Ascites were obtained after 7 to 10 days, and antibodies were purified by protein A/G affinity columns. Plasma samples. (IVM) during mass SB 415286 drug administration (MDA) campaigns (3, 4). As a result, IVM-based MDA campaigns have been interrupted or delayed in areas of Central Africa where is usually coendemic with either or (5). Diagnostic methods that can accurately identify individuals that are at high risk of developing SAEs during IVM-based treatment are thus needed to achieve lymphatic filariasis and onchocerciasis elimination goals by 2020/2025 as targeted by the WHO. With the recent findings of an association between very high mf loads and mortality, independent of the effect of IVM treatment (6), identifying those at risk becomes all the more important. Traditional methods of mf identification and quantification are based on the microscopic examination of midday blood samples (7), a tedious and sometimes inaccurate process that is neither point of care (POC) nor high throughput (8). Real-time quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) methods are credible alternatives to microscopy since they are high throughput and combine a high degree of sensitivity and specificity with the ability to accurately quantify mf levels (9,C11). However, they require a well-equipped laboratory (for qPCR), relatively expensive reagents, and time-intensive DNA/RNA extraction processes. More recently, the CellScope Loa (or LoaScope), a mobile phone-based video microscopy system, has been described to identify individuals at high risk of developing SAEs (12) and is being used for the rapid and very accurate counting of mf at the POC. However, such a device has not yet been commercialized. We have recently developed an antigen capture immunoassay that is also capable of quantitating microfilaria-derived antigen(s) (13); SB 415286 however, it has not yet been developed SB 415286 as a POC tool because of some constraints (time-consuming protein expression and expensive reagents CEACAM6 and gear) associated with the luciferase immunoprecipitation system (LIPS) technology used. With the recent advances in genomics, a variety of pathogens, including the filarial parasites (14), (15), and (16, 17), have been fully sequenced. It has, therefore, become possible to describe the transcriptomes and proteomes of different stages of these filarial helminth parasites (18). When coupled with newer bioinformatic tools, they lead to the SB 415286 relatively rapid identification of potential vaccine, drug, and biomarker candidates (18). The present study aimed to identify new biomarkers through transcriptomics and bioinformatics that can be the basis of an antigen capture immunoassay for the detection and quantification of mf at the POC. RESULTS Biomarker candidates for immunoassays. A total of 12,277 mRNAs (of 15,444 open reading frames predicted) were identified in transcriptome sequencing (RNA-seq) analyses of mf derived from mf (16). Filtering the data set for putative proteins with no or little sequence homology with human, proteins and with significant SecretomeP scores ( 0.6) resulted in the identification of 11 SB 415286 mf-specific proteins. All of the mf-specific proteins were annotated as hypothetical proteins with variable expression levels in mf (fragments per kilobase per million [FPKM] ranging from 1 to 3,877) (Table 1). TABLE 1 Details of specific selected proteins specifically identified in the transcriptome of microfilariae mf-infected sera, with the detected signals significantly increased when sera were pretreated with glycine (see Fig. S2 in the supplemental material). Open in a separate windows FIG 1 Development of capture ELISAs for microfilaria proteins. Serial dilutions (spiked with 0.005 g/ml to 5 g/ml) of each of the recombinant proteins (LOAG_14221 [red], LOAG_15846 [blue], LOAG_03292 [purple], and LOAG_11259 [green]) in PBS (A) and in human AB serum (B) were tested with their corresponding polyclonal antisera. The seroreactivities of the polyclonal sera are plotted with the protein concentrations (g/ml) around the axes and net optical densities (O.D.) around the axes. Sensitivity and specificity of polyclonal immunoassays. Using pooled human AB serum samples spiked with increasing concentrations of the appropriate antigen, we generated standard curves (see Fig. 2A and ?andB)B) to estimate the levels of circulating LOAG_14221 and LOAG_15846 in glycine-pretreated sera. As can be seen, 14 of the 25 tested mf-positive (mf+) samples had detectible levels (range, 20 to 642 ng/ml) of LOAG_14221,.