Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Abstract

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....

Serology-Enabled Discovery of Genetically Diverse Hepaciviruses in a New Host

“…Plasmid DNA was then prepared from these two different pREN2 expression vectors using a Qiagen Midi preparation kit. Following transfection of mammalian expression vectors, crude protein extracts were obtained as described for use as antigen (8).…”

“…Similarly, although the demonstration of specific adaptive immune responses against viral structural and nonstructural proteins cannot in itself prove a causal relationship to disease, it does provide definitive evidence of host infection (2,7). Here, we describe the usefulness of a sensitive serological assay (7)(8)(9)(10)(11) that can be quickly established for new pathogens to screen different animal species for evidence of virus infection and thereby for identification of a novel virus's natural reservoir and host range. Our serology data indicated the presence of CHV-like viruses in horses and led to the genetic characterization of eight novel and genetically diverse nonprimate hepaciviruses (NPHVs).…”

Anti-cytokine autoantibodies are associated with opportunistic infection in patients with thymic neoplasia

“…For LIPS testing, a master plate was first constructed by diluting patient sera 1:10 in assay buffer A (50mM Tris, pH 7.5, 100mM NaCl, 5mM MgCl 2 , 1% Triton X-100) in a 96-well polypropylene microtiter plate, which was then used for dispensing diluted plasma samples for testing in a 96-well plate format as described. 23 For evaluating antibody titers by LIPS, 40 L of buffer A, 10 L of diluted human sera (1 L equivalent), and 1 ϫ 10 7 light units (LUs) of Ruc-antigen Cos1 cell extract, diluted in buffer A to a volume of 50 L, were added to each well of a polypropylene plate at room temperature for 1 hour. The mixture was then transferred to 96-well filter plates containing protein A/G beads for 1 hour.…”

“…After transfection into COS1 cells, crude protein extracts were obtained as described. 23 Plasma was obtained from heparinized venous whole blood by centrifugation and stored in aliquots at Ϫ80°C. All samples were coded to prevent observer bias before assay.…”

Discovery of a novel nidovirus in cattle with respiratory disease

“…To examine the seroprevalance of BoNV, we generated a luciferase immunoprecipitation assay (LIPS) to measure antibody levels against BoNV (Burbelo et al, 2009). We implemented this assay to examine 127 bovine sera originating from the central and western USA (Kansas, Idaho, Missouri and Texas).…”