UltramicroELISA indirecto para la deteccion de anticuerpos totales a citomegalovirus en suero humano

Laferte, Jose; Marrero, Miguel; Alvarez, Maritza; Jomarron, Liliana; Garcia, Serafina; Vazquez, Susana; Morier, Luis; Ulacia, Mario; Melchor, Antonio

doi:10.1590/S0036-46651992000100008

Resúmenes

Se normalizó un ultramicroELISA indirecto para la detección de anticuerpos a Citomegalovirus (CMV) humano (UMELISA CMV). Se determinó la concentración óptima de antígeno en 30 ug/ml, la dilución de los sueros fue de 1:40 y la dilución de trabajo del conjugado fue de 1:1500. El UMELISA CMV fue comparado con las técnicas de aglutinación de latex para anticuerpos anti-CMV (Dupont de Neumors) y la inmunofluorescencia indirecta (EFT). Los resultados mostraron un alto grado de concordancia y elevada copositividad y conegatividad del UMELISA con respecto a estos dos ensayos. El método es válido para el pesquisaje de anticuerpos en banco de sangre asi como para el diagnóstico de la infección mediante sueros pareados.

UltramicroELISA; Citomegalovirus

We have standardized an indirect ultramicro ELISA assay for detecting antibodies to human Cytomegalovirus (CMV) using human serum samples (UMELISA CMV). The optimal concentration of coating antigen (30 ug/ml), serum dilution (1:40) and anti-human conjugate working dilution (1:1500), were determined by a check board titration method. The UMELISA CMV was compared with the latex agglutination test for antibodies to CMV (Dupont de Nemours) and with an indiret immunofluorescent method. The results have showed the high coincidence, sensitivity and especificity of the proposed assay regarding the two methods compared with, and supporting its use either for a blood donors screening or in the serological diagnosis of this infection by paired serum samples.

SOROLOGIA

UltramicroELISA indirecto para la deteccion de anticuerpos totales a citomegalovirus en suero humano

Indirect Ultramicroelisa assay for the detection of human antibodies to cytomegalovirus using human serum samples

Jose Laferte^I; Miguel Marrero^I; Maritza Alvarez^I; Liliana Jomarron^I; Serafina Garcia^I; Susana Vazquez^I; Luis Morier^I; Mario Ulacia^II; Antonio Melchor^III

^IDepartamento de Virología. Instituto de Medicina Tropical "Pedro Kouri". Siboney. Ciudad Habana, Cuba

^IIBanco de sangre provincial. Ciudad Habana, Cuba

^IIICentro de Inmunoensayo. Ciudad Habana, Cuba

^{Dirección para correspondencia} Dirección para correspondencia: Lic. José Laferté Instituto de Medicina Tropical "Pedro Kouri" Apartado Postal Nº 601 Ciudad Habana, Cuba

RESUMEN

Se normalizó un ultramicroELISA indirecto para la detección de anticuerpos a Citomegalovirus (CMV) humano (UMELISA CMV). Se determinó la concentración óptima de antígeno en 30 ug/ml, la dilución de los sueros fue de 1:40 y la dilución de trabajo del conjugado fue de 1:1500. El UMELISA CMV fue comparado con las técnicas de aglutinación de latex para anticuerpos anti-CMV (Dupont de Neumors) y la inmunofluorescencia indirecta (EFT). Los resultados mostraron un alto grado de concordancia y elevada copositividad y conegatividad del UMELISA con respecto a estos dos ensayos. El método es válido para el pesquisaje de anticuerpos en banco de sangre asi como para el diagnóstico de la infección mediante sueros pareados.

Unitermos: UltramicroELISA; Citomegalovirus.

SUMMARY

We have standardized an indirect ultramicro ELISA assay for detecting antibodies to human Cytomegalovirus (CMV) using human serum samples (UMELISA CMV). The optimal concentration of coating antigen (30 ug/ml), serum dilution (1:40) and anti-human conjugate working dilution (1:1500), were determined by a check board titration method. The UMELISA CMV was compared with the latex agglutination test for antibodies to CMV (Dupont de Nemours) and with an indiret immunofluorescent method. The results have showed the high coincidence, sensitivity and especificity of the proposed assay regarding the two methods compared with, and supporting its use either for a blood donors screening or in the serological diagnosis of this infection by paired serum samples.

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Recebido para publicação em 16/5/1990.

Aceito para publicação em 06/12/1991.

1. ADLER, S.P. - Transfusion associated cytomegalovirus infection. Rev. infect. Dis., 5: 933-977, 1983.
2. ADLER, S.P.; LAWRENCE, L.T & BAGGET, J. - Prevention of transfusion associated CMV infection in very low birthweigth infants using frozen blood and donors seronegative for CMV. Transfusion, 24: 333-335, 1984.
3. ADLER, S.P.; Mc VOY, M.; BIRO, B.G.; BRITT, W.J.; HIDER, P. & MERSHALL, D. - Detection of cytomegalovirus antibody with latex agglutination. J. clin. Microbiol., 22: 68-70, 1985.
4. ALVAREZ, M.; MARRERO, M.; SOLER, M.; PUIG, L. & MORENO, D. - Diagnóstico rápido de citomegalovirus en pacientes inmunocomprometidos mediante AcM que reconocen proteinas precoces virales. Mem. Inst. Oswaldo Cruz, 84: 265-268, 1989.
5. BAUMGARTNER, J.D.; GLAUSER, M.P.; BURGO-BLACK, A.L.; BLACK, R.D.; PYNDIAH, N. & CHIOLERO, R. - Severe cytomegalovirus infection in multiply transfused, spleneotomised, trauma patients. Lancet, 2: 63-66, 1982.
6. BECKWITH, D.G.; HALSTEAD, D.C.; ALPAUGH, K.; SCHWEDEN, A.; BLOONT-FRONEFIELD, D.A. & TOTH, K. - Comparison of latex aglutination test with five other methods for determining the presence of antibody against cytomegalovirus. J. clin. Microbiol., 21: 328-331, 1986.
7. BENEKE, J.S.; TEGTMEYER, G.E.; ALTER, H.J.; LUETKEMEYER, R.; SOLOMEN, R. & BAYER, W.L. - Relation of titres of antibodies to CMV in blood donors to the transmition of CMV infection. J. infect. Dis., 150: 883-888, 1984.
8. BOOTH, J.C.; HANNINGTON, G.; BAKER, T.M.F.; STERN, H.; KANGRO, H.; GRIFFITH, P.D. & HEATH, R.B. - Comparison of Enzime-Linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and pasive hemaglutination techniques for detecting cytomegalovirus IgG antibody. J. clin. Path., 35: 1345-1348, 1982.
9. CASTELLANO, G.A.; HAZZAR, G.T.; MADDEN. D.L. & SEVER, J.L. - Comparison of the ELISA and the indirect hemaglutination test for detection of antibody to cytomegalovirus. J. infect. Dis., 136: 5337-5340, 1977.
10. DUMMER, J.S.; WHITE, L.T.; HO, M.; GRIFFITH, B.T.; HARDESTY, R.L. & BAHNSON, H.T. - Morbity of cytomegalovirus infection in recipients of heart or heart-lung transplants who received cyclosporine. J. infect. Dis., 152: 1182-1191, 1985.
11. FORBES, B.A. - Acquisition of cytomegalovirus infection. An update. Clin. Microbiol. Rev., 2: 204-216, 1989.
12. HORN, A.; FERNANDEZ YERO, J.L. & SCHULZE, M. - UltramicroELISA for alphafetoprotein with the chambern analytical technique. J. clin. Chem. clin. Biochem., 19: 702, 1981.
13. LANDINI, M.P.; R.E.M.C. & COPPOLECCHIA, P. - Comparison of the occurrence of antibodies to human cytomegalovirus as demonstrated by complement fixation. Enzime Linked immunosorbent assay and indirect immunofluorescence. Boll. Inst. sieroter. milan., 61: 320-323, 1982.
14. LOWRY, D.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.
15. ONORATO, I.M.; MOVENS, D.M.; MARTONE, W.J. & STANSFIELD, S.K. - Epidemiology of cytomegalovirus infections: Recomendations for prevention and control. Rev. infect. Dis., 7: 479-496, 1985.
16. OTERO, A.J.; SARRACENT, J.; FERNANDEZ YERO, J.L. & RODRIGUEZ, I. - A 10 microliter ultramicroELISA for detection of monoclonal antibodies against human alphafeto protein. Hybridoma, 3: 391, 1984.
17. PHIPPS, P.H.; GREGOIRE, L.; ROSSIER, E. & PERRY, C. - Comparison of five methods of cytomegalovirus antibody screening of blood donors. J. clin. Microbiol., 18: 1296-1300, 1983.
18. PRINCE, A.M.; SZMUNESS, W.; WILLIAN, S.J. & DAVID, D.S. - A serologic study of cytomegalovirus infection associated with blood transfusion. New Engl. J. Med., 284: 1125-1131, 1971.
19. RIGGS, J.L. - Immunofluorescent staining. In: LENNETTE, E.H. & SCHMIDT, N.J., ed. - Diagnostic procedures for viral, rickettsial and chlamydial infections. 5th. ed. Washington, Am. Pub. Health Association, 1979. p. 141-151.
20. YOW, M.D. - Congenital cytomegalovirus diseases. A new problem. J. infect. Dis., 159: 163-167, 1989.

Dirección para correspondencia:

Lic. José Laferté

Instituto de Medicina Tropical "Pedro Kouri"

Apartado Postal Nº 601

Ciudad Habana, Cuba

Fechas de Publicación

Publicación en esta colección
06 Set 2006
Fecha del número
Feb 1992

Histórico

Recibido
16 Mayo 1990
Acepto
06 Dic 1991

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

[1] 1. ADLER, S.P. - Transfusion associated cytomegalovirus infection. Rev. infect. Dis., 5: 933-977, 1983.

[2] 2. ADLER, S.P.; LAWRENCE, L.T & BAGGET, J. - Prevention of transfusion associated CMV infection in very low birthweigth infants using frozen blood and donors seronegative for CMV. Transfusion, 24: 333-335, 1984.

[3] 3. ADLER, S.P.; Mc VOY, M.; BIRO, B.G.; BRITT, W.J.; HIDER, P. & MERSHALL, D. - Detection of cytomegalovirus antibody with latex agglutination. J. clin. Microbiol., 22: 68-70, 1985.

[4] 4. ALVAREZ, M.; MARRERO, M.; SOLER, M.; PUIG, L. & MORENO, D. - Diagnóstico rápido de citomegalovirus en pacientes inmunocomprometidos mediante AcM que reconocen proteinas precoces virales. Mem. Inst. Oswaldo Cruz, 84: 265-268, 1989.

[5] 5. BAUMGARTNER, J.D.; GLAUSER, M.P.; BURGO-BLACK, A.L.; BLACK, R.D.; PYNDIAH, N. & CHIOLERO, R. - Severe cytomegalovirus infection in multiply transfused, spleneotomised, trauma patients. Lancet, 2: 63-66, 1982.

[6] 6. BECKWITH, D.G.; HALSTEAD, D.C.; ALPAUGH, K.; SCHWEDEN, A.; BLOONT-FRONEFIELD, D.A. & TOTH, K. - Comparison of latex aglutination test with five other methods for determining the presence of antibody against cytomegalovirus. J. clin. Microbiol., 21: 328-331, 1986.

[7] 7. BENEKE, J.S.; TEGTMEYER, G.E.; ALTER, H.J.; LUETKEMEYER, R.; SOLOMEN, R. & BAYER, W.L. - Relation of titres of antibodies to CMV in blood donors to the transmition of CMV infection. J. infect. Dis., 150: 883-888, 1984.

[8] 8. BOOTH, J.C.; HANNINGTON, G.; BAKER, T.M.F.; STERN, H.; KANGRO, H.; GRIFFITH, P.D. & HEATH, R.B. - Comparison of Enzime-Linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and pasive hemaglutination techniques for detecting cytomegalovirus IgG antibody. J. clin. Path., 35: 1345-1348, 1982.

[9] 9. CASTELLANO, G.A.; HAZZAR, G.T.; MADDEN. D.L. & SEVER, J.L. - Comparison of the ELISA and the indirect hemaglutination test for detection of antibody to cytomegalovirus. J. infect. Dis., 136: 5337-5340, 1977.

[10] 10. DUMMER, J.S.; WHITE, L.T.; HO, M.; GRIFFITH, B.T.; HARDESTY, R.L. & BAHNSON, H.T. - Morbity of cytomegalovirus infection in recipients of heart or heart-lung transplants who received cyclosporine. J. infect. Dis., 152: 1182-1191, 1985.

[11] 11. FORBES, B.A. - Acquisition of cytomegalovirus infection. An update. Clin. Microbiol. Rev., 2: 204-216, 1989.

[12] 12. HORN, A.; FERNANDEZ YERO, J.L. & SCHULZE, M. - UltramicroELISA for alphafetoprotein with the chambern analytical technique. J. clin. Chem. clin. Biochem., 19: 702, 1981.

[13] 13. LANDINI, M.P.; R.E.M.C. & COPPOLECCHIA, P. - Comparison of the occurrence of antibodies to human cytomegalovirus as demonstrated by complement fixation. Enzime Linked immunosorbent assay and indirect immunofluorescence. Boll. Inst. sieroter. milan., 61: 320-323, 1982.

[14] 14. LOWRY, D.H.; ROSEBROUGH, N.J.; FARR, A.L. & RANDALL, R.J. - Protein measurement with the Folin phenol reagent. J. biol. Chem., 193: 265-275, 1951.

[15] 15. ONORATO, I.M.; MOVENS, D.M.; MARTONE, W.J. & STANSFIELD, S.K. - Epidemiology of cytomegalovirus infections: Recomendations for prevention and control. Rev. infect. Dis., 7: 479-496, 1985.

[16] 16. OTERO, A.J.; SARRACENT, J.; FERNANDEZ YERO, J.L. & RODRIGUEZ, I. - A 10 microliter ultramicroELISA for detection of monoclonal antibodies against human alphafeto protein. Hybridoma, 3: 391, 1984.

[17] 17. PHIPPS, P.H.; GREGOIRE, L.; ROSSIER, E. & PERRY, C. - Comparison of five methods of cytomegalovirus antibody screening of blood donors. J. clin. Microbiol., 18: 1296-1300, 1983.

[18] 18. PRINCE, A.M.; SZMUNESS, W.; WILLIAN, S.J. & DAVID, D.S. - A serologic study of cytomegalovirus infection associated with blood transfusion. New Engl. J. Med., 284: 1125-1131, 1971.

[19] 19. RIGGS, J.L. - Immunofluorescent staining. In: LENNETTE, E.H. & SCHMIDT, N.J., ed. - Diagnostic procedures for viral, rickettsial and chlamydial infections. 5th. ed. Washington, Am. Pub. Health Association, 1979. p. 141-151.

[20] 20. YOW, M.D. - Congenital cytomegalovirus diseases. A new problem. J. infect. Dis., 159: 163-167, 1989.

Brasil

Brasil

UltramicroELISA indirecto para la deteccion de anticuerpos totales a citomegalovirus en suero humano

Indirect Ultramicroelisa assay for the detection of human antibodies to cytomegalovirus using human serum samples

Resúmenes

Dirección para correspondencia:

Fechas de Publicación

Histórico