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Korean J Parasitol > Volume 33(2):1995 > Article

Original Article
Korean J Parasitol. 1995 Jun;33(2):107-115. English.
Published online Jun 20, 1995.  http://dx.doi.org/10.3347/kjp.1995.33.2.107
Copyright © 1995 by The Korean Society for Parasitology
Diagnosis of bovine cryptosporidiosis by indirect immunofluorescence assay using monoclonal antibody
Sung-Hwan Wee,*1Chung-Gil Lee,2 and Hoo-Don Joo1
1National Veterinary Research Institute, RDA, Anyang 430-016, Korea.
2College of Veterinary Medicine, Chonnam National University, Kwangju 500-757, Korea.
Received February 09, 1995; Accepted May 09, 1995.

Abstract

Two hybridoma cell lines against Cryptosporidium parvum oocysts (VRI- CN91) were produced. The isotype of these 2 monoclonal antibodies (mAbs) was IgG2b (1E7.2) and and IgM (C6). Enzyme immuno-transfer blotting analysis showed that 1E7.2 reacted specifically to 36 kDa protein and C6 reacted to 67 and 70 kDa proteins. C. parvum was bound specifically to the surface region of oocysts by these mAbs. No cross- reactivity was observed with tachyzoites of Toxoplasma gondii and oocysts of Eimeria zuernii, E. bovis and E. canadensis of bovine origin. The indirect immunofluorescence assay (IIF) using mAb C6 was successful with counterstain. With the IIF using mAb C6, oocysts appeared as 3 to 5 µm spherical objects fluorescing bright apple green against a reddish dark background. The IIF using mAb C6 was agreed in specificity and sensitivity with those of a commercial diagnostic kit. These results demonstrated that the produced mAbs were specific to C. parvum and that the mAb C6 could be used for diagnosis of cryptosporidiosis.

Figures


Fig. 1
The proteins of Cryptosporidium parvum shown on 5-15% gradient SDS-PAGE using Coomassie brilliant blue G staining technique; standard molecular weights are indicated on the left.


Fig. 2
Antigenic profiles of Cryptosporidium parvum oocysts detected by enzyme immuno-transfer blotting. Lane 1: polyclonal anti-C. parvum rabbit serum. Lane 2: hybridoma supernatants containing monoclonal antibody 1E7.2 followed by a goat anti-mouse horseradish peroxidase-conjugated IgG; standard molecular weights ard indicated on th left.


Fig. 3
Enzyme immuno-transfer blotting of 5-15% gradients SDS-PAGE of oocyst antigens detected with hybridoma supernatants containing monoclonal antibody C6 followed by a goat anti-mouse horseradish peroxidase-conjugated IgM; standard molecular weights are indicated on the left.


Fig. 4
Cryptosporidium parvum oocysts labeled by monoclonal antibody 1E7.2, showing the antibody reacted specifically with the surface region of the oocysts (× 500).


Fig. 5
Cryptosporidium parvum oocysts in calf diarrheal fecal specimens diagnosed by indirect immunofluorescent assay using monoclonal antibody C6 (× 250).

Tables


Table 1
Comparison of diagnostic efficacy between modified acid-fast stain and immunofluorescent assay for detection of Cryptosporidium parvum oocysts in diarrheal feces of an experimentally infected calf


Table 2
Comparison of th relative specificity and sensitivity between modified acid-fast stain and immunofluorescent assay for detection of Cryptosporidium parvum oocysts in diarrheal feces of an experimentally infected calf

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