Multiplex Bead-based Assay for Neglected Tropical Diseases

Staff of the Ministry of Health of Cambodia selected antigens to be included in the multiplex. The following parasite-specific recombinant antigens were used in the MBA (Table 1): NIE for Strongyloides stercoralis [8 (link)]; SAG2A for Toxoplasma gondii [9 (link), 10 (link)]; T24H for cysticercosis [11 (link)]; PfMSP-1₁₉ (3D7 strain) and PfMSP-1₄₂ (3D7 strain and FVO strain) for P. falciparum malaria [12 (link), 13 (link)]; and PvMSP-1₁₉ (Belem strain) for P. vivax malaria [14 (link), 15 (link)]. For lymphatic filariasis, Brugia malayi Bm14 (SXP-1) [16 (link)], B. malayi Bm33 (Bm-AP-1) [17 (link)], and W. bancrofti Wb123 [18 (link)] antigens were used. Wb123 is reported to be largely species specific [18 (link), 19 (link)], while the Bm14 and Bm33 antigens cross react with sera from W. bancrofti infected patients as well as with sera from some patients infected with other filarial worm species [17 (link), 20 (link)].
Recombinant Bm14 [21 (link)], SAG2A [22 (link)], and NIE [23 (link)] proteins tagged with Schistosoma japonicum glutathione-S-transferase (GST) and control GST with no fusion partner [24 (link)] were expressed and purified as described elsewhere. Bm33 [25 (link)] and T24H [26 (link)] were expressed with GST fused to the amino terminus and with six histidines (His₆) on the carboxy terminus and purified as previously described. Following purification, the His₆ tag was removed from T24H by Factor Xa cleavage [26 (link)]. Recombinant PfMSP-1₁₉-GST (3D7 parasite strain) fusion protein and PfMSP-1₄₂ proteins (3D7 and FVO parasite strains) lacking fusion tags were provided by C. Kauth and H. Bujard (Heidelberg University, Heidelberg, Germany) [27 (link)]. Wb123-GST fusion protein was provided by Dr. T. Nutman (NIH, Bethesda, MD).
The P. vivax PvMSP1₁₉-GST was cloned, expressed, and purified for the MBA. The coding sequence (including the carboxy-terminal, hydrophobic anchor sequence) was amplified from Belem strain DNA (provided by J. Barnwell, CDC, Atlanta, GA) using the following forward and reverse deoxyoligonucleotide PCR primers: 5’-CGC GGA TCC ACT ATG AGC TCC GAG CAC ACA TG-3’ and 5’-GCG GAA TTC TTA AAG CTC CAT GCA CAG GAG-3’, respectively. BamHI and EcoRI restriction endonuclease sites used for directional cloning into pGEX 4T-2 plasmid (GE Healthcare, USA) are underlined in the primer sequences. Polymerase chain reaction amplification conditions and protocols for cloning into Escherichia coli BL21 cells (Stratagene, USA) have previously been described [28 (link)]. The sequence of the resulting PvMSP1₁₉ clone was confirmed to match that found in GenBank (accession number AF435594.1) [29 (link)]. Recombinant PvMSP1₁₉-GST fusion protein was expressed and purified on a glutathione Sepharose 4B affinity column as directed by the manufacturer (GE Healthcare). Glutathione-eluted proteins were dialyzed overnight against 300 volumes of 25 mM Tris buffer at pH 7.5 using Spectra-Por3 dialysis membrane (3,500-Da cutoff, Spectrum Laboratories, Rancho Dominguez, CA). Proteins were bound to a Mono Q HR5/5 strong anion exchange column (GE Healthcare) and eluted with a 20 min linear gradient from 0 to 0.25 M NaCl in 25 mM Tris buffer at pH 7.5. Protein fractions collected between 0.15 and 0.21 M NaCl were mostly free of contaminants by SDS polyacrylamide gel analysis and were combined. The final protein product was dialyzed against 300 volumes of PBS and then concentrated to approximately 1 mg/ml using a Centricon-10 centrifugal filter device (Millipore Corporation, Bedford, MA). The yield from 2 L of E. coli cells was approximately 1.5 mg of purified PvMSP1₁₉-GST protein.