Recombinant glutathione S-transferase (GST)-tagged rp17 (Chembio Diagnostic Systems, Inc., Medford, NY) and full-length recombinant TmpA (ViroGen, Watertown, MA) were provided as a 114-kDa fusion at the N terminus with β-galactosidase. Both rp17 and TmpA were dialyzed overnight at 4°C into phosphate-buffered saline (PBS). Antigens were coupled to polystyrene microspheres (SeroMap beads; Luminex Corporation, Austin, TX) as previously described (1 (link)). Briefly, carboxyl groups on the beads were chemically modified to ester groups by 1-ethyl-3(3-dimethlaminopropyl) carbodiimide (EDC) (Calbiochem, San Diego, CA) in the presence of N-hydroxysulfosuccinimide (NHS) (Pierce, Rockford, IL). Primary amine groups on the antigens were then reacted with ester groups on the beads to create amide covalent bonds. Antigen-coupled beads were quantified by hemocytometer and stored at 4°C with PBS containing 1% bovine serum albumin (BSA) plus protease inhibitors.
Seromap beads
Manufactured by DiaSorin
Sourced in United States
SeroMAP beads are a type of magnetic micro-particles used in immunoassay-based diagnostic tests. They serve as a solid support for the capture and detection of target analytes in biological samples.
Automatically generated - may contain errors
7 protocols using seromap beads
1
Antigen Coupling to Polystyrene Beads
2
Coupling Viral Antigens to Polystyrene Beads
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1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (Calbiochem, Woburn, USA) was used to convert the carboxyl groups on carboxylated polystyrene beads (SeroMap Beads; Luminex Corporation, Austin, USA) to esters. The esters on the so-called activated beads could then react with amine groups on the antigens to form covalent amide bonds between the beads and the antigens. We coupled 12.5 million activated beads – in phosphate-buffered saline at pH 7.2 – with 30 µg of each dengue virus-like particle and 7.5 µg of the chikungunya virus antigen. For the malaria antigens and glutathione-S-transferase, we coupled 12.5 million activated beads, in 50 mmol/L 2-(N-morpholino) ethanesulfonic acid buffer with 0.85% (w/v) NaCl, at pH 5.0 – with 28 µg of the 19-kDa fragment, from clone 3D7, linked to glutathione-S-transferase, 15 µg of the 42-kDa fragment from clone 3D7, 15 µg of the 42-kDa fragment from clone FVO, and 12 µg of glutathione-S-transferase. Coupling efficiency was evaluated using monoclonal antibodies and reference sera that were known to be highly reactive to the antigens.31 (link),32 (link)
3
Quantifying Anti-FIX Antibodies in Hemophilia
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Modified αVIII-FLI Assay for Anti-FVIII Antibodies
5
Anti-FVIII Antibodies Detection Assay
6
Optimizing Malaria Antigen Quantification
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The MAP assay was previously optimized for quantification of Ab to multiple malarial antigens [30] (link), including VAR2CSA sequences [24] (link). To determine the optimal concentration of ID1-ID2a, different amounts of the ID1-ID2a recombinant proteins were coupled to 1 million microspheres (SeroMAP beads, Luminex Corp., Austin, TX), including 1.6, 5, and 10 µg of the 3D7 protein and 1, 5, 10, and 15 µg of the FCR3 protein. ID1-ID2 (3D7) at 1.6 µg/million microspheres and ID1-ID2 (FCR3) at 5 µg/million microspheres were found to be optimal. DBL1 (3D7 and 7G8), DBL1+2 FCR3, DBL2 FCR3, DBL3 (FCR3) and DBL5 (FCR3) were coupled at 1 µg/million microspheres [24] (link); FV2 was coupled at 3 µg protein/million microspheres based on previous optimization studies [23] (link).
7
Covalent Coupling of Antigens to Polystyrene Beads
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Antigens were covalently coupled to polystyrene beads (SeroMap Beads; Luminex Corporation, Austin, TX) by modifying carboxyl groups on the beads to ester groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (EMD Millipore Calbiochem, USA) in the presence of N-hydroxysulfosuccinimide (sulfo-NHS) (Thermo Scientific Pierce, USA)40 (link). Ester groups on the beads bind to primary amine groups on antigens to create stable amide covalent bonds. Beads were briefly sonicated in a water bath and washed with 0.1 M sodium phosphate buffer, pH 6.2 (NaP) in preparation for bead activation. Beads were protected from light and rotated for 20 min in NaP with 5 mg/ml each EDC and NHS. After activation, activated beads were washed and suspended in coupling buffer, antigen added, and rotated at room temperature for 2 h. Coupling buffers and antigen amounts were previously determined for each antigen (Supplementary Table 5 ). After 2 h, antigen-coupled beads were washed with PBS and unreacted sites were blocked with 1% bovine serum albumin (BSA) in PBS for 30 min. Antigen-coupled beads were resuspended in storage buffer (PBS, 1% BSA, 0.05% Tween-20, 0.02% NaN3, protease inhibitors) and kept at 4 °C until use in assays.
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