Multiplex Assay Detection of Immunoglobulin G Antibodies That Recognize Giardia intestinalis and Cryptosporidium parvum Antigens

Abstract

Giardiasis and cryptosporidiosis are common enteric parasitic diseases that have similar routes of transmission. In this work, we have identified epitopes within the Giardia variant-specific surface protein (VSP) sequences that are recognized by IgG antibodies from 13 of 14 (93%) sera from patients with stool-confirmed giardiasis. The conserved epitopes are shared among VSPs from both of the assemblages that commonly infect humans, and they are likely to be structural, as both sodium dodecyl sulfate treatment and dithiothreitol reduction decrease antibody recognition. In a multiplex bead assay (MBA), we used three VSP fragments from an assemblage A Giardia strain, three VSP fragments from assemblage B strains, and the ␣-1 giardin structural antigen to detect IgG antibodies to Giardia and used the recombinant 17-and 27-kDa antigens to simultaneously detect IgG antibodies to Cryptosporidium. The MBA differentiated between sera from Giardia and Cryptosporidium outbreaks and also identified a giardiasis outbreak that may have included cryptosporidiosis cases. Approximately 40% of cryptosporidiosis outbreak samples had high MBA responses for both the 27-and 17-kDa antigens, while <10% of nonoutbreak and giardiasis outbreak samples had high responses. At least 60% of giardiasis outbreak samples were positive for antibodies to multiple Giardia antigens, while <12% of nonoutbreak samples and samples from U.S. and British Columbia cryptosporidiosis outbreaks met our definition for Giardia seropositivity. A MBA using multiple parasite antigens may prove useful in the epidemiologic analysis of future waterborne or food-borne outbreaks of diarrheal disease.

Systemic Antibody Responses to the Immunodominant p23 Antigen and p23 Polymorphisms in Children with Cryptosporidiosis in Bangladesh

“…A number of previous studies have reported serum antibody responses to p23 [30][31][32][33][34][35][36] in immunocompetent and immunocompromised hosts. However, most studies were serosurveys to estimate prevalence and investigate outbreaks 32,33,[56][57][58][59][60] or to demonstrate the utility of p23 as an antigen for ELISAs.…”

“…However, most studies were serosurveys to estimate prevalence and investigate outbreaks 32,33,[56][57][58][59][60] or to demonstrate the utility of p23 as an antigen for ELISAs. 30,35 Priest and others investigated serum antibody responses to a 27-kDa antigen (same as p23) in a birth cohort of children in Peru by using the same recombinant C. parvum protein we used as antigen for the ELISA. As in our study, most children in their study were infected with C. hominis.…”

“…The plasmid containing the p23 insert cloned into the pGEX 4T-2 vector was obtained from Dr. Jeffrey Priest (Centers for Disease Control and Prevention, Atlanta, GA) under a Materials Transfer Agreement with North Carolina State University (Raleigh, NC) and overexpressed in Escherichia coli BL21 cells as described. 30 The recombinant p23 (rp23)-glutathione-S-transferase fusion protein was purified by affinity chromatography using a glutathione Sepharose 4B column (Amersham Biosciences, Piscataway, NJ). The glutathione-S-transferase tag was cleaved off with thrombin as described.…”

“…The glutathione-S-transferase tag was cleaved off with thrombin as described. 30 Protein concentration was measured using the Micro BCA protein assay kit (Pierce, Rockford, IL).…”

“…[27][28][29] The p23 antigen is one of the most immunodominant Cryptosporidium antigens and is consistently recognized by serum from actively infected or previously exposed humans. [30][31][32][33][34][35][36] The presence of pre-existing antibodies to this antigen was associated with reduced oocyst shedding and protection from diarrhea in infected human volunteers. 37 In addition, serum antibody responses to p23 were associated with a reduced risk of diarrheal illness in immunosuppressed persons.…”