- INTRODUCTION
- MATERIALS AND METHODS
- Human sera.
- Purification of mRNA and rapid amplification of cDNA polymerase chain reaction (RACE PCR).
- Cloning of cDNA and DNA sequencing.
- Expression and purification of the expressed protein.
- Protein determination.
- Analytical SDS-PAGE and immunoblotting.
- Preparation of recombinant protein antigen for the cystatin capture ELISA.
- Cystatin capture ELISA.
- Statistical analysis.
- RESULTS
- DISCUSSION
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Reference Manager
BibTeX
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-
1
World Health Organization, 1995. Control of foodborne trematode infections. World Health Organ Tech Rep Ser 849 :1–157.
-
2
Mas-Coma S, Bargues MD, Esteban JG, 1998. Human fasciolosis. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 411–434.
-
3
Andrews SJ, 1998. The life cycle of Fasciola hepatica. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 1–29.
-
4
Maleewong W, Wongkham C, Intapan PM, Pipitgool V, 1999. Fasciola gigantica-specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis. Am J Trop Med Hyg 61 :648–651.
-
5
Grams R, Vichasri-Grams S, Sobhon P, Upatham ES, Viyanant V, 2001. Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica. Parasitol Int 50 :105–114.
-
6
O’Neill SM, Parkinson M, Strauss W, Angles R, Dalton JP, 1998. Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg 58 :417–423.
-
7
O’Neill SM, Parkinson M, Dowd AJ, Strauss W, Angles R, Dalton JP, 1999. Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg 60 :749–751.
-
8
Strauss W, O’Neill SM, Parkinson M, Angles R, Dalton JP, 1999. Diagnosis of human fascioliasis: detection of anti-cathepsin L antibodies in blood samples collected on filter paper. Am J Trop Med Hyg 60 :746–748.
-
9
Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO, 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41 :43–49.
-
10
Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ, 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int J Parasitol 31 :728–737.
-
11
Rokni MB, Massoud J, O’Neill SM, Parkinson M, Dalton JP, 2002. Diagnosis of human fasciolosis in the Gilan province of northern Iran: application of cathepsin L-ELISA. Diagn Microbiol Infect Dis 44 :175–179.
-
12
Erdman DD, 1981. Clinical comparison of ethyl acetate and di-ethyl ether in the formalin-ether sedimentation technique. J Clin Microbiol 14 :483–485.
-
13
Chomczynski P, Sacchi N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162 :156–159.
-
14
Cohen SN, Chang AC, 1977. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol 132 :734–737.
-
15
Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 :248–254.
-
16
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 :680–685.
-
17
Towbin H, Staehelin T, Gordon J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76 :4350–4354.
-
18
Ikeda T, 1998. Cystatin capture enzyme-linked immunosorbent assay for immunodiagnosis of human paragonimiasis and fascioliasis. Am J Trop Med Hyg 59 :286–290.
-
19
Galen RS, 1980. Predictive value and efficiency of laboratory testing. Pediatr Clin North Am 27 :861–869.
-
20
Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA, Sunter DC, Barrett AJ, 1983. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J 211 :129–138.
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SERODIAGNOSIS OF HUMAN FASCIOLIASIS BY A CYSTATIN CAPTURE ENZYME-LINKED IMMUNOSORBENT ASSAY WITH RECOMBINANT FASCIOLA GIGANTICA CATHEPSIN L ANTIGEN
CHAIRAT TANTRAWATPAN
CHAIRAT TANTRAWATPAN
Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan
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WANCHAI MALEEWONG
WANCHAI MALEEWONG
Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan
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CHAISIRI WONGKHAM
CHAISIRI WONGKHAM
Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan
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SOPIT WONGKHAM
SOPIT WONGKHAM
Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan
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PEWPAN M. INTAPAN
PEWPAN M. INTAPAN
Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan
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KUNIO NAKASHIMA
KUNIO NAKASHIMA
Department of Biochemistry, and Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Faculty of Human Health, Tokai Gakuen University, Tenpaku-ku, Nagoya, Aichi, Japan
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Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola gigantica, was expressed in Escherichia coli as a calmodulin binding peptide fusion protein with a molecular mass of approximately 35 kD. The recombinant cathepsin L1 (rCTL1) was tested for its antigenic potential in a cystatin capture enzyme-linked immunosorbent assay (ELISA) to diagnose human fascioliasis. The ELISA plates were sensitized with chicken egg cystatin and incubated with bacterial lysates containing the recombinant protein before the standard ELISA procedures were performed. Analysis of the sera of 13 patients infected with F. gigantica (group 1), 204 patients with other parasitic infections (group 2), 32 cholangiocarcinoma patients (group 3), and 42 healthy controls (group 4) showed that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of this ELISA using rCTL1 were 100%, 98.92%, 98.97%, 81.25%, and 100%, respectively. These results indicate that this assay has high sensitivity and specificity in the diagnosis of human fascioliasis. In addition, we have produced sufficient amounts of antigen for use in diagnosis.
Author Notes
- INTRODUCTION
- MATERIALS AND METHODS
- Human sera.
- Purification of mRNA and rapid amplification of cDNA polymerase chain reaction (RACE PCR).
- Cloning of cDNA and DNA sequencing.
- Expression and purification of the expressed protein.
- Protein determination.
- Analytical SDS-PAGE and immunoblotting.
- Preparation of recombinant protein antigen for the cystatin capture ELISA.
- Cystatin capture ELISA.
- Statistical analysis.
- RESULTS
- DISCUSSION
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
World Health Organization, 1995. Control of foodborne trematode infections. World Health Organ Tech Rep Ser 849 :1–157.
-
2
Mas-Coma S, Bargues MD, Esteban JG, 1998. Human fasciolosis. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 411–434.
-
3
Andrews SJ, 1998. The life cycle of Fasciola hepatica. Dalton JP, ed. Fasciolosis. Wallingford, United Kingdom: CABI Publishing, 1–29.
-
4
Maleewong W, Wongkham C, Intapan PM, Pipitgool V, 1999. Fasciola gigantica-specific antigens: purification by a continuous-elution method and its evaluation for the diagnosis of human fascioliasis. Am J Trop Med Hyg 61 :648–651.
-
5
Grams R, Vichasri-Grams S, Sobhon P, Upatham ES, Viyanant V, 2001. Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica. Parasitol Int 50 :105–114.
-
6
O’Neill SM, Parkinson M, Strauss W, Angles R, Dalton JP, 1998. Immunodiagnosis of Fasciola hepatica infection (fascioliasis) in a human population in the Bolivian Altiplano using purified cathepsin L cysteine proteinase. Am J Trop Med Hyg 58 :417–423.
-
7
O’Neill SM, Parkinson M, Dowd AJ, Strauss W, Angles R, Dalton JP, 1999. Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase. Am J Trop Med Hyg 60 :749–751.
-
8
Strauss W, O’Neill SM, Parkinson M, Angles R, Dalton JP, 1999. Diagnosis of human fascioliasis: detection of anti-cathepsin L antibodies in blood samples collected on filter paper. Am J Trop Med Hyg 60 :746–748.
-
9
Carnevale S, Rodriguez MI, Guarnera EA, Carmona C, Tanos T, Angel SO, 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagn Microbiol Infect Dis 41 :43–49.
-
10
Cornelissen JB, Gaasenbeek CP, Borgsteede FH, Holland WG, Harmsen MM, Boersma WJ, 2001. Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease. Int J Parasitol 31 :728–737.
-
11
Rokni MB, Massoud J, O’Neill SM, Parkinson M, Dalton JP, 2002. Diagnosis of human fasciolosis in the Gilan province of northern Iran: application of cathepsin L-ELISA. Diagn Microbiol Infect Dis 44 :175–179.
-
12
Erdman DD, 1981. Clinical comparison of ethyl acetate and di-ethyl ether in the formalin-ether sedimentation technique. J Clin Microbiol 14 :483–485.
-
13
Chomczynski P, Sacchi N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162 :156–159.
-
14
Cohen SN, Chang AC, 1977. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol 132 :734–737.
-
15
Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 :248–254.
-
16
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 :680–685.
-
17
Towbin H, Staehelin T, Gordon J, 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76 :4350–4354.
-
18
Ikeda T, 1998. Cystatin capture enzyme-linked immunosorbent assay for immunodiagnosis of human paragonimiasis and fascioliasis. Am J Trop Med Hyg 59 :286–290.
-
19
Galen RS, 1980. Predictive value and efficiency of laboratory testing. Pediatr Clin North Am 27 :861–869.
-
20
Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA, Sunter DC, Barrett AJ, 1983. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J 211 :129–138.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 473 | 452 | 23 |
| Full Text Views | 247 | 3 | 0 |
| PDF Downloads | 43 | 5 | 0 |