After enrollment in October 1998, a finger prick blood specimen was collected between 7:30 pm and 9:30 pm for MF density using a 20-L thickC blood smear, and this was repeated in February and May 1999. is an excellent serologic technique for multiple antigens that offers substantial advantages over single-antigen based enzyme-linked immunosorbent assay in mass drug administration studies for monitoring changes in antibody levels. Introduction Numerous multiplex bead assays (MBAs) have been used to detect or quantify analytes in serum, culture supernatants, oral fluids, or other biological fluids from humans or animals.1C10 The popularity of the MBA is the result of 1) the relative ease of covalently coupling of an analyte-capture ligand to spectrally classified carboxylated microspheres; 2) the simultaneous collection of multiple data points from a single specimen, eliminating the one-data point per well enzyme-linked immunosorbent assay (ELISA); 3) the direct proportionality of the fluorescence intensity of the reporter fluorochrome to the amount of captured analyte; and 4) the use of a 96-well format with up to 100 differently classified beads per well, which yield a potential of 9,600 data points per plate. These attractive features of the MBA conserve specimens and save on labor, time, and cost when compared with the ELISA format. Furthermore, the MBA has been shown to be Labetalol HCl at least as sensitive as the ELISA.8,11 Thus far, the MBA has not been used to determine specific immunoglobulin antibody levels in humans infected with or Labetalol HCl antigens.14C16 For monitoring MDA programs, microfilaremia, antigenemia, and antibody levels provide measures of program impact. Antibody levels to relevant antigens can be assessed quantitatively by ELISA. Fortunately, for the evaluation of MDA success, antifilarial antibodies decrease after filarial antigens decrease, generally approximately 6C8 weeks post-drug administration, which is unlike some parasitic infections, such as schistosomiasis, in which antibody responses are not useful in differentiating past and present infections.17 Although the ELISA has provided useful information, it is not only expensive and laborious but requires relatively large volumes of serum or plasma to detect antibodies against multiple antigens. The need for improved serologic test for filariasis has been noted.18,19 We used MBA to measure antibody levels in serum specimens collected longitudinally from a subset of children in Haiti who were enrolled in a large, single-dose, placebo-controlled drug study that was initiated in October 1998 and monitored through May 1999.20 The MBA was originally a 23-plex bead assay consisting of recombinant antigens from blood-borne and enteric-borne protozoans and helminths, which is too large to describe in this report. In this study, Bm14 and Rabbit Polyclonal to ARNT Bm33 from were coupled to beads and used for the detection of IgG. In addition, Bm14- and Bm33-coupled beads were used for the detection of IgG4. Materials and Methods Study population and design. The study population and design have been described.20 The original study was reviewed and approved by the Centers for Disease Control and Prevention Institutional Review Board and by the Ethics Committee of H?pital Sainte Croix, Leogane, Haiti. Briefly, our subset of 148 children, each with complete set of serum samples obtained at time points A, B, and C (described below) were 5C11 years of age at enrollment and were from the original study of 1 1,292 children who lived in Leogane, Haiti, a coastal town with a population of 10,000C15,000. After enrollment in October 1998, a finger prick blood specimen was collected between 7:30 pm and 9:30 pm for MF density using a 20-L thickC blood Labetalol HCl smear, and this was repeated in February and May 1999. These collections were designated time points A, B, and C. Upon enrollment and collection of pretreatment specimens, each child randomly received a single dose of a placebo, albendazole (ALB), diethylcarbamazine (DEC), or combined DEC/ALB as described.20 At the end of the study in May 1999, those children who had received placebo or ALB alone were treated with DEC and ALB, and those children who received the DEC/ALB combination or DEC alone received ALB. For the 148 children, levels of IgG responses to the five antigens were determined. Of the 148 children, sufficient beads were available for a subset of 95 children who were used to determined IgG4 levels to Bm14 and Bm33 antigens. For antigenemia, the Og4C3 assay (James Cook University Tropical Biotechnology Pty. Ltd., Townsville, Queensland, Australia) was used at time points A and C as described.20 Recombinant antigen preparation and purification. A recombinant Bm14 antigen fused with six histidines (His6) was provided by the National Institutes of Health/National Institute of Allergy and Infectious Diseases Filariasis Research Reagent Repository Center (FR3), Molecular Resources Division (Smith College, Northampton, MA). Bm33 from fused with His6 and GST was cloned, expressed, and purified as described below. The cloned DNA sequence included amino acids 18C224 of.