Using place specific primers containing BamH1 and Xho1 modifications, each of these 19 DNA inserts was amplified and cloned into the BamH1/Xho1 site of pREN2, a mammalian Ruc expression vector explained previously [17]. basis for the quick detection of IgG and IgG4 reactivity inside a luciferase immunoprecipitation system (LIPS) assay. These anti-Wb123 IgG and IgG4 antibodies were only present in those with Wb infection and not in those infected with closely related filarial parasites (e.g. (Wb), (Bm) and may lead to disfiguring and disabling lymphedema and elephantiasis. Recent and ongoing control actions, aimed at interrupting transmission by eliminating the reservoir of illness (through mass drug administration [MDA] wherever possible, e.g., Global System for the Removal of Lymphatic 1-Methyladenosine Filariasis [GPELF]) offers led to considerable decreases in the prevalence of illness and the risk of disease [1]. Despite these steps, estimates suggest that 120 million people remain infected with Wb or Bm with an additional 1 billion people being at risk in the tropics and subtropics worldwide [2]. Superimposed on this estimate of Wb- and Bm-infected individuals is the concern about the severe adverse events associated with MDA in West and Central Africa in areas where another filarial parasite, [Ll], is usually co-endemic [3], [4]. MDA programs are in progress in more than 50 of the 72 LF-endemic countries, with 13 having halted following at least 5 annual MDA treatments [1]. Currently, WHO guidelines for assessment of transmission interruption are based on the monitoring of antigenemia (for Wb at least) in children; however, antibody responses C particularly to antigens expressed in L3s (the infective larvae) are likely to provide much earlier steps of ongoing transmission [5], [6] than the 1-Methyladenosine presence of microfilariae or circulating filarial antigen. Such an approach has been used quite successfully in onchocerciasis-endemic regions of Central America where the absence of antibody responses to an (Ov) L3-expressed antigen, Ov16 [7], has been used as one of the criteria to certify areas 1-Methyladenosine free of Ov transmission [8]C[11]. A number of immunoassays using a variety of different filarial SHCC Ags have been proposed for use as surveillance tools in LF; these include Bm14/BmSXP-1 [12], BmR1 [13], WbSXP-1 [14], and Bm33 [15]. The sensitivity of these assays has generally been high but limited specificity with respect to non-LF causing filariae (Ov, Ll, L3 ESTs and L3 ESTs (downloaded as FASTA files from Genbank, NCBI, NLM) were put together into contigs using the Desktop cDNA Annotation System (dCAS 1.4.3) software package [16]. The producing output, and Excel table with hyperlinks, was used to identify potential proteins that were specific for the lymphatic filariae (Wb) and/or (Bm) and that were without significant homology to the related filariae ([Ll] and [Ov]). Contigs were selected for further evaluation as candidate assay targets based on: 1) length of at least 200 bp with a predicted open reading frame (ORF); and 2) lack of sequence homology to the nonredundant 1-Methyladenosine protein database (nr) and other stages (mf, adult males, adult females) of Bm or Wb (Table S1). Plasmid and primers Each of the 19 potential targets (full length or longest put together contig) was synthesized commercially (Genscript, Piscataway, NJ) with codon usage optimized for expression in mammalian 1-Methyladenosine cells. Using place specific primers made up of BamH1 and Xho1 modifications, each of these 19 DNA inserts was amplified and cloned into the BamH1/Xho1 site of pREN2, a mammalian Ruc expression vector explained previously [17]. The producing pREN2 expression vector was prepared using a Qiagen Midi kit (Qiagen, Gaithersburg, MD). Automated DNA sequencing was used to confirm the integrity of the DNA constructs. Ruc-antigen fusion extracts were prepared from transfected COS1 cells as previously explained [17]. Serum samples Serum samples (Table 1 and Table S2) from patients with filarial infections were chosen in such a way that only a single infecting species of filariae was present either because of geographic constraints and/or by definitive identification of a particular parasite and exclusion of LF (circulating antigen unfavorable by TropBio ELISA). For the blinded analyses, well-characterized de-identified serum selections were used (kindly provided by Drs. Patrick Lammie and Shenoy). Table 1 Samples used to assess sensitivity and specificity of Wb123 LIPS. (mf+)Ecuador24Guatemala10Sierra Leone2Cameroon* 2Total38 (mf+)Benin55Cameroon3Gabon2Central African Republic2Total62 (mf+)Mali15Uganda5Total20Other helminth infections test was utilized for comparison of antibody levels in different groups. The Spearman rank correlation was utilized for correlation analyses. All statistical analyses were performed using GraphPad (v5). Results L3-derived ESTs from (n?=?2048), (n?=?5068), (n?=?3351) and (n?=?4168) were each assembled into contigs by dCAS analysis (906.