Malária humana: padronização e optimização de testes sorológicos para diagnóstico individual e inquéritos soroepidemiológicos

Ferreira, Antonio Walter; Sanchez, Maria Carmen Arroyo

doi:10.1590/S0036-46651988000300003

Resumo

O teste de imunofluorescência indireta (IFI) é considerado teste de referência na soroiogia da malária. Neste trabalho procuramos optimizar o teste empregando P. falciparum obtido de sangue humano e de cultura e P. vivax obtido de sangue de paciente como antígenos, para pesquisa de anticorpos IgG e IgM. Das variáveis técnicas estudadas melhores resultados foram obtidos quando os soros foram diluidos ern PBS contendo 1% de Tween 80 e as lâminas contendo a suspensão antigênica foram estabilizadas em dessecadores ou fixadas com acetona. Foi também padronizado o teste imunoenzimático ELISA com antigenos de P. falciparum obtidos em cultura. O estudo comparativo com o teste de imunofluorescência indireta para pesquisa de anticorpos IgG mostrou: a) nos pacientes primo infectados por P. falciparum a sensibilidade para ambos os testes foi de 71%; b) nos pacientes primo infectados pelo P. vivax a sensibilidade foi de 40% para ambos os testes; c) nos pacientes não primo infectados e com malária atual pelo P. falciparum a sensibilidade para ambos os testes foi de 100%; d) nos pacientes não primo infectados e com malária atual pelo P. vivax a sensibilidade foi de 85% para o teste ELISA e de 92% para a IFI; e) nos pacientes com malária mista a sensibilidade para ambos os testes foi de 100%. A especificidade da IFI foi de 100% e do teste ELISA 95% nos casos de indivíduos não maláricos. Os resultados obtidos sugerem ser o teste ELISA, uma boa alternativa para o teste de IFI, para a pesquisa de anticorpos IgG anti P. falciparum, na soroiogia da malária.

Malária; IFI; Teste imunoenzimático ELISA

ARTIGOS ORIGINAIS

Malária humana: padronização e optimização de testes sorológicos para diagnóstico individual e inquéritos soroepidemiológicos

Human malaria: standardization of serologic tests for individual diagnostic and seroepidemiologic surveys

Antonio Walter Ferreira^{I, II}; Maria Carmen Arroyo Sanchez^I

^ILaboratório de Soroepidemiologia do Instituto de Medicina Tropical de São Paulo. São Paulo, SP, Brasil

^IIDepartamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina da Universidade de São Paulo. São Paulo, SP, Brasil

^{Endereço para correspondência} Endereço para correspondência: Dr. Antonio Walter Ferreira Laboratório de Imunologia do Instituto de Medicina Tropical de São Paulo Av. Dr. Enéas de Carvalho Aguiar, 470 CEP 05403 São Paulo, SP, Brasil

RESUMO

O teste de imunofluorescência indireta (IFI) é considerado teste de referência na soroiogia da malária. Neste trabalho procuramos optimizar o teste empregando P. falciparum obtido de sangue humano e de cultura e P. vivax obtido de sangue de paciente como antígenos, para pesquisa de anticorpos IgG e IgM. Das variáveis técnicas estudadas melhores resultados foram obtidos quando os soros foram diluidos ern PBS contendo 1% de Tween 80 e as lâminas contendo a suspensão antigênica foram estabilizadas em dessecadores ou fixadas com acetona. Foi também padronizado o teste imunoenzimático ELISA com antigenos de P. falciparum obtidos em cultura. O estudo comparativo com o teste de imunofluorescência indireta para pesquisa de anticorpos IgG mostrou: a) nos pacientes primo infectados por P. falciparum a sensibilidade para ambos os testes foi de 71%; b) nos pacientes primo infectados pelo P. vivax a sensibilidade foi de 40% para ambos os testes; c) nos pacientes não primo infectados e com malária atual pelo P. falciparum a sensibilidade para ambos os testes foi de 100%; d) nos pacientes não primo infectados e com malária atual pelo P. vivax a sensibilidade foi de 85% para o teste ELISA e de 92% para a IFI; e) nos pacientes com malária mista a sensibilidade para ambos os testes foi de 100%. A especificidade da IFI foi de 100% e do teste ELISA 95% nos casos de indivíduos não maláricos.

Os resultados obtidos sugerem ser o teste ELISA, uma boa alternativa para o teste de IFI, para a pesquisa de anticorpos IgG anti P. falciparum, na soroiogia da malária.

Unitermos: Malária; IFI; Teste imunoenzimático ELISA.

SUMMARY

The indirect immunofluorescence antibody test (IFA) is normally employed as reference test in the serology of malaria. In this report we standardized and optimized the test, for our condition, utilizing P. falciparum obtained from human blood on culture and P. vivax obtained from human blood as antigens, for detection of IgG and IgM antibodies. Some technical variables, were tested and best resulto were obtained when sera were diluted in PBS containing 1% Tween-80 and the slides, containing the antigenic preparation were fixed in cold acetone or stabilized on dried air with silica. The ELISA test was standardized for P. falciparum antibodies and the comparison of the IFA and ELISA showed: a) in P. falciparum prime infected patient the sensitivity was 71% for both tests; b) in P. vivax prime infected patients the sensitivity was 40% for both tests; c) in non prime infected patients with P. falciparum malaria the sensitivity was 100% for both tests; d) in non prime infected patients with P. vivax malaria the sensitivity was 85% for ELISA and 92% for IFA; e) in patients with P. vivax and P. falciparum malaria the sensitivity was 100% for both tests. The specificity was 95% for ELISA and 100% for IFA in non malaria individuals.

The results showed that the ELISA test could be an alternative for IFA for IgG antibodies in the serology of malaria.

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AGRADECIMENTOS

Ao CNPq - Projeto do Polonoroeste - Processo n? 70.0350/85 pelo suporte financeiro.

Ao Laboratório de Malária da SUCEN pelo fornecimento de cultura de P. falciparum no início do trabalho e pelo fornecimento de soros de pacientes com malária.

A Vera de Paula Quartier de Oliveira, Rivaldo de Souza Cardoso e Paulo de Oliveira pela assistência técnica na execução do projeto.

1. AMBROISE-THOMAS, P. - Diagnosis and seroepidemiologic studies of malaria by immunofluorescence, and indirect haemagglutination and immunoenzymology. Israel J. med. Sci., 14: 690-691, 1978.
2. AMBROISE-THOMAS, P. - L'immunofluorescence dans la sérologie du paludisme. WHO/MAL, (953): 1-6, 1981.
3. AMBROISE-THOMAS, P.; BILLIAULT, X.; DESGEORGES, P.T. & BOUTTAZ, M. - Mise en evidence, par une micromethode immuno-enzymologique (ELISA), d'antigènes métaboliques produits "in vitro" par Plasmodium falciparum en culture. WHO/MAL, (930): 1-5, 1981.
4. BEAUDOIN, R.L.; RAMSEY, J.M. & PACHECO, N.D. - Antigens employed in immunodiagnostic tests for the detection of malarial antibodies. WHO/MAL, (952): 1-11, 1981.
5. B1DWELL, D.E. & VOLLER, A. - Malaria diagnosis by enzyme-linked immunosorbent assays. Brit. med. J., 282: 1747-8, 1981.
6. BROWN, G.V.; STACE, J.D. & ANDERS, R.F. - Specificities of antibodies boosted by acute Plasmodium falciparum infection in man. Amer. J. trop. Med. Hyg., 32: 1221-1228, 1983.
7. CLARK, H.F. & SHEPPARD, C.C. - A dialysis technique for preparing fluorescent antibody. Virology, 20: 642-644, 1963.
8. COLLINS, W.E.; LUNDE, M.N. & SKINNER, J.C. - Development of antibodies to P. vivax as measured by two different techniques. Amer. J. trop. Med. Hyg., 24: 412-416, 1975.
9. COLLINS, W.E. & SKINNER, J.C. - The indirect fluorescent antibody test for malaria. Amer. J. trop. Med. Hyg., 21: 690-695, 1972.
10. DRUILHE, P. & MONJOUR, L. - Sérodiagnostic du paludisme par immuno-précipitation. Valeur comparée de différentes techniques d'extraction des antigènes solubles. Sensibilité par rapport à l'immunofluorescence indirecte. C.R. Soc. Biol. (Paris), 169: 1089-1095, 1975.
11. FRANCIS, V.S.; GUPTA, M.M. & BHAT, P. - Evaluation of a crude soluble antigen from "in vitro" culture of Plasmodium falciparum for ELISA. Tropen-med. Parasit., 33: 240-242, 1982.
12. FRANCO, E.L.F. - Immunoserology of malaria. Apostila. Julho de 1984. 22p.
13. GUPTA, M.M.; SEBASTIAN. M.J.; BHAT, P. & LOBEL, H.O. - Evaluation of "in'vitro" cultured P. falciparum as antigen for malaria serology. J. trop. Med. Hyg., 84: 165-170, 1981.
14. JENSEN, J.B. - Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum Amer. J. trop. Med. Hyg., 27: 1274-1276, 1978.
15. JENSEN, J.B. & TRAGER, W. - Some recent advances in the cultivation of Plasmodium falciparum Israel J. med. Sci., 14: 563-570, 1978.
16. KREIER, J.P. - The isolation and fractionation of malaria-infected cells. Bull. Wld. Hlth. Org., 55: 317-331, 1977.
17. LOBEL, H.O. - Indications for and usefulness of serological techniques in epidemiological investigation and assessment. WHO/MAL, (967): 1-11, 1981.
18. LÓPEZ-ANTUÑANO, F.J. - Falciparum malaria antigen slides for indirect immuno-fluorescence test made from "in vitro" cultures. Trans. roy. Soc. trop. Med. Hyg., 68: 257, 1974.
19. LÓPEZ-ANTUÑANO, F.J. - Estadarización de las pruebas de Inmuno-Fluorescencia Indirecta (IFI) para malaria. PAHO/WHO Interoffice Memorandum, 1984.
20. LUNDE, M.N. & POWERS, K.G. - The preparation of malaria haemagglutination antigen. Ann. trop. Med. Parasit., 70: 283-291, 1976.
21. MANAWADU, B.R. & VOLLER, A. - Standardization of the indirect fluorescence antibody test for malaria. Trans. roy. Soc. trop. Med. Hyg., 72: 456-462, 1978.
22. NAKANE, P.K. & KAWAOI, A. - Peroxidase-labeled antibody a new method of conjugation. J. Histochem. Cytochem., 22: 1084-1091, 1974.
23. PASVOL, G.; WILSON, R.J.M.; SMALLEY, M.E. & BROWN, J. - Separation of viable schizont-infected red cells of Plasmodium falciparum form human blood. Ann. trop. Med. Parasit., 72: 87-88, 1978.
24. QUAKYI, I.A. - The development and validation of an enzyme-linked immunosorbent assay for malaria. Tropenmed. Parasit., 31: 325-333, 1980.
25. QUILICI, M.; DELMONT, J.; RANQUE, J.; ROUGEMONT, A.; MARTARESCHE, B. & BOISSON, M.E. - Évaluation de l'antigène de Plasmodium falciparum d'origine humaine pour la réaction d'immunofluorescence indirecte dans le paludisme. Bull. Soc. Path, exot., 68: 515-521, 1975.
26. REESE, R.T.; LANGRETH, S.G. & TRAGER, W. - Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood. Bull. Wld. Hlth. Org., 57 (suppl. 1): 53-61, 1979.
27. REPORT on the Merieux Foundation/WHO Meeting on Immunological methods in malariology. WHO/MAL, (948): 1-17, 1981.
28. ROFFI, J.; LAFABRIE, B. & STACH, J.L. - Utilisation d'antigènes purifies pour le sérodiagnostic et les études épidémiologiques du paludisme humain. Intérêt de la technique ELISA pour la mise en évidence des IgG et des IgM spécifiques. Bull. Soc. Path, exot., 76: 49-68, 1983.
29. SAUL, A.; MYLER, P.; ELLIOTT, T.; T. & KIDSON, C. - Purification of mature schizonts of P. falciparum on colloidal silica gradients. Bull. Wld. Hlth. Org., 60: 755-759, 1982.
30. SPENCER, H.C.; COLLINS, W.E.; CHIN, W. & SKINNER, J.C. - The enzyme-linked immunosorbent assay (ELISA) for malaria. I. The use of in vitrocultured P. falciparum as antigen. Amer. J. trop. Med. Hyg., 28: 927-938, 1979.
31. SPENCER, H.C.; COLLINS, W.E. & SKINNER, J.C. - The enzyme-linked immunosorbent assay (ELISA) for malaria. II. Comparison with the malaria indirect fluorescent antibody test (IFA). Amer. J. trop. Med. Hyg., 28: 933-936, 1979.
32. SPENCER, H.C.; COLLINS, W.E.; WARREN, M.; JEFFERY, G.M.; MASON, J.; HUONG, A.Y.; STANFILL, P.S. & SKINNER, J.C. - The enzymelinked immunosorbent assay (ELISA) for malaria. III. Antibody response in documented P. falciparum infections. Amer. J. trop. Med. Hyg., 30: 747-750, 1981.
33. SULZER, A.J.; WILSON, M. & HALL, E.C. - Indirect fluorescent antibody tests for parasitic diseases. V. An evaluation of a thick-smear antigen in the IFA test for malaria antibodies. Amer. J. trop. Med. Hyg., 18: 199-205, 1969.
34. TANDOM, A.; SAXENA, R.P.; BHATIA, B. & SAXENA, K.C. - The enzyme-linked immunosorbent assay in the immunodiagnosis of human malaria. Trans. roy. Soc. trop. Med. Hyg., 76: 371-372, 1982.
35. TARGETT, G.A.T. - Antibody response to Plasmodium falciparum malaria: comparison of immunoglobulin concentration antibody titers and antigenicity of different assexual stages of the parasite. Clin. exp. Immunol., 7: 501-517, 1970.
36. TRAGER, W. - Plasmodium falciparum in culture: improved continuous flow method. J. Protozool., 26: 125-129, 1979.
37. TRAGER, W. - Recent developments in enlarging the scale of production of Plasmodium falciparum "in vitro". Bull. Wld. Hlth. Org., 57 (suppl. 1): 85-86, 1979.
38. TRAGER, W. & JENSEN, J.B. - Human malaria parasites in continuous culture. Science, 193: 673-675, 1976.
39. VOLLER, A.; BARTLETT, A. & BIDWELL, D.E. - Enzyme immunoassays for parasitic diseases. Trans. roy. Soc. trop. Med. Hyg., 70: 98-106, 1976.
40. VOLLER, A.; BIDWELL, D.E.; BARTLETT, A. & EDWARDS, R. - A comparison of isotopic and enzyme immunoassays for tropical parasitic diseases. Trans. roy. Soc. trop. Med. Hyg., 71: 431-437, 1977.
41. VOLLER, A.; BIDWELL, D.; HULT, G. & ENGVALL, E. - A microplate of enzyme-linked immunosorbent assay and its application to malaria. Bull. Wld. Hlth. Org., 51: 209-211, 1974.
42. VOLLER, A.; CORNILLE-BROGGER, R.; STOREY, J. & MOLINEAUX, L. - A longitudinal study of P. falciparum malaria in the West African savanna using the ELISA technique. Bull. Wld. Hlth. Org., 58: 429-438, 1980.
43. VOLLER, A.; HOLDT, G.; THORS, C. & ENGVALL, E. - New serological test for malaria antibodies. Brit. med. J., 1: 61, 1975.
44. WILLET, G.P. & CANFIELD, C.J. - Plasmodium falciparum: continuous cultivation of erythrocyte stages in plasma-free culture medium. WHO/MAL, (1002): 1-8, 1983.
45. WILSON, R.J.M. & LING, I. - Fractionation and characterization of Plasmodium falciparum antigens. Bull. Wld. Hlth. Org., 57 (suppl. 1): 123-133, 1979.
46. ZUCKERMAN, A. - Current status of the immunology of blood and tissue Protozoa. II. Plasmodium. Exp. Parasit., 42: 374-446, 1977.

Endereço para correspondência:

Dr. Antonio Walter Ferreira

Laboratório de Imunologia do Instituto de Medicina Tropical de São Paulo

Av. Dr. Enéas de Carvalho Aguiar, 470

CEP 05403 São Paulo, SP, Brasil

Datas de Publicação

Publicação nesta coleção
17 Fev 2011
Data do Fascículo
Jun 1988

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

[1] 1. AMBROISE-THOMAS, P. - Diagnosis and seroepidemiologic studies of malaria by immunofluorescence, and indirect haemagglutination and immunoenzymology. Israel J. med. Sci., 14: 690-691, 1978.

[2] 2. AMBROISE-THOMAS, P. - L'immunofluorescence dans la sérologie du paludisme. WHO/MAL, (953): 1-6, 1981.

[3] 3. AMBROISE-THOMAS, P.; BILLIAULT, X.; DESGEORGES, P.T. & BOUTTAZ, M. - Mise en evidence, par une micromethode immuno-enzymologique (ELISA), d'antigènes métaboliques produits "in vitro" par Plasmodium falciparum en culture. WHO/MAL, (930): 1-5, 1981.

[4] 4. BEAUDOIN, R.L.; RAMSEY, J.M. & PACHECO, N.D. - Antigens employed in immunodiagnostic tests for the detection of malarial antibodies. WHO/MAL, (952): 1-11, 1981.

[5] 5. B1DWELL, D.E. & VOLLER, A. - Malaria diagnosis by enzyme-linked immunosorbent assays. Brit. med. J., 282: 1747-8, 1981.

[6] 6. BROWN, G.V.; STACE, J.D. & ANDERS, R.F. - Specificities of antibodies boosted by acute Plasmodium falciparum infection in man. Amer. J. trop. Med. Hyg., 32: 1221-1228, 1983.

[7] 7. CLARK, H.F. & SHEPPARD, C.C. - A dialysis technique for preparing fluorescent antibody. Virology, 20: 642-644, 1963.

[8] 8. COLLINS, W.E.; LUNDE, M.N. & SKINNER, J.C. - Development of antibodies to P. vivax as measured by two different techniques. Amer. J. trop. Med. Hyg., 24: 412-416, 1975.

[9] 9. COLLINS, W.E. & SKINNER, J.C. - The indirect fluorescent antibody test for malaria. Amer. J. trop. Med. Hyg., 21: 690-695, 1972.

[10] 10. DRUILHE, P. & MONJOUR, L. - Sérodiagnostic du paludisme par immuno-précipitation. Valeur comparée de différentes techniques d'extraction des antigènes solubles. Sensibilité par rapport à l'immunofluorescence indirecte. C.R. Soc. Biol. (Paris), 169: 1089-1095, 1975.

[11] 11. FRANCIS, V.S.; GUPTA, M.M. & BHAT, P. - Evaluation of a crude soluble antigen from "in vitro" culture of Plasmodium falciparum for ELISA. Tropen-med. Parasit., 33: 240-242, 1982.

[12] 12. FRANCO, E.L.F. - Immunoserology of malaria. Apostila. Julho de 1984. 22p.

[13] 13. GUPTA, M.M.; SEBASTIAN. M.J.; BHAT, P. & LOBEL, H.O. - Evaluation of "in'vitro" cultured P. falciparum as antigen for malaria serology. J. trop. Med. Hyg., 84: 165-170, 1981.

[14] 14. JENSEN, J.B. - Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum Amer. J. trop. Med. Hyg., 27: 1274-1276, 1978.

[15] 15. JENSEN, J.B. & TRAGER, W. - Some recent advances in the cultivation of Plasmodium falciparum Israel J. med. Sci., 14: 563-570, 1978.

[16] 16. KREIER, J.P. - The isolation and fractionation of malaria-infected cells. Bull. Wld. Hlth. Org., 55: 317-331, 1977.

[17] 17. LOBEL, H.O. - Indications for and usefulness of serological techniques in epidemiological investigation and assessment. WHO/MAL, (967): 1-11, 1981.

[18] 18. LÓPEZ-ANTUÑANO, F.J. - Falciparum malaria antigen slides for indirect immuno-fluorescence test made from "in vitro" cultures. Trans. roy. Soc. trop. Med. Hyg., 68: 257, 1974.

[19] 19. LÓPEZ-ANTUÑANO, F.J. - Estadarización de las pruebas de Inmuno-Fluorescencia Indirecta (IFI) para malaria. PAHO/WHO Interoffice Memorandum, 1984.

[20] 20. LUNDE, M.N. & POWERS, K.G. - The preparation of malaria haemagglutination antigen. Ann. trop. Med. Parasit., 70: 283-291, 1976.

[21] 21. MANAWADU, B.R. & VOLLER, A. - Standardization of the indirect fluorescence antibody test for malaria. Trans. roy. Soc. trop. Med. Hyg., 72: 456-462, 1978.

[22] 22. NAKANE, P.K. & KAWAOI, A. - Peroxidase-labeled antibody a new method of conjugation. J. Histochem. Cytochem., 22: 1084-1091, 1974.

[23] 23. PASVOL, G.; WILSON, R.J.M.; SMALLEY, M.E. & BROWN, J. - Separation of viable schizont-infected red cells of Plasmodium falciparum form human blood. Ann. trop. Med. Parasit., 72: 87-88, 1978.

[24] 24. QUAKYI, I.A. - The development and validation of an enzyme-linked immunosorbent assay for malaria. Tropenmed. Parasit., 31: 325-333, 1980.

[25] 25. QUILICI, M.; DELMONT, J.; RANQUE, J.; ROUGEMONT, A.; MARTARESCHE, B. & BOISSON, M.E. - Évaluation de l'antigène de Plasmodium falciparum d'origine humaine pour la réaction d'immunofluorescence indirecte dans le paludisme. Bull. Soc. Path, exot., 68: 515-521, 1975.

[26] 26. REESE, R.T.; LANGRETH, S.G. & TRAGER, W. - Isolation of stages of the human parasite Plasmodium falciparum from culture and from animal blood. Bull. Wld. Hlth. Org., 57 (suppl. 1): 53-61, 1979.

[27] 27. REPORT on the Merieux Foundation/WHO Meeting on Immunological methods in malariology. WHO/MAL, (948): 1-17, 1981.

[28] 28. ROFFI, J.; LAFABRIE, B. & STACH, J.L. - Utilisation d'antigènes purifies pour le sérodiagnostic et les études épidémiologiques du paludisme humain. Intérêt de la technique ELISA pour la mise en évidence des IgG et des IgM spécifiques. Bull. Soc. Path, exot., 76: 49-68, 1983.

[29] 29. SAUL, A.; MYLER, P.; ELLIOTT, T.; T. & KIDSON, C. - Purification of mature schizonts of P. falciparum on colloidal silica gradients. Bull. Wld. Hlth. Org., 60: 755-759, 1982.

[30] 30. SPENCER, H.C.; COLLINS, W.E.; CHIN, W. & SKINNER, J.C. - The enzyme-linked immunosorbent assay (ELISA) for malaria. I. The use of in vitrocultured P. falciparum as antigen. Amer. J. trop. Med. Hyg., 28: 927-938, 1979.

[31] 31. SPENCER, H.C.; COLLINS, W.E. & SKINNER, J.C. - The enzyme-linked immunosorbent assay (ELISA) for malaria. II. Comparison with the malaria indirect fluorescent antibody test (IFA). Amer. J. trop. Med. Hyg., 28: 933-936, 1979.

[32] 32. SPENCER, H.C.; COLLINS, W.E.; WARREN, M.; JEFFERY, G.M.; MASON, J.; HUONG, A.Y.; STANFILL, P.S. & SKINNER, J.C. - The enzymelinked immunosorbent assay (ELISA) for malaria. III. Antibody response in documented P. falciparum infections. Amer. J. trop. Med. Hyg., 30: 747-750, 1981.

[33] 33. SULZER, A.J.; WILSON, M. & HALL, E.C. - Indirect fluorescent antibody tests for parasitic diseases. V. An evaluation of a thick-smear antigen in the IFA test for malaria antibodies. Amer. J. trop. Med. Hyg., 18: 199-205, 1969.

[34] 34. TANDOM, A.; SAXENA, R.P.; BHATIA, B. & SAXENA, K.C. - The enzyme-linked immunosorbent assay in the immunodiagnosis of human malaria. Trans. roy. Soc. trop. Med. Hyg., 76: 371-372, 1982.

[35] 35. TARGETT, G.A.T. - Antibody response to Plasmodium falciparum malaria: comparison of immunoglobulin concentration antibody titers and antigenicity of different assexual stages of the parasite. Clin. exp. Immunol., 7: 501-517, 1970.

[36] 36. TRAGER, W. - Plasmodium falciparum in culture: improved continuous flow method. J. Protozool., 26: 125-129, 1979.

[37] 37. TRAGER, W. - Recent developments in enlarging the scale of production of Plasmodium falciparum "in vitro". Bull. Wld. Hlth. Org., 57 (suppl. 1): 85-86, 1979.

[38] 38. TRAGER, W. & JENSEN, J.B. - Human malaria parasites in continuous culture. Science, 193: 673-675, 1976.

[39] 39. VOLLER, A.; BARTLETT, A. & BIDWELL, D.E. - Enzyme immunoassays for parasitic diseases. Trans. roy. Soc. trop. Med. Hyg., 70: 98-106, 1976.

[40] 40. VOLLER, A.; BIDWELL, D.E.; BARTLETT, A. & EDWARDS, R. - A comparison of isotopic and enzyme immunoassays for tropical parasitic diseases. Trans. roy. Soc. trop. Med. Hyg., 71: 431-437, 1977.

[41] 41. VOLLER, A.; BIDWELL, D.; HULT, G. & ENGVALL, E. - A microplate of enzyme-linked immunosorbent assay and its application to malaria. Bull. Wld. Hlth. Org., 51: 209-211, 1974.

[42] 42. VOLLER, A.; CORNILLE-BROGGER, R.; STOREY, J. & MOLINEAUX, L. - A longitudinal study of P. falciparum malaria in the West African savanna using the ELISA technique. Bull. Wld. Hlth. Org., 58: 429-438, 1980.

[43] 43. VOLLER, A.; HOLDT, G.; THORS, C. & ENGVALL, E. - New serological test for malaria antibodies. Brit. med. J., 1: 61, 1975.

[44] 44. WILLET, G.P. & CANFIELD, C.J. - Plasmodium falciparum: continuous cultivation of erythrocyte stages in plasma-free culture medium. WHO/MAL, (1002): 1-8, 1983.

[45] 45. WILSON, R.J.M. & LING, I. - Fractionation and characterization of Plasmodium falciparum antigens. Bull. Wld. Hlth. Org., 57 (suppl. 1): 123-133, 1979.

[46] 46. ZUCKERMAN, A. - Current status of the immunology of blood and tissue Protozoa. II. Plasmodium. Exp. Parasit., 42: 374-446, 1977.