Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

Abstract

<jats:p>INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.</jats:p>

Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle ProgressionIn Vitro

“…Antibodies have also been made against specific proteins ( 15 , 17 , 18 ); however, this method of antibody production does not typically allow for the discovery of novel antigens. Additionally, many antibodies previously raised against Cryptosporidium were made in rabbits ( 15 , 18 , 19 ), which is a nonrenewable source of antibody. Collectively, these available reagents do not allow the specific life cycle stages to be clearly delineated with unique markers, confounding attempts to track development during in vitro growth.…”

Prevalência e espécies de Cryptosporidium em humanos

“…Ensaios imunológicos, tais como imunofluorescência (direta ou indireta), podem também ser usados para identificar Cryptosporidium spp. em amostras de água e fezes (OSAKI et al, 2011;CO-DICES et al, 2013;FERNANDES et al, 2020). O método PCR também pode ser usado em análises de amostra de água (OSAKI et al, 2013).…”

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

“…Current techniques for the environmental detection of Cryptosporidium require the fi ltration of large volumes, elution, isolation, and enumeration of the oocysts by immunofl uorescent microscopy 10,29 . However, this approach cannot determine whether Cryptosporidium oocysts are viable or whether they belong to a human-infective genotype.…”