Multiplex Bead Assay for Serum Samples from Children in Haiti Enrolled in a Drug Study for the Treatment of Lymphatic Filariasis

Abstract

A multiplex bead assay (MBA) was used to analyze serum samples collected longitudinally from children enrolled in a drug trial for treatment of filariasis in Leogane, Haiti. Recombinant antigens Bm14 and Bm33 from Brugia malayi, third polar tube protein (PTP3) from Encephalitozoon cuniculi, and merozoite surface protein-119 (MSP-119) from Plasmodium falciparum were coupled to carboxylated polystyrene microspheres. IgG responses to PTP3 and MSP-119 were not affected by albendazole (ALB), diethylcarbamazine (DEC), or combination of diethylcarbamazine and albendazole (DEC/ALB). However, IgG and IgG4 responses to Bm14 and Bm33 were significantly decreased (P < 0.001) by DEC and DEC/ALB treatment. Antibody responses to Bm14 and Bm33 decreased after DEC treatment (but not placebo) among children who were negative for microfilaremia and antigenemia at baseline, suggesting that these children harbored early stages of infection. The MBA 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.

Longitudinal Evaluation of Enteric Protozoa in Haitian Children by Stool Exam and Multiplex Serologic Assay

“…Over the 6 years of stool collection, the total number of GIC and EHC stool episodes from the 135 children was 178 and 39, respectively. No significant differences in the prevalence of children with at least one stool episode were observed between children 4.5 years and children 4.5 years of age: for GIC, 60.0% (75 of 125) and 57.1% (40 of 70), respectively; and for EHC, 16.0% (20 of 125) and 21.3% (13 of 61), respectively.…”

“…The esters readily react with available primary amine groups on the antigen, forming a stable covalent amide bond between antigen and bead. All couplings were performed in phosphate buffered saline, pH 7.2, as previously described 16 ; the coupling efficiency was determined by polyclonal goat anti-GST antibodies and by sera known to be highly reactive to the antigens.…”

“…The procedure for the MBA was slightly modified from a previous study by extending the incubation period of the serum specimens (dilution 1:400) to the antigen-coupled beads from 45 minutes to 1.5 hours. 16,17 Data were acquired using a reader (Luminex Corp) equipped with Bio-Plex Manager 6.0 software (Bio-Rad, Hercules, CA) and was reported as the average of the median fluorescence intensity from duplicate wells minus the background (MFI-bg). For each antigen, a serum blank and a negative control serum were run on each plate along with a positive control serum that was diluted to yield mid-range fluorescence intensity.…”

“…Serology has been used in the assessment of treatment programs for the elimination of filariasis, comparing pre-and post-administration of anti-filarial drugs when antibody levels are high and low, respectively. [14][15][16] We used a MBA to assess immunoglobulin G (IgG) responses to 28 antigens in longitudinal serum specimens collected from Haitian children enrolled in a filariasis study from 1990 to 1999. 17 The MBA technique was selected because of its proven performance, its conservation of serum specimens (only 125 nL/well) and its relatively low labor and low cost, about $100 per plate per 10-plex assay, excluding labor and cost of antigens.…”

“…17 The MBA technique was selected because of its proven performance, its conservation of serum specimens (only 125 nL/well) and its relatively low labor and low cost, about $100 per plate per 10-plex assay, excluding labor and cost of antigens. 16 Here, we report IgG responses from these children to the recombinant E. histolytica lectin adhesion antigen (LecA), to the recombinant C. parvum antigens 17-and 27-kDa (Cry17 and Cry27) from invasive sporozoites, to the glutathione-S-transferase (GST) from Schistosoma japonicum, a control antigen that elicits no appreciable human IgG activity even in persons infected with S. japonicum (personal observation), and to five recombinant variant-specific surface proteins (VSP1-VSP5) from G. intestinalis. For comparison, IgG responses to these antigens from adult Haitian individuals were also examined.…”