Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Urdaneta, Haydeé; Rangel, Antonio; Martins, Maria Sonia; Muñoz, Jose Francisco; Hernández M, Manuel

doi:10.1590/S0036-46651996000100008

Abstracts

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Entamoeba histolytica; Amebiasis; Immunodiagnostic; Monoclonal antibodies; Capture ELISA

A amebíase mantém sua importância epidemiológica em países subdesenvolvidos onde sua prevalência a converteu na parasitose de maior morbidade e mortalidade após malaria e esquistosomose. Em regra, tanto o diagnóstico clínico como os levantamentos epidemiológicos assentam na identificação microscópica de cistos e/ou trofozoítos em extractos fecais. Este procedimento requer pessoal muito bem treinado, é laborioso, e frequentemente fornece resultados contraditórios. Para obviar estas dificuldades, no presente trabalho montamos uma técnica de diagnóstico imunoenzimático baseado na captura de um antígeno de 96 kDa presente nas fezes de indivíduos infectados pela E. hystolytica (COPROELISA-Eh). Triplicatas de 117 amostras fecais processadas pelo método de concentração do formol-éter foram definidas como positivas ou negativas por três microscopistas especialistas em amebíase. Outra alíquota foi submetida ao teste de captura de antígeno por um anticorpo monoclonal contra um antígeno de 96 kDa específico de membranas de cepas patogênicas de E. histolytica. Consideramos como positivos aqueles resultados em que densidades óticas estavam acima do valor dos controles negativos mais 2 desvios padrões. COPROELISA-Eh mostrou 94.4% de sensibilidade, 98.3% de especificidade, 96.2% de valor preditivo positivo e 97.6% de valor preditivo negativo para a deteção de E. hystolytica nas fezes. Em conclusão, o método COPROELISA-Eh é mais sensível e específico do que o exame microscópico dos extratos fecais, não requer pessoal especializado na identificação morfológica das amebas, e permite o processamento de um grande número de amostras simultâneamente.

IMMUNODIAGNOSIS

Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Entamoeba histolytica: imunodiagnóstico, através de captura de antígeno fecal em amebíase entérica por um anticorpo monoclonal

Haydeé Urdaneta^I; Antonio Rangel^I; Maria Sonia Martins^II; Jose Francisco Muñoz^I; Manuel Hernández M.^I

^IInstitute of Clinical Immunology, Los Andes University, Venezuela

^IIInstitute of Biological Sciences. Department of Parasitology, Federal University of Minas Gerais, Brasil

^{Correspondence to} Correspondence to: Haideé Urdaneta Instituto de Inmunologia Clinica, Edificio Louis Pasteur Apartado 566, Universidad de Los Andes Mérida, Venezuela 5101 FAX 58-074-403212 E-mail: uhaydee@ing.ula.ve

SUMMARY

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Keywords:Entamoeba histolytica; Amebiasis, Coproantigens; Immunodiagnostic; Monoclonal antibodies; Capture ELISA.

RESUMO

A amebíase mantém sua importância epidemiológica em países subdesenvolvidos onde sua prevalência a converteu na parasitose de maior morbidade e mortalidade após malaria e esquistosomose. Em regra, tanto o diagnóstico clínico como os levantamentos epidemiológicos assentam na identificação microscópica de cistos e/ou trofozoítos em extractos fecais. Este procedimento requer pessoal muito bem treinado, é laborioso, e frequentemente fornece resultados contraditórios. Para obviar estas dificuldades, no presente trabalho montamos uma técnica de diagnóstico imunoenzimático baseado na captura de um antígeno de 96 kDa presente nas fezes de indivíduos infectados pela E. hystolytica (COPROELISA-Eh). Triplicatas de 117 amostras fecais processadas pelo método de concentração do formol-éter foram definidas como positivas ou negativas por três microscopistas especialistas em amebíase. Outra alíquota foi submetida ao teste de captura de antígeno por um anticorpo monoclonal contra um antígeno de 96 kDa específico de membranas de cepas patogênicas de E. histolytica. Consideramos como positivos aqueles resultados em que densidades óticas estavam acima do valor dos controles negativos mais 2 desvios padrões. COPROELISA-Eh mostrou 94.4% de sensibilidade, 98.3% de especificidade, 96.2% de valor preditivo positivo e 97.6% de valor preditivo negativo para a deteção de E. hystolytica nas fezes. Em conclusão, o método COPROELISA-Eh é mais sensível e específico do que o exame microscópico dos extratos fecais, não requer pessoal especializado na identificação morfológica das amebas, e permite o processamento de um grande número de amostras simultâneamente.

Full text available only in PDF format.

Texto completo disponível apenas em PDF.

ACKNOWLEDGEMENTS

We are indebted to Dr. Edward Felix Silva and Mr. João da Costa Vianna for kindly donating some strains of E. histolytica.

This work was supported by grants M-438-93-C, CDCHT Universidad de Los Andes, Venezuela.

Recebido para publicação em 10/11/1995

Aceito para publicação em 28/02/1996

1. ALLAN, J.C.; CRAIG, P.S.; GARCIA-NOVAL, J. et al. - Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans. Parasitology, 104: 347-355, 1992.
2. ALLAN, J.C.; MENCOS, F.; GARCIA-NOVAL, J. et al. - Dipstick dot ELISA for the detection of Taenia coproantigens in humans. Parasitology, 107: 79-82, 1993.
3. ARVIND, A.S.; SHETTY, M.D. & FARTHING, M.J. - Serodiagnosis of amoebiasis. Serodiagnosis and immunotherapy. Infect. Dis., 2: 79-84, 1988.
4. ASH, L.R. & ORIHEL, T.C. - Parasites: a guide to laboratory procedures and identification. Chicago, American Society of Clinical Pathology, 1987. p. 191-199.
5. BARRETO, M.P. - Diagnóstico da amebiasis intestinal pelo exame coprológico. Rev. Inst. Med. trop. S. Paulo., 4: 269, 1962.
6. BEAVER, P.C.; JUNG, R.C. & CUPP, E.W. Amebiasis. In: CRAIG, C.F.; BEAVER, P.C. & JUNG, R.C. - Clinical Parasitology. 9 .ed. Philadelphia, Lea & Febiger, 1984. p. 101.
7. BRUCKNER, D.A. - Amebiasis. Clin. Microbiol. Rev., 5: 356-369, 1992.
8. CUNHA, A.S. - Amebiase. In: CASTRO, L.P.; ROCHA, P.S. & CUNHA, A.S. Tópicos em gastroenterología. 2. ed. Rio de Janeiro, Médica Científica, 1991. p. 287.
9. CHENG, X.J.; TACHIBANA, H.; KOBAYASHI, S.; KANEDA, Y. & HUANG, M.Y. - Analysis of pathogenicity of Entamoeba histolytica by polymerase chain reaction. Chun. Kuo. chi. sheng. Chung. Hsuch. Yu. Chi Sheng. Chung. Ping. Tsa. Chin., 11: 172-175, 1993.
10. DEPLAZES, P.; ECKERT, J.; PAWLOWKI, S.Z; MACHOWSKA, L. & GOTTSTEIN, B. - An enzyme linked immunosorbent assay for diagnostic detection of Taenia saginata copro-antigen in humans. Trans. roy. Soc. trop. Med. Hyg., 85: 391-396, 1991.
11. FLORES, B.M.; REED, S.L.; RAVDIN, J.I. & TORIAN, B.E. - Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica. J. clin. Microbiol., 31: 1403-1407, 1993.
12. GARDUŃO, E.J.; MARTINEZ, M.C.; MEJÍA, J.M. et al. - Pruebas de diagnóstico inmunológico en amebiasis. Evaluación de la literatura. In: SEMINÁRIO SOBRE AMIBIASIS, 11, México, 1989. p. 67.
13. GARFINKEL, L.I.; GILARD, M.; HUBER, M. et al. - DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect. Immunol., 57: 926, 1989.
14. GOLDIN, A.J.; APT, W.; AGUILERA, X. et al. - A capture ELISA detects Giardia lamblia antigens in formalin-treated faecal samples. Trans. roy. Soc. trop. Med. Hyg., 86: 164, 1992.
15. GONZÁLEZ, A. - Revisión del estado actual del diagnóstico diferencial de las amibas en México. Salud publ. Méx., 32: 589, 1990.
16. GUERRANT, R. L. - The global problem of amebiasis: current status research needs, and opportunities for progress. Rev. infect. Dis., 8: 218-227, 1986.
17. GUIMARĂES, S.; URDANETA, H.; SILVA, E. & TAVARES, C. - Entamoeba histolytica: antigenic characterization of axenic strains from Brazil. Rev. Inst. Med. trop. S. Paulo, 33: 6-11, 1991.
18. HAQUE, R.; KRESS, K.; WOOD, S. et al. - Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin. J. infect. Dis., 167: 247-249, 1993.
19. HAQUE, R.; NEVILLE, L.M.; WOOD, S. & PETRI, W. A. - Detection of Entamoeba histolytica and E. dispar in stool. Amer. J. trop. Med. Hyg., 50: 595-596, 1994.
20. JACKSON, T.F.H.G.; GATHIRAM, V. & SIMJEE, A.E. - Seroepidemiological study of antibody responses to the zymodemes of Entamoeba histolytica. Lancet, 1: 716, 1985.
21. LOTTER, H.E.; MANNWEILER, M.S. & TANNICH, E. - Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica. J. clin. Microbiol., 30: 3163-3167, 1992.
22. MATHUR, T.N. & KAUR, J. - The frequency of excretion of cysts of Entamoeba histolytica in known cases of non-dysenteric amoebic colitis based on 21 stool examinations. Indian J. med. Res., 61: 330, 1973.
23. MERINO, E.W.; DEL MURO, G.R. & ORTIZ-ORTIZ, L. - Evaluation of the ELISA test for the detection of Entamoeba histolytica in feces. J. clin. lab. Anal., 4: 39-42, 1990.
24. NAGERWARAN, C.; CRAIG, P.S. & DEVANEY, E. - Coproantigen detection in rats experimentally infected with Strongyloides ratti. Parasitology, 108: 335, 1994.
25. NAYEEN, M.A. & HABIBULLAH, C.M. - A comparative study of different isolate fractions of Entamoeba histolytica in amoebic liver abscess. Trop. Gastroent., 14: 144-147, 1993.
26. NEAL, R.A. - Phylogeny: the relationship of Entamoeba histolytica to morphologically similiar amebae of the four-nucleated cyst group. In: RAVDIN, J.I., ed. Amebiasis: human infection by Entamoeba histolytica. New York, Churchill Livingstone, 1988. p. 13-26.
27. RAVDIN, J.I.; JACKSON, T.F.G.H.; PETRI, W.A. et al. - Association of serum antibodies to adherence lectin with invasive amebiasis and asymptomatic infection with pathogenic Entamoeba histolytica. J. infect. Dis., 162: 768, 1990.
28. SENGUPTA, K.; DAS, P.; JOHNSON, T.M. et al. - Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH:200) and their application in the detection of current amoebic infection. J. Eukaryot. Microbiol., 40: 722-726, 1993.
29. SHANDIL, R.K. & VINAYAK, V.K. - Immunochemical characterization of a 29 kDa surface-associated molecule of Entamoeba histolytica and its recognition by serum from patients with amoebiasis. J. med. Microbiol., 36: 41, 1992.
30. SILVA, E.F.; CARVALHO, M.G. & BRANDĂO, M.G. - Monoxenizaçăo, axenizaçăo e cultura de amostras de Entamoeba histolytica isoladas no Brasil de pacientes sintomáticos e assintomáticos. In: CONGRESSO BRASILEIRO DE PARASITOLOGÍA, 6., Belo Horizonte, 1981. Resumos. p. 18.
31. STANLEY, S.L.; JACKSON, T.F.H.G.; REED, S.L. et al. - Serodiagnoses of invasive amebiasis using a recombinant Entamoeba histolytica protein. J. Amer. med. Ass., 266: 1984-1986, 1991.
32. TORIAN, B.E.; REED, S.L.; FLORES, B.R. et al. - The 96 kDa antigen as an integral membrane protein in pathogenic Entamoeba histolytica. Potential differences in pathogenic and non-pathogenic isolates. Infect. Immun., 58: 753-760, 1990.
33. UNGAR, B.L.P.; YOLKEN, R.H. & QUINN, T.C. - Use of monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens. Amer. J. trop. Med. Hyg., 34: 465-472, 1985.
34. URDANETA, H.R.; MARTINS, M.S.; SILVA, E.F. & TAVAREZ, C.A.P. - Identification and purification of an isolate-specific 96 kDa antigen of Entamoeba histolytica by using a monoclonal antibody. Mem. Inst. Oswaldo Cruz, 86: 276, 1991.
35. URDANETA, H.; GUIMARĂES, S.; SILVA, E. & TAVARES, C.A.P. - Entamoeba histolytica: detection of coproantigens by purified antibody in the capture sandwich ELISA. Rev. Inst. Med. trop. S. Paulo, 36: 539-545, 1994.
36. URDANETA, H.; RONDON, M.; MUŃOZ, M. & HERNANDEZ, M. - Isolation and axanization of two Entamoeba histolytica strains. GEN, 49: 23-28, 1995.
37. VINAYAK, V.K.; DUTT, P. & PURI, M. - An immunoenzymatic dot ELISA for the detection Giardia lamblia antigen in stool eluates of clinical cases of giardiasis. J. immunol. Meth., 137: 245, 1991.
38. WALSH, J.A. - Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev. infect. Dis., 8: 228-238, 1986.
39. WONSIT, R.; THAMMAPALERD, N.; THARAVANIJ, S.; RADOMYOS, P. & BUNNAG, D. - Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens. Trans. roy. Soc. trop. Med. Hyg., 86: 166-169, 1992.
40. ZHANG, Y.; LI, E.; JACKSON, T.F.H.G. et al. - Use of a recombinant 170 kilodalton surface antigen of Entamoeba histolytica for serodiagnosis of amebiasis and identification of immunodominant domains of the native molecule. J. clin. Microbiol., 30: 2788-2792, 1992.

Correspondence to:

Haideé Urdaneta

Instituto de Inmunologia Clinica, Edificio Louis Pasteur

Apartado 566, Universidad de Los Andes

Mérida, Venezuela 5101

FAX 58-074-403212

E-mail:

uhaydee@ing.ula.ve

Publication Dates

Publication in this collection
22 Sept 2006
Date of issue
Feb 1996

History

Accepted
28 Feb 1996
Received
10 Nov 1995

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] 1. ALLAN, J.C.; CRAIG, P.S.; GARCIA-NOVAL, J. et al. - Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans. Parasitology, 104: 347-355, 1992.

[2] 2. ALLAN, J.C.; MENCOS, F.; GARCIA-NOVAL, J. et al. - Dipstick dot ELISA for the detection of Taenia coproantigens in humans. Parasitology, 107: 79-82, 1993.

[3] 3. ARVIND, A.S.; SHETTY, M.D. & FARTHING, M.J. - Serodiagnosis of amoebiasis. Serodiagnosis and immunotherapy. Infect. Dis., 2: 79-84, 1988.

[4] 4. ASH, L.R. & ORIHEL, T.C. - Parasites: a guide to laboratory procedures and identification. Chicago, American Society of Clinical Pathology, 1987. p. 191-199.

[5] 5. BARRETO, M.P. - Diagnóstico da amebiasis intestinal pelo exame coprológico. Rev. Inst. Med. trop. S. Paulo., 4: 269, 1962.

[6] 6. BEAVER, P.C.; JUNG, R.C. & CUPP, E.W. Amebiasis. In: CRAIG, C.F.; BEAVER, P.C. & JUNG, R.C. - Clinical Parasitology. 9 .ed. Philadelphia, Lea & Febiger, 1984. p. 101.

[7] 7. BRUCKNER, D.A. - Amebiasis. Clin. Microbiol. Rev., 5: 356-369, 1992.

[8] 8. CUNHA, A.S. - Amebiase. In: CASTRO, L.P.; ROCHA, P.S. & CUNHA, A.S. Tópicos em gastroenterología. 2. ed. Rio de Janeiro, Médica Científica, 1991. p. 287.

[9] 9. CHENG, X.J.; TACHIBANA, H.; KOBAYASHI, S.; KANEDA, Y. & HUANG, M.Y. - Analysis of pathogenicity of Entamoeba histolytica by polymerase chain reaction. Chun. Kuo. chi. sheng. Chung. Hsuch. Yu. Chi Sheng. Chung. Ping. Tsa. Chin., 11: 172-175, 1993.

[10] 10. DEPLAZES, P.; ECKERT, J.; PAWLOWKI, S.Z; MACHOWSKA, L. & GOTTSTEIN, B. - An enzyme linked immunosorbent assay for diagnostic detection of Taenia saginata copro-antigen in humans. Trans. roy. Soc. trop. Med. Hyg., 85: 391-396, 1991.

[11] 11. FLORES, B.M.; REED, S.L.; RAVDIN, J.I. & TORIAN, B.E. - Serologic reactivity to purified recombinant and native 29-kilodalton peripheral membrane protein of pathogenic Entamoeba histolytica. J. clin. Microbiol., 31: 1403-1407, 1993.

[12] 12. GARDUŃO, E.J.; MARTINEZ, M.C.; MEJÍA, J.M. et al. - Pruebas de diagnóstico inmunológico en amebiasis. Evaluación de la literatura. In: SEMINÁRIO SOBRE AMIBIASIS, 11, México, 1989. p. 67.

[13] 13. GARFINKEL, L.I.; GILARD, M.; HUBER, M. et al. - DNA probes specific for Entamoeba histolytica possessing pathogenic and nonpathogenic zymodemes. Infect. Immunol., 57: 926, 1989.

[14] 14. GOLDIN, A.J.; APT, W.; AGUILERA, X. et al. - A capture ELISA detects Giardia lamblia antigens in formalin-treated faecal samples. Trans. roy. Soc. trop. Med. Hyg., 86: 164, 1992.

[15] 15. GONZÁLEZ, A. - Revisión del estado actual del diagnóstico diferencial de las amibas en México. Salud publ. Méx., 32: 589, 1990.

[16] 16. GUERRANT, R. L. - The global problem of amebiasis: current status research needs, and opportunities for progress. Rev. infect. Dis., 8: 218-227, 1986.

[17] 17. GUIMARĂES, S.; URDANETA, H.; SILVA, E. & TAVARES, C. - Entamoeba histolytica: antigenic characterization of axenic strains from Brazil. Rev. Inst. Med. trop. S. Paulo, 33: 6-11, 1991.

[18] 18. HAQUE, R.; KRESS, K.; WOOD, S. et al. - Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin. J. infect. Dis., 167: 247-249, 1993.

[19] 19. HAQUE, R.; NEVILLE, L.M.; WOOD, S. & PETRI, W. A. - Detection of Entamoeba histolytica and E. dispar in stool. Amer. J. trop. Med. Hyg., 50: 595-596, 1994.

[20] 20. JACKSON, T.F.H.G.; GATHIRAM, V. & SIMJEE, A.E. - Seroepidemiological study of antibody responses to the zymodemes of Entamoeba histolytica. Lancet, 1: 716, 1985.

[21] 21. LOTTER, H.E.; MANNWEILER, M.S. & TANNICH, E. - Sensitive and specific serodiagnosis of invasive amebiasis by using a recombinant surface protein of pathogenic Entamoeba histolytica. J. clin. Microbiol., 30: 3163-3167, 1992.

[22] 22. MATHUR, T.N. & KAUR, J. - The frequency of excretion of cysts of Entamoeba histolytica in known cases of non-dysenteric amoebic colitis based on 21 stool examinations. Indian J. med. Res., 61: 330, 1973.

[23] 23. MERINO, E.W.; DEL MURO, G.R. & ORTIZ-ORTIZ, L. - Evaluation of the ELISA test for the detection of Entamoeba histolytica in feces. J. clin. lab. Anal., 4: 39-42, 1990.

[24] 24. NAGERWARAN, C.; CRAIG, P.S. & DEVANEY, E. - Coproantigen detection in rats experimentally infected with Strongyloides ratti. Parasitology, 108: 335, 1994.

[25] 25. NAYEEN, M.A. & HABIBULLAH, C.M. - A comparative study of different isolate fractions of Entamoeba histolytica in amoebic liver abscess. Trop. Gastroent., 14: 144-147, 1993.

[26] 26. NEAL, R.A. - Phylogeny: the relationship of Entamoeba histolytica to morphologically similiar amebae of the four-nucleated cyst group. In: RAVDIN, J.I., ed. Amebiasis: human infection by Entamoeba histolytica. New York, Churchill Livingstone, 1988. p. 13-26.

[27] 27. RAVDIN, J.I.; JACKSON, T.F.G.H.; PETRI, W.A. et al. - Association of serum antibodies to adherence lectin with invasive amebiasis and asymptomatic infection with pathogenic Entamoeba histolytica. J. infect. Dis., 162: 768, 1990.

[28] 28. SENGUPTA, K.; DAS, P.; JOHNSON, T.M. et al. - Production and characterization of monoclonal antibodies against a highly immunogenic fraction of Entamoeba histolytica (NIH:200) and their application in the detection of current amoebic infection. J. Eukaryot. Microbiol., 40: 722-726, 1993.

[29] 29. SHANDIL, R.K. & VINAYAK, V.K. - Immunochemical characterization of a 29 kDa surface-associated molecule of Entamoeba histolytica and its recognition by serum from patients with amoebiasis. J. med. Microbiol., 36: 41, 1992.

[30] 30. SILVA, E.F.; CARVALHO, M.G. & BRANDĂO, M.G. - Monoxenizaçăo, axenizaçăo e cultura de amostras de Entamoeba histolytica isoladas no Brasil de pacientes sintomáticos e assintomáticos. In: CONGRESSO BRASILEIRO DE PARASITOLOGÍA, 6., Belo Horizonte, 1981. Resumos. p. 18.

[31] 31. STANLEY, S.L.; JACKSON, T.F.H.G.; REED, S.L. et al. - Serodiagnoses of invasive amebiasis using a recombinant Entamoeba histolytica protein. J. Amer. med. Ass., 266: 1984-1986, 1991.

[32] 32. TORIAN, B.E.; REED, S.L.; FLORES, B.R. et al. - The 96 kDa antigen as an integral membrane protein in pathogenic Entamoeba histolytica. Potential differences in pathogenic and non-pathogenic isolates. Infect. Immun., 58: 753-760, 1990.

[33] 33. UNGAR, B.L.P.; YOLKEN, R.H. & QUINN, T.C. - Use of monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens. Amer. J. trop. Med. Hyg., 34: 465-472, 1985.

[34] 34. URDANETA, H.R.; MARTINS, M.S.; SILVA, E.F. & TAVAREZ, C.A.P. - Identification and purification of an isolate-specific 96 kDa antigen of Entamoeba histolytica by using a monoclonal antibody. Mem. Inst. Oswaldo Cruz, 86: 276, 1991.

[35] 35. URDANETA, H.; GUIMARĂES, S.; SILVA, E. & TAVARES, C.A.P. - Entamoeba histolytica: detection of coproantigens by purified antibody in the capture sandwich ELISA. Rev. Inst. Med. trop. S. Paulo, 36: 539-545, 1994.

[36] 36. URDANETA, H.; RONDON, M.; MUŃOZ, M. & HERNANDEZ, M. - Isolation and axanization of two Entamoeba histolytica strains. GEN, 49: 23-28, 1995.

[37] 37. VINAYAK, V.K.; DUTT, P. & PURI, M. - An immunoenzymatic dot ELISA for the detection Giardia lamblia antigen in stool eluates of clinical cases of giardiasis. J. immunol. Meth., 137: 245, 1991.

[38] 38. WALSH, J.A. - Problems in recognition and diagnosis of amebiasis: estimation of the global magnitude of morbidity and mortality. Rev. infect. Dis., 8: 228-238, 1986.

[39] 39. WONSIT, R.; THAMMAPALERD, N.; THARAVANIJ, S.; RADOMYOS, P. & BUNNAG, D. - Enzyme-linked immunosorbent assay based on monoclonal and polyclonal antibodies for the detection of Entamoeba histolytica antigens in faecal specimens. Trans. roy. Soc. trop. Med. Hyg., 86: 166-169, 1992.

[40] 40. ZHANG, Y.; LI, E.; JACKSON, T.F.H.G. et al. - Use of a recombinant 170 kilodalton surface antigen of Entamoeba histolytica for serodiagnosis of amebiasis and identification of immunodominant domains of the native molecule. J. clin. Microbiol., 30: 2788-2792, 1992.

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