Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log 10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens. Video LinkThe video component of this article can be found at https://www.jove.com/video/1549/ Protocol Schematic Overview:This video demonstrates the steps involved in performing the LIPS assay. Antigens are expressed in Cos1 cells as recombinant Renilla luciferase (Ruc)-antigen fusions, and crude extracts are obtained and used without purification. The LIPS assay is initiated by incubating crude Ruc-antigen extracts with patient sera in microtiter wells. The antibody-antigen mixture is then transferred to a 96-well filter plate containing protein A/G beads to capture IgG molecules. After washing the filter plate containing the protein A/G beads, antibody bound Ruc-antigen is measured by the addition of coelenterazine substrate and light units are measured with a luminometer. PART 1: Transfection of Plasmids for production of Renilla-Antigen Fusion ProteinsSet-up: Cos1 cells are cultured in DMEM-10% FCS using standard tissue culture protocols. Plasmids for Renilla luciferase fusions have been described previously 1 . DNA for these plasmids is prepared using a Midiprep kit from Qiagen. The yield should be approximately 1 -3 mg. Measure the DNA concentration and store as a 1000 μg/ml stock solution at -20°C. Procedure:1. One day before transfection, split Cos-1 cells into new 100 x 20 mm dishes at approximately 2 X 10 6 per plate and incubate at 37 °C.2. On the following day, the Cos-1 cells should be 80-95% confluent. Label 1.5 ml polypropylene microfuge tubes for each plasmid DNA to be transfected. Allow the FuGENE-6 transfection reagent, which is stored at 4° C, to warm up to room temp. 3. Add 94 μl of Opti-MEM media to each microfuge tube. Next add 6 μl of FuGENE 6 to the Opti-MEM media without touching the side wall....

Patients with thymic malignancy have high rates of autoimmunity leading to a variety of autoimmune diseases, most commonly myasthenia gravis caused by anti-acetylcholine receptor autoantibodies. High rates of autoantibodies to cytokines have also been described, although prevalence, spectrum, and functionality of these anti-cytokine autoantibodies are poorly defined. To better understand the presence and function of anti-cytokine autoantibodies, we created a luciferase immunoprecipitation system panel to search for autoantibodies against 39 different cytokines and examined plasma from controls (n ‫؍‬ 30) and patients with thymic neoplasia (n ‫؍‬ 17). In this screen, our patients showed statistically elevated, but highly heterogeneous immunoreactivity against 16 of the 39 cytokines. Some patients showed autoantibodies to multiple cytokines.

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