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An Overview Cryptosporidium Antigen Detection Microwell ELISA
Summary
Cryptosporidium is a coccidian parasite that is recognized as an important enteric pathogen. The organism causes an acute, though self-limiting infection in immunocompetent individuals. Incubation periods of 1 to 12 days have been reported with most oocyst shedding ending by day 21. Symptoms range from mild to severe diarrhea with a variety of complications.
The infection in immunocompromised patients is much more severe and may often be life threatening. Passage of fluid, up to 12 liters per day, has been reported.
Multiple pathways of Cryptosporidium transmission have been implicated. These include animal to human, water contamination and person-to-person. The latter may include contact between members of the same household, day care centers, and homosexual men.
Diagnosis of Cryptosporidium infections was done originally by direct detection techniques. Of these, microscopic examination of stools using stains or fluorescence labeled antibodies has been the most common. However, this method relies on an experienced technician and subsequent observation of intact organisms. Because of the historically low proficiency of correct microscopic examinations, alternative diagnostic methods have been investigated.
One important alternative has been the development of an antigen capture enzyme linked immunosorbent assay (ELISA) for use with stools. These tests, which have shown comparable sensitivity to experienced microscopic examinations, are fairly simple to perform and do not require the observation of intact organisms.
Principle of Procedure
During the first incubation, antibodies attached to the wells capture Cryptosporidium antigens present in the stool supernatant. The second incubation adds an additional anti- Cryptosporidium antibody that “sandwiches” the antigen. The third incubation attaches horseradish peroxidase to the sandwich. After washings to remove unbound enzyme, a chromogen is added which develops a color in the presence of the enzyme complex and peroxide. The stop solution ends the reaction. The reaction may then be read visually or with an ELISA reader. |