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


CONCLUSIONS
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.
Burbelo
Peter D
PD
Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University Medical Center, Washington, DC 20057, USA. burbelpd@georgetown.edu
Goldman
Radoslav
R
Mattson
Thomas L
TL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000941:Antigens / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D001709:Biotechnology / Q000379:methods*; D001798:Blood Proteins / Q000032:analysis*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D049410:Luciferases, Renilla / Q000378:metabolism*; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000097:blood; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D048790:Renilla; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D001798:Blood Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D049410:Luciferases, Renilla