Measuring Anti-HIV-1 Env Antibody Titers
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Serum IgG titers against HIV-1 C.1086 Env gp140 were determined by enzyme-linked immunosorbent assays (ELISAs) using standard protocols (21 (link)). Baseline sera from each animal served as negative control, and optical density (OD) values twofold above baseline were considered positive and extrapolated to determine anti-Env antibody concentrations. Antibody levels in rectal secretions were measured using a binding antibody multiplex assay using C.1086 gp140 K160N-labeled magnetic beads (MagPlex; Bio-Rad) as previously described (62 (link)). C.1086 gp120 (from B. Haynes) coated on the magnetic beads was used.
3
Multiplex Cytokine Profiling in Rat Samples
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Rat plasma and brain homogenates were collected as previously described. All samples were kept on ice during the setup process. Protein concentration of brain homogenates was calculated using a BCA assay. Bio-Plex Pro Assay rat cytokine plates were purchased from Bio-Rad (Hercules, CA, United States). A 1:4 dilution of plasma and brain homogenate was tested following the Bio-Plex Pro cytokine plate protocol. The plates were read on the Bio-Rad MagPlex.
4
Recombinant Norwegian Antigen Production
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Inorganic chemicals and organic solvents used were reagent grade or better. Production of recombinant Norwegian antigens included the use of MagNA Pure External Lysis Buffer from Roche, Basel, Switzerland, a PCR clean-up extraction kit from Macherey-Nagel, Düren, Germany, restriction enzymes from Thermo Fisher Scientific, Waltham, MA, USA, pGEX-6P vector and glutathione sepharose 4B resin from Cytiva, Marlborough, MA, USA, and Quick Start Bradford protein assay from Bio-Rad, Hercules, CA, USA. After buffer exchange for some of the antigens (the Norwegian recombinant proteins and the commercial p25 and p16–25), protein quantification was performed using Qubit™ Protein Assay Kit from Thermo Fisher Scientific. For coupling of antigens, magnetic beads (MagPlex) and Bio-Plex Amine Coupling Kit from Bio-Rad, were used. Coupling of antigens with GST was confirmed using the anti-GST antibody clone vpg66 and streptavidin R-phycoerythrin (SAPE), both from Thermo Fisher Scientific. For running the immunoassay, protein G conjugated to phycoerythrin (protein G/PE) from AcZon Pharma, Bologna, Italy was used. During optimisation and verification, a sheep serum from In3diagnostic, Turin, Italy and a reference serum from Innovative Diagnostics, Grabels, France, were included.
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