The use of mammalian cells produces antigens free of contaminating bacterial proteins

The use of mammalian cells produces antigens free of contaminating bacterial proteins. NIE LIPS assay. When SsIR was used in combination with NIE in the LIPS format, sensitivity and specificity improved to 100%, with a 7-fold difference between positive RU 58841 and negative values. No advantage was found in using a LIPS assay based on IgG4. At post-treatment follow-up, a significant decline in antibody titers was detected using the NIE ELISA (P< .0017) and the NIE LIPS assay (P< .0001). == Conclusions == LIPS addresses several limitations of current ELISAs and represents a major advance in the diagnosis ofS. stercoralisinfection. AlthoughStrongyloides stercoralisoften causes chronic and clinically asymptomatic contamination, the number ofStrongyloidesparasites can increase substantially in immunocompromised hosts, leading to hyperinfection, dissemination, and death if unrecognized [1]. Early recognition ofS. stercoralisinfection is usually challenging because of scanty and intermittent excretion of larvae in chronically infected immunocompetent hosts [2]. Despite this, the mainstay of diagnostic testing forS. stercoralisinfection has been stool examination, although more recently ELISAs have been used to measure antibodies to crude larval antigen. Serologic approaches to the diagnosis ofS. stercoralisinfection, however, have been hampered by poor specificity, reliance on crude parasite extracts, and the time needed to perform the assays [35]. A major drawback to ELISA-based diagnosis ofS. stercoralisinfection has been a reliance on crude antigen that must be prepared by isolating worms from the feces of heavily infected patients or experimental animals. Thus, investigators have turned to recombinant antigens, which can be purified easily and produced in large amounts [6]. Indeed, a 31-kDa recombinant antigen (termed NIE) derived from anS. stercoralisL3 cDNA library provided the basis for an ELISA that approaches the sensitivity and specificity of the crude antigenbased ELISA [5]. A stylish alternative to ELISA-based methods, luciferase immunoprecipitation systems (LIPS), has been successfully applied to the characterization of antibody responses toPneumocystis jirovecii,HIV, and hepatitis viruses [7]. LIPS is a relatively straightforward technology for identifying serum made up of antigen-specific antibodies and for generating quantitative antibody response profiles. Briefly, this approach involves fusion of a protein RU 58841 antigen to the enzyme reporterRenillaluciferase (Ruc), expression of the Ruc-antigen fusion in mammalian COS RU 58841 cells, immobilization of the Ruc-antigen fusion on protein A/G beads, and quantitation of antigen-specific antibody by the addition of a coelenterazine substrate and the measurement of light production [8]. This assay represents a major improvement over ELISA technology in that it produces a low background often with a 7-log dynamic range, thereby generating values with substantial separation between negative and positive antibody responses. The low background and high signal seen in the LIPS method can be credited, in part, to the use of a solution-phase immunoprecipitation assay that allows detection of a large number of conformational epitopes. The use of mammalian cells produces antigens free of contaminating bacterial proteins. An additional advantage of LIPS is that, once the Ruc-antigen constructs are made, relatively little time is needed to perform the assay. Recently, we reported the use of Ruc-antigen fusion proteins, produced in COS1 monkey RU 58841 kidney cells, in an immunoprecipitation assay to measure human antibody responses to tumor-associated proteins [8] and to a variety of infectious brokers [7]. In this study, we have broadened the application of LIPS to the diagnosis and monitoring ofS. stercoralisinfection. To develop a more rapid, specific, and standardized assay, we first developed a LIPS assay based on IgG (or IgG4) antibody to NIE and compared it with a standard NIE ELISA. Our data, generated using serum samples fromS. stercoralisinfected patients, filaria-infected patients, and uninfected control subjects, demonstrated the clear benefit of the LIPS method. Moreover, when a second antigen,S. stercoralisimmunoreactive antigen (SsIR), was used in combination with NIE in Rabbit polyclonal to CD80 the LIPS format, we found an even greater degree of sensitivity and specificity. Finally, we assessed the ability of LIPS to evaluate the success of treatment in the follow-up ofS. stercoralisinfected patients. == METHODS == == Patient populations == Informed consent was obtained from all patients. Protocols involving adult human subjects were approved by the Institutional Review Board at the National Institutes of Health. Serum samples were obtained from patients (n= 31) within 1 month afterS. stercoralislarvae were found in their stools. Healthy, uninfected control subjects (n= 36) had no history of travel to an area of endemicity. Filaria-infected patients (n= 39) had confirmed loiasis or onchocerciasis with at least 1 stool sample unfavorable forS. stercoralislarvae. Six of.