A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins

Abstract

Background: Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data.

Results: We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically.

Conclusion: This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.

Figure 1

Immunoprecipitation experiments with commercial antibodies. Various amounts of anti-FLAG monoclonal, anti-p53 polyclonal or control (anti-WASP) polyclonal antibodies were mixed with 5 μl of a Cos1 extract containing Ruc-p53 for 1 h in the presence of protein A/G beads, processed and light units measured. The data shown is from one of three independent experiments giving similar results.

Figure 2

The immunoprecipitation assay with Ruc-p53 and a clinical serum sample. A: The immunoprecipitation activity is proportional to incubation time. Tubes containing identical amounts of Ruc-p53 fusion protein extract (5 μl), patient 34 sera (1 μl) and protein A/G beads were incubated for 5, 30, 60, 90 and 120 min and processed for luciferase activity. B: Immunoprecipitation activity with various amounts of total crude patient 34 sera. Different amounts of patient sera (0.002 to 2 μl) were mixed with 5 μl of the Ruc-p53 fusion protein extract and incubated for 1 hour in the presence of protein A/G beads, processed and light units measured. The data shown is from one of three independent experiments giving similar results.

Figure 3

Competition assays blocking Ruc-p53 immunoprecipitation using bacterially-produced antigen. Different amounts of E. coli-produced MBP-p53 were incubated with patient sera 34 (0.5 μl) or commercial anti-p53 antibody (25 ng) for 1 h. Protein A/G beads and Ruc-p53 extract were then added and incubated for an additional 1 h, processed and light units measured. The data shown is from one of two independent experiments giving similar results.

Figure 4

Structure of the pREN2 mammalian expression vector. Features indicated are CMV (cytomegalovirus) promoter, the N-terminal FLAG epitope and Ruc. Sequences for Ruc are in bold. cDNAs for tumor antigens were cloned downstream of Ruc between the BamH1-Xho1 sites.