Four-Antigen Mixture Containing V-Cyclin for Serological Screening of Human Herpesvirus 8 Infection

Burbelo, Peter D.; Leahy, Hannah P.; Groot, Sandra; Bishop, Lisa R.; Miley, Wendell; Iadarola, Michael J.; Whitby, Denise; Kovacs, Joseph A.

doi:10.1128/cvi.00474-08

Cited by 34 publications

(51 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This highly scalable system allows for the parallel LIPS profiling of a panel of human autoantigens 3 or the whole proteome of a virus 4 . It is likely that many different antibody profiles generated by LIPS will be useful for diagnostics [3][4][5][6][7][8][9] , antigen discovery 9 , following the course of a treatment 10 , vaccine monitoring and has broad implications for specific disease states and for public health in general.…”

Section: Discussionmentioning

confidence: 99%

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Burbelo

Ching

Klimavicz

et al. 2009

JoVE

Self Cite

118

170

View full text Add to dashboard Cite

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....

show abstract

Section: Discussionmentioning

confidence: 99%

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Burbelo

Ching

Klimavicz

et al. 2009

JoVE

Self Cite

118

170

View full text Add to dashboard Cite

show abstract

“…The reason for this discrepancy may relate to horse immune differences as well as the likelihood that many of the horse serum samples represent late Lyme infection. Although not explored here, an antigen mixture of different B. burgdorferi strains by LIPS also might be useful for diagnostic screening (6,13,14). For example, based on the comparably low background signals of VOVO, DbpA, and DbpB, these antigens could be employed as a mixture, thereby simplifying data collection and analysis.…”

Section: Discussionmentioning

confidence: 99%

Antibody Profiling of Borrelia burgdorferi Infection in Horses

Burbelo

Bren

Ching

et al. 2011

Clin. Vaccine Immunol.

Self Cite

View full text Add to dashboard Cite

Infection with Borrelia burgdorferi is common in horses and ponies from the New England and mid-Lyme disease caused by the spirochete Borrelia burgdorferi represents an important problem in veterinary medicine due to the wide-spread infection of dogs, cats, and horses in areas where the organism is endemic (3, 30). While most cases of equine B. burgdorferi infection remain asymptomatic, some horses show signs of illness, including sporadic lameness, weight loss, arthritis, and encephalitis (16). Unlike humans, where the first sign of B. burgdorferi infection is erythema migrans skin lesions, the diagnosis of Lyme disease in horses is complex, and diagnostic testing is only one parameter for confirmation (16).A variety of serological tests, including immunofluorescence assays (IFA), Western blotting, and enzyme-linked immunosorbent assays (ELISAs), have been employed to detect human antibodies to B. burgdorferi (29). Considerable progress has been made in employing defined antigenic targets, such as the C6 peptide (1, 20, 27) and other recombinant B. burgdorferi antigens, including BBK07 (18, 19) and decorin binding proteins (Dbp) DbpA and DbpB (21,22,24,28,31,32). Although extensive testing of different antigens and assay formats has been performed for human Lyme disease (29), less is known about the optimal serological diagnosis of equine B. burgdorferi infection. Recently, the C6 SNAP test has been used for the serological diagnosis of equine Lyme disease (23, 26). Unfortunately, Chang and colleagues found that the C6 SNAP test detected only 63% of known, experimentally B. burgdorferiinfected horses, suggesting that this test is suboptimal for the diagnosis of equine infection (26). In light of the poor sensitivity of the currently available C6 SNAP test, a better understanding of humoral responses in B. burgdorferi-infected horses is needed.Luciferase immunoprecipitation systems (LIPS) comprise a relatively new approach for the serological testing of antibodies associated with many different pathogens (5). LIPS is based on employing light-emitting Renilla luciferase-fusion proteins in a liquid-phase immunoprecipitation assay. Serologic testing by LIPS for a variety of human pathogens provides the full exploitation of antibody profiles because of its robustness, multiplex capacity, and high sensitivity and specificity (6,11,12,14,15). Recently, a LIPS test employing a synthetic VOVO antigen consisting of two variants of immunodominant epitopes of the C6 peptide and OspC achieved 98% sensitivity and 100% specificity for the diagnosis of human Lyme disease, but the findings were not statistically different from those of the C6 ELISA (10). LIPS antibody profiling of recombinant DbpA and DbpB also was highly informative (10). In this study, LIPS tests for VOVO, DbpA, and DbpB antigens were used for measuring B. burgdorferi antibodies in horses, and the results were compared to IFA results for the diagnosis of equine B. burgdorferi infection.

show abstract

“…For LIPS testing, only a small amount of serum (1 microliter per test or less) is needed. Upon testing twenty different proteins from the Kaposi Sarcoma-associated herpes virus (KSHV/HHV-8) proteome, v-cyclin was identified as a new, informative antigen for diagnosis of KSHV infection in Kaposi Sarcoma (KS) patients [13]. Figure 1 shows LIPS detection of antibodies against the established K8.1 KSHV antigen and v-cyclin in KS patients.…”

Section: Infectious Disease Diagnostics and Antigen Discovery By Lipsmentioning

confidence: 99%

Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems

Burbelo¹,

Ching²,

Bren³

et al. 2011

Expert Review of Proteomics

View full text Add to dashboard Cite

B-cell mediated humoral responses are triggered in many human diseases including autoimmune, cancer, neurologic, and infectious diseases. However, the full exploitation of the information contained within a patient's antibody repertoire, for diagnosis, monitoring and even disease prediction has been limited due to the poor diagnostic performance of many immunoassay formats. We have developed Luciferase immunoprecipitation systems (LIPS) that harnesses light emitting proteins to generate high definition antibody profiles optimal for both diagnostics and biomarker discovery. Here we describe the results and implications from a range of LIPS antibody profiling studies performed in our laboratory. These include highly sensitive diagnostics for domestic and global pathogens, insights into infection-related diseases, discovery of new biomarkers for human diseases, subcategorization of symptoms and identification of pathogenic autoantibodies against self-proteins. These investigations highlight the types of humoral response profiles associated with different diseases, provide new information related to disease pathogenesis, and provide a framework for incorporating LIPS antibody profiling into global health initiatives and disease monitoring.

show abstract

Four-Antigen Mixture Containing V-Cyclin for Serological Screening of Human Herpesvirus 8 Infection

Cited by 34 publications

References 27 publications

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Antibody Profiling of Borrelia burgdorferi Infection in Horses

Searching for biomarkers: humoral response profiling with luciferase immunoprecipitation systems

Contact Info

Product

Resources

About