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Title: COMPARISON OF IMMUNOFLUORESCENT ANTIBODY ASSAY (IFA) AND IMMUNOMAGNETIC ELECTROCHEMILUMINESCENCE (IM-ECL) IN DETECTION OF CRYPTOSPORIDIUM PARVUM IN KARST WATER SAMPLES

Author
item Kuczynska, Ewa
item Boyer, Douglas
item Shelton, Daniel

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/8/2002
Publication Date: 2/24/2003
Citation: KUCZYNSKA, E., BOYER, D.G., SHELTON, D.R. COMPARISON OF IMMUNOFLUORESCENT ANTIBODY ASSAY (IFA) AND IMMUNOMAGNETIC ELECTROCHEMILUMINESCENCE (IM-ECL) IN DETECTION OF CRYPTOSPORIDIUM PARVUM IN KARST WATER SAMPLES. JOURNAL OF MICROBIOLOGICAL METHODS. 2003. v. 53. p. 17-26.

Interpretive Summary: Cryptosporidiosis is an increasingly important waterborne disease caused by ingestion of small numbers of oocysts of the protozoan Cryptosporidium parvum. The disease usually causes fever and diarrhea in normally healthy individuals, but can be fatal to infants, the elderly, and individuals with weakened immune systems. Current methods for identifying Cryptosporidium parvum oocysts in environmental samples require large sample volumes and are difficult, time-consuming, and suffer from low recoveries. We tested several established techniques and new technologies for identifying and enumerating Cryptosporidium parvum oocysts in low-volume (one to ten liters) environmental water samples. Our investigations showed that the new technologies appear to be promising for the qualitative and quantitative detection of Cryptosporidium parvum from the environmental water samples, however further development to improve sensitivity is required.

Technical Abstract: Immunofluorescent antibody assay (IFA) and immunomagnetic electrochemiluminescence (IM-ECL) were used for comparison of the percent of recovery of Cryptosporidium parvum in environmental water samples obtained from a spring draining a karst basin. The monoclonal antibodies to C. parvum, isotype IgG3 were used for optimization of the IM-ECL protocol. The combination of biotinylated and TAG - labeled anti- C. parvum antibodies with the streptavidin beads gave a linear regression slope for logarithm ECL vs. logarithm fresh oocysts of 0.79 (from 5 to 5,000 oocysts), which indicates a constant ECL signal per oocyst. Standard curves gave a dynamic range of 5 to 5,000 oocysts/ml (fresh) and 10 to 100,000 cells/ml (4 months old oocysts) with the maximum limit of linear detection higher than 100,000. The linear slope of four months old oocysts decreased to 0.62, which indicates that ECL signal is a function of oocyst age. The experiment associated with bead storage time shows that even after 4 months of storage of the biotinylated antibodies, the complex retains the ability for binding the oocysts and generating the ECL signal. Based on the IFA results in the experiment concerning oocysts recovery with different methods of debris removal, the most efficient method involved dispersion, followed by flotation and immunomagnetic separation (24% recovery). The ECL results obtained in that experiment were very similar to the results obtained in the IFA method, which indicates that the IM-ECL method is accurate. Results of the IFA in the study of the prevalence of C. parvum in the underground water showed that oocysts were present in 78% of water samples with average number of oocysts of 6.4 ± 5.5 and ranged from 0 (13 samples) to 23.3 (2 samples). The ECL signal generated from water samples ranged from 3771 to 622 (average 1620 ± 465). However, the background value estimated in groundwater samples with low number of oocysts detected by IFA was highly variable and elevated (from 3702 to 272, average 1503 ± 475). The background value as a result of nonspecific binding to beads by unidentified organic components in the water can inhibit or even completely mask the signal generated by oocysts. Our investigations showed that the IM-ECL method appears to be promising for the qualitative and quantitative detection of C. parvum from the environmental water, however the method requires further development to improve its sensitivity.