A luciferase immunoprecipitation systems assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis infection

Although Strongyloides stercoralis often causes chronic and clinically asymptomatic infection, the number of Strongyloides parasites can increase substantially in immunocompromised hosts, leading to hyperinfection, dissemination, and death if unrecognized [1]. Early recognition of S. stercoralis infection is challenging because of scanty and intermittent excretion of larvae in chronically infected immunocompetent hosts [2]. Despite this, the mainstay of diagnostic testing for S. stercoralis infection has been stool examination, although more recently ELISAs have been used to measure antibodies to crude larval antigen. Serologic approaches to the diagnosis of S. stercoralis infection, however, have been hampered by poor specificity, reliance on crude parasite extracts, and the time needed to perform the assays [3–5]. A major drawback to ELISA-based diagnosis of S. stercoralis infection 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 an S. stercoralis L3 cDNA library provided the basis for an ELISA that approaches the sensitivity and specificity of the crude antigen–based ELISA [5]. An attractive alternative to ELISA-based methods, luciferase immunoprecipitation systems (LIPS), has been successfully applied to the characterization of antibody responses to Pneumocystis jirovecii, HIV, and hepatitis viruses [7]. LIPS is a relatively straightforward technology for identifying serum containing antigen-specific antibodies and for generating quantitative antibody response profiles. Briefly, this approach involves fusion of a protein antigen to the enzyme reporter Renilla luciferase (Ruc), expression of the Ruc-antigen fusion in mammalian COS 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 kidney cells, in an immunoprecipitation assay to measure human antibody responses to tumor-associated proteins [8] and to a variety of infectious agents [7]. In this study, we have broadened the application of LIPS to the diagnosis and monitoring of S. stercoralis infection. 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 from S. stercoralis–infected patients, filaria-infected patients, and uninfected control subjects, demonstrated the clear benefit of the LIPS method. Moreover, when a second antigen, S. stercoralis immunoreactive antigen (SsIR), was used in combination with NIE in 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 of S. stercoralis–infected patients.