The Luciferase immunoprecipitation system (LIPS) was performed as previously described [12 (link)]. Serum samples were diluted 1:10 in buffer A (50 mN Tris [pH 7.5], 100 mM NaCl, 5 mM MGCL2, and 1% Triton X-100). 1 × 107 relative light units (RLU) of a Renilla-luciferase-NS3-antigen fusion (RLUC-NS3) was mixed into 40 μL buffer A and subsequently added to 10 μL of diluted sera for 1 h shaking at room temperature. For antigen precipitation: 30% Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL) were added to a 96-well filter HTS plate (Millipore, Bedford, MA), before 100 μL of the RLUC-NS3 antigen serum mixture was added for 1 h shaking at RT. Afterwards the filter plate was washed on a vacuum plate washer. Antigen-precipitation was quantified by adding 100 μL coelenterazine (p.j.k. GmbH, Kleinblittersdorf, Germany) on a Berthold LB960 centro XS3 plate luminometer (Berthold, Freiburg). The threshold for NS3-antibody positive samples was set at three standard deviations above the average of the negative controls.
Ultralink protein a g beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ultralink Protein A/G Beads are agarose-based affinity beads designed for the purification of immunoglobulins and other Fc-containing proteins. The beads feature a covalently coupled Protein A/G ligand, which binds to the Fc region of antibodies with high affinity. This allows for the efficient capture and purification of antibodies from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Lb960 centro xs3 plate luminometer, by Berthold Technologies (3 mentions)
Hts plate, by Merck Group (2 mentions)
Coelenterazine, by Promega (1 mentions)
Furimazine, by Promega (1 mentions)
Filter hts plate, by Merck Group (1 mentions)
Centro lb 960 microplate luminometer, by Berthold Technologies (1 mentions)
4 protocols using ultralink protein a g beads
1
Luciferase Immunoprecipitation for NS3 Antibodies
2
Quantification of Anti-EqHV NS3 Antibodies
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Anti-EqHV NS3 antibodies were semi-quantitatively measured by luciferase immunoprecipitation system (LIPS) as previously described (27 (link)). Serum samples diluted 1:10 in buffer A (50 mM Tris [pH 7.5], 100 mM NaCl, 5 mM MGCL2, and 1% Triton X-100) were measured in duplicates. Luciferase-NS3 fusion antigen (107 relative light units) was mixed in 40 μL buffer A and then added to 10 μL of diluted sera. The mixture was shaken at room temperature for 1 h. Antigen was precipitated using 30% Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL) added to a 96-well filter HTS plate (Millipore, Bedford, MA). Afterwards, 100 μL of the RLUC-NS3 antigen serum mixture was added and incubated for 1 h shaking at room temperature. The filter plate was then washed with phosphate-buffered saline on a vacuum plate washer. Antigen-precipitation was quantified by adding 100 μL coelenterazine (PJK GmbH, Kleinblittersdorf, Germany) on a Berthold LB960 Centro XS3 plate luminometer (Berthold, Freiburg, Germany). The threshold for NS3-antibody positive samples was set at three standard deviations above the average of the negative controls.
3
Quantification of Anti-EqHV NS3 Antibodies
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Anti-EqHV NS3 antibodies were semi-quantitatively measured by luciferase immunoprecipitation system (LIPS) as previously described (27 (link)). Serum samples diluted 1:10 in buffer A (50 mM Tris [pH 7.5], 100 mM NaCl, 5 mM MGCL2, and 1% Triton X-100) were measured in duplicates. Luciferase-NS3 fusion antigen (107 relative light units) was mixed in 40 μL buffer A and then added to 10 μL of diluted sera. The mixture was shaken at room temperature for 1 h. Antigen was precipitated using 30% Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL) added to a 96-well filter HTS plate (Millipore, Bedford, MA). Afterwards, 100 μL of the RLUC-NS3 antigen serum mixture was added and incubated for 1 h shaking at room temperature. The filter plate was then washed with phosphate-buffered saline on a vacuum plate washer. Antigen-precipitation was quantified by adding 100 μL coelenterazine (PJK GmbH, Kleinblittersdorf, Germany) on a Berthold LB960 Centro XS3 plate luminometer (Berthold, Freiburg, Germany). The threshold for NS3-antibody positive samples was set at three standard deviations above the average of the negative controls.
4
High-throughput Luciferase Immunoprecipitation Assay
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Essentially as described, the LIPS assays were performed in approximately 2.5 h using a 96-well plate format at room temperature [19 (link),20 (link),21 (link)]. Briefly, recombinant luciferase antigen lysates were produced from transfection of DNA plasmids constructs into Cos1 cells and their activity in light units (LU) was determined with a tube luminometer (Turner Design 20/20). To initiate testing, 40 μL of buffer A, 10 μL of diluted human sera (1 μL equivalent), and the luciferase-antigen cell extract (input of approximately 10 million LU), diluted in buffer A, were added to each well of a microtiter plate for 1 h. Next, a 30% suspension (6 μL) of Ultralink protein A/G beads (Pierce Biotechnology, Rockford, IL, USA) was added to the bottom of each well of a 96-well filter HTS plate (Millipore, Bedford, MA, USA). The 100 μL antigen-antibody reaction mixture was moved to a filter plate and incubated for 1 h on a rotary shaker. Multiple washing steps of the retained protein A/G beads were then performed. After completion of the washing steps, LU were measured in a Berthold LB 960 Centro microplate luminometer (Berthold Technologies, Bad Wildbad, Germany) using either coelenterazine or furimazine substrate (Promega, Madison, WI, USA) for detecting Renilla luciferase and NanoLuc activity, respectively.
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