Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

Abstract

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27-and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27-and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.Cryptosporidium parvum, a protozoan parasite that invades the intestinal epithelium of a wide range of mammalian hosts, causes a self-limiting but sometimes severe diarrheal illness in immunocompetent humans (2). However, in those with compromised immune systems, the disease can be debilitating, chronic, and life threatening (4, 23). Because of its ubiquitous distribution in the environment, its small size, and its resistance to standard chlorination techniques, C. parvum contamination of drinking water may pose a significant public health risk (1,11,12). Numerous outbreaks have been traced to contaminated water, and waterborne outbreaks have even occurred in communities served by state-of-the-art water treatment facilities (3,8,9,15). To conduct epidemiologic studies to assess the risks of C. parvum infection that may be associated with drinking water and other potential sources of exposure, new serologic assays that are rapid, sensitive, and specific are needed.Characteristic serum immunoglobulin G (IgG) antibody responses have been shown to develop in humans after C. parvum infection. As shown by Western blot analysis of serum samples collected from outbreak patients and human volunteers, the antibody response is consistently directed toward two low-molecular-weight antigen families: one in the 27-kDa size range and a second in the 17-kDa size range (16-19, 25, 26).We recently reported the development of two enzyme-linked immunosorbent assays (ELISAs) for the detection ...

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.…”

“…[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.…”

Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum

“…In previous work, we demonstrated that human infection with C. parvum elicits an IgG antibody response to the 17-and 27-kDa surface antigens within 10 -14 days of symptom onset (19,40). Although both of these antigens include post-translational carbohydrate and/or lipid modifications, we demonstrated that much of the antibody response was directed against the protein component (19,21).…”

Changes in Serum Immunoglobulin G Levels as a Marker for Cryptosporidium sp. Infection in Peruvian Children

“…However, because microscopy is relatively insensitive when small numbers of oocysts are excreted (acid-fast stool microscopy is 83.7% sensitive and 98.9% specific relative to PCR) and because the period of oocyst shedding can be short, many infections may escape microscopic detection (2,5,9). Earlier work demonstrated that serum immunoglobulin G antibody responses to two immunodominant sporozoite surface antigens develop upon infection and can be detected for some time after resolution (half-life of 12 weeks) (4,6,7,8). To assess the usefulness of these antibody assays as epidemiologic tools, we assayed longitudinally collected serum samples from children who participated in a birth cohort study of diarrheal disease in Lima, Peru, between 1995 and 1998 (1, 3, 10).…”