Evaluacion de la tecnica de contraimmunoelectroforesis para determinar la potencia antigena de las vacunas antirrabicas

Miceli, Graciela; Torroba, Jorge E.; Diaz, Ana María

doi:10.1590/S0036-46651993000600011

Resúmenes

El método recomendado por la Organización Mundial de la Salud (OMS) para la prueba de potencia de vacunas antirrábicas como producto final es la prueba NIH. Algunas técnicas in vitro se han propuesto para el control durante el proceso de produción y complementan el ensayo in vivo antes mencionado. Este trabajo presenta los resultados obtenidos cuando se utilizó la técnica de contrainmunoelectroforésis (CIE) para determinar el contenido de antígenos en muestras de 84 y 40 lotes de vacunas antirrábicas producidas en tejido nervioso de cerebro de ratón lactante mediante cultivo de tejidos, respectivamente. La evaluación de las muestras en, y en torno de, las 0.3 UI por ambos métodos muestran que, en la práctica, un título CIE de 1:4 cumpliría con un mínimo de potencia de la prueba NIH. Un bajo grado de variabilidad de la prueba CIE fue observada en nuestro laboratorio cuando dos lotes de vacunas de referencia de trabajo y 7 lotes de vacunas antirrábicas, de diferente origen y actividad, fueron ensayadas en cinco pruebas independientes. Todos los títulos se ubicaron dentro de una dilución doble, lo que es indicativo de su reproducibilidad. Se observó buena sensibilidad para detectar el deterioro del antígeno en el ensayo de degradación térmica, cuando muestras de 3 lotes de vacuna líquida de cerebro de ratón lactante fueron mantenidas a4 y 37ºC cada una, por 28 días. Se evaluaron semanalmente por los ensayos de CIE y NIH. Finalmente, se observó que el ensayo de CIE podría ser utilizado por los productores para estimar el punto final de los procesos de concentración para que se corresponda con un valor antigénico deseado en la prueba de potencia NIH.

Rabia; Vacunas; Control de potencia "in vitro"

The method recommended by the World Health Organization (WHO) for the potency assay of human and animal rabies vaccines as final product is the NIH test. Some in vitro techniques have been proposed for in process testing and supplement the in vivo test mentioned above. This paper presents the results obtained when using a counterimmunoelectrophoresis (CIE) techniques to determine the antigens content in samples of 84 and 40 samples of lots of suckling mouse rabies and tissue culture rabies vaccines, respectively. The evaluation of rabies vaccines under, at, near and up to 0.3 UI by the NIH and CIE tests showed that for practical use, a CIE titer of 1:4 would match the minimum of NIH potency test requirement. Low degree of CIE test variability within our laboratory was observed, when two working reference and 7 lots of rabies vaccines from different origin and activity were tested in five independent assays. All titres fell within one double dilution what is an indication of its reproducibility. Good sensitivity to detect antigen deterioration in thermal degradation test was observed when samples of 3 lots of liquid suckling mouse rabies vaccine were held stored at 4ºC or at 37ºC for 4 weeks and assays by CIE and NIH tests. Finally, CIE test could apply by manufactures to follow the concentration procedures to determine the final end point which could match a desired antigenic value in the NIH potency test.

IMUNOLOGIA

Evaluacion de la tecnica de contraimmunoelectroforesis para determinar la potencia antigena de las vacunas antirrabicas

Evaluation ot the counterimmunoelectrophoresis technique to determine the antigenic potency of antirrabic vaccines

Graciela Miceli^I; Jorge E. Torroba^II; Ana María Diaz^II

^ICátedra de Inmunología Veterinaria. Facultad de Ciencias Veterinarias. UNLP. 60 y 118 - (1900) La Plata - Buenos Aires - Argentina

^IIInstituto Panamericano para la Protección de Alimentos y Zoonosis (PAHO/WHO) - Talcahuano 1660 - (1640) Martínez - Buenos Aires - Argentina

RESUMEN

El método recomendado por la Organización Mundial de la Salud (OMS) para la prueba de potencia de vacunas antirrábicas como producto final es la prueba NIH. Algunas técnicas in vitro se han propuesto para el control durante el proceso de produción y complementan el ensayo in vivo antes mencionado.

Este trabajo presenta los resultados obtenidos cuando se utilizó la técnica de contrainmunoelectroforésis (CIE) para determinar el contenido de antígenos en muestras de 84 y 40 lotes de vacunas antirrábicas producidas en tejido nervioso de cerebro de ratón lactante mediante cultivo de tejidos, respectivamente.

La evaluación de las muestras en, y en torno de, las 0.3 UI por ambos métodos muestran que, en la práctica, un título CIE de 1:4 cumpliría con un mínimo de potencia de la prueba NIH. Un bajo grado de variabilidad de la prueba CIE fue observada en nuestro laboratorio cuando dos lotes de vacunas de referencia de trabajo y 7 lotes de vacunas antirrábicas, de diferente origen y actividad, fueron ensayadas en cinco pruebas independientes. Todos los títulos se ubicaron dentro de una dilución doble, lo que es indicativo de su reproducibilidad.

Se observó buena sensibilidad para detectar el deterioro del antígeno en el ensayo de degradación térmica, cuando muestras de 3 lotes de vacuna líquida de cerebro de ratón lactante fueron mantenidas a4 y 37ºC cada una, por 28 días. Se evaluaron semanalmente por los ensayos de CIE y NIH. Finalmente, se observó que el ensayo de CIE podría ser utilizado por los productores para estimar el punto final de los procesos de concentración para que se corresponda con un valor antigénico deseado en la prueba de potencia NIH.

Unitermos: Rabia; Vacunas; Control de potencia "in vitro"

SUMMARY

The method recommended by the World Health Organization (WHO) for the potency assay of human and animal rabies vaccines as final product is the NIH test. Some in vitro techniques have been proposed for in process testing and supplement the in vivo test mentioned above.

This paper presents the results obtained when using a counterimmunoelectrophoresis (CIE) techniques to determine the antigens content in samples of 84 and 40 samples of lots of suckling mouse rabies and tissue culture rabies vaccines, respectively.

The evaluation of rabies vaccines under, at, near and up to 0.3 UI by the NIH and CIE tests showed that for practical use, a CIE titer of 1:4 would match the minimum of NIH potency test requirement. Low degree of CIE test variability within our laboratory was observed, when two working reference and 7 lots of rabies vaccines from different origin and activity were tested in five independent assays. All titres fell within one double dilution what is an indication of its reproducibility.

Good sensitivity to detect antigen deterioration in thermal degradation test was observed when samples of 3 lots of liquid suckling mouse rabies vaccine were held stored at 4ºC or at 37ºC for 4 weeks and assays by CIE and NIH tests. Finally, CIE test could apply by manufactures to follow the concentration procedures to determine the final end point which could match a desired antigenic value in the NIH potency test.

Texto completo disponible sólo en PDF.

Full text available only in PDF format.

AGRADECIMIENTOS

Los autores agradecen a la Dra. Isabel N. de Kantor sus valiosas sugerencias en la preparación de este manuscrito.

Recebido para publicação em 01/12/1992

Aceito para publicação em 22/07/1993

1. ACHA, P.N. & ARAMBULO, P.V.- Rabies in the tropics. History and current status. In: KUWERT, E., ed. Rabies in the tropics. Berlin, Springer Verlag, 1984. p. 343-359.
2. ARKO, R.J.; WIKTOR, T.J. & SIKES, R.K. - The antibody binding test for vaccine potency. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. Geneva, World Health Organization, 1973. p. 292-294.
3. BARTH, R.; GROSS-ALBENHAUSEN, E.; JAEGER, O. & MILCKE, L. - The antibody-binding test, a useful method for quantitative determination of inactivated rabies virus antigen. J. biol. Stand., 9: 81-89, 1981.
4. BARTH, R.; GRUSCHKAN, H.; JAEGER, O. & MILCKE, L. - Concentration of rabies antigen and potency test. In: KUWERT, E.K.; WIKTOR, T.J. & KOPROWSKI, H., ed. Cell culture rabies vaccines and their protective effect in man. Geneva, International Green Cross, 1981. p. 60-63.
5. DIAZ, A.M.O. - Rabies neutralizating antibodies determination by the modified counterimmunoelectrophoresis test and the rapid fluorescent focus inhibition test. Zbl. Bakt. Hyg. A., 256: 1-6, 1983.
6. DIAZ, A.M.O. - The counterimmunoelectrophoresis techniques for "in vitro" detection of antigens in rabies vaccines. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. 4. ed. Geneva, World Health Organization, 1992. (In press).
7. DIAZ, A.M.O.; ASSAD MILLER, R.; CORTES, M.A.; DELLEPIANE, N.; ELBERGER, D.; FABREGA, F.; MICELI, G.S.; MENDOZA, G.; OBARRIO, M.E.; OCHOA, M.T.; BECCO, O. & PERDOMO, G. - Rabies reference vaccine for use as regional standard for Latin America and the Caribbean Countries. Biologicals, 18: 281-287, 1990.
8. DIAZ, A.M.O.; NEBEL, A.E. & MICELI, G.S. - A counterimmunoelectrophoresis technique for in vitro detection of antigens in rabies vaccines. In: THRAENHART, O.; KOPROWSKI, H.; BOGEL, K. & SUREAU, P., ed. Progress in rabies control. England, Wells Medical, 1989. p. 305-310.
9. DIAZ, A.M.O.; PERDOMO, G. & BECCO, O. - Estabilidad de la vacuna antirrábica de cerebro de ratón lactante almacenada a distintas temperaturas. Bol. Ofic.sanit. panamer., 104: 261-271, 1988.
10. FERGUSON, M.; SEAGROATT, V. & SCHILD, G.C. - Single radial immunodiffusion assays for the standardization of the antigenic content of rabies vaccines. Develop. biol. Stand., 64: 81-86,1986.
11. FERGUSON, M. & SCHILD, G.C. - A single-radial-immunodiffusion technique for the assay of rabies glycoprotein antigen: application for potency tests of vaccines against rabies. J. gen. Virol., 59: 197-201, 1982.
12. FERGUSON, M.; WAXHMANN, B.; NEEDY, C.F. & FITZGERALD, E.A. - The effect of strain difference on the assay of rabies virus glycoprotein by single radial immunodiffusion. J. biol. Stand., 5: 73-77, 1987.
13. FITZGERALD, E.A.; GALLAGHER, M.; HUNTER, W.S. & SELIGMAN Jr., E.B. - Use of the antibody assay in immunized mice for the determination of rabies vaccine potency. In: HENESSEN, W. & REGAMEY, R.H., ed. Developments in biological standardization. Basel, Karger, 1978. v.40, p. 183-186.
14. FITZGERALD, E.A.; GREEN, O.L. & SELIGMAN Jr., E.B. - Rabies vaccine potency testing: a comparison between the antibody binding test and the NHI test. Symp. Ser. Immunobiol. Stand., 21: 300-307, 1974.
15. FITZGERALD, E.A. & NEEDY, C.F. - Use of the single-radial-immunodiffusion test as a replacement for the NIH mouse potency test for rabies vaccines. In: IABS SYMPOSIUM ON REDUCTION OF ANIMAL USAGE IN THE DEVELOPMENT AND CONTROL OF BIOLOGICAL PRODUCTS. London, 24-26 April 1985. Develop. biol. Stand., 64: 73-97, 1986.
16. KUWERT, E.K.; WERNER, J.; MARCUS, I. & SCHEIERMANN, N. - Introductory remarks: stumbles and heaps in rabies vaccination of man with special regard to the F.R. Germany. In: KUWERT, E.K.; WIKTOR, T.J. & KOPROWSKI, H., ed. Cell culture rabies vaccines and their protective effect in man. Geneva, International Green Cross, 1981. p. 31-51.
17. LAFON, M.; PERRIN, P. & SUREAU, P. - Enzyme-immunoassays (EIA) for potency control of rabies vaccines. In: THRAENHART, G.; KOPROWSKI, H.; BOGEL, K. & SUREAU, P, ed. Progress in rabies control. England, Wells Medical, 1989. p. 315-319.
18. LARGHI, O.P. & NEBEL, A.E. - Rabies virus inactivation by binary ethylenimine: new method for inactivated vaccine production. J. clin. Microbiol., 11: 120-122, 1980.
19. MAYNER, R.E. & NEEDY, C.F. - Evaluation of the single radial immunodiffusion assay for measuring the glycoprotein content of rabies vaccines. J. biol. Stand., 15: 1-10, 1987.
20. ORGANIZACIÓN MUNDIAL DE LA SALUD. Comité de Expertos de la OMS en Rabia. 7º Informe. Ginebra, 1984. Org. Mund. Salud Ser. Inf. Técn., (709), 1984.
21. ROUMIANTZEFF, M.; AJJAN, N.; BRANCHE, R.; FOURNIER, P.; MONTAGNON, B.; TROTEMANN, P. & VINCENT-FALQUET, J.C. - Rabies vaccine produced in cell culture: production, control and clinical results. In: KURSTAK, E., ed. Applied virology. New York, Academic Press, 1984. p. 241-296.
22. SELIGMAN, E.B. - The NIH test for potency. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. Geneva, World Health Organization, 1973. p. 279-286.
23. SCHNEIDER, L. - Aluminium phosphate method for rabies virus purification. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. 3. ed. Geneva, World Health Organization, 1973. v. 1b, p. 179-181.
24. SPIEGEL, M.R. - Teoría de la correlación. In: SPIEGEL, M.R., ed. Estadística. 5. ed. México, Mc Graw-Hill, 1969. p. 247-264.
25. THRAENIIART, O. & RAMAKRISHNAN, K. - Standardization of an enzymed immunoassay for the in vitro potency assay of inactivated tissue cultures rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera. J. biol. Stand., 17: 291-309, 1989.
26. WORLD HEALTH ORGANIZATION - Expert Committee on Biological Standardization. Thirty-first Report, 1981. Wld. Hlth. Org. techn. Rep. Ser., (658), 1981.
27. WORLD HEALTH ORGANIZATION - Report on the inventory of techniques for potency testing of rabies vaccines. Geneva, 1991.

Fechas de Publicación

Publicación en esta colección
03 Jul 2006
Fecha del número
Dic 1993

Histórico

Acepto
22 Jul 1993
Recibido
01 Dic 1992

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

[1] 1. ACHA, P.N. & ARAMBULO, P.V.- Rabies in the tropics. History and current status. In: KUWERT, E., ed. Rabies in the tropics. Berlin, Springer Verlag, 1984. p. 343-359.

[2] 2. ARKO, R.J.; WIKTOR, T.J. & SIKES, R.K. - The antibody binding test for vaccine potency. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. Geneva, World Health Organization, 1973. p. 292-294.

[3] 3. BARTH, R.; GROSS-ALBENHAUSEN, E.; JAEGER, O. & MILCKE, L. - The antibody-binding test, a useful method for quantitative determination of inactivated rabies virus antigen. J. biol. Stand., 9: 81-89, 1981.

[4] 4. BARTH, R.; GRUSCHKAN, H.; JAEGER, O. & MILCKE, L. - Concentration of rabies antigen and potency test. In: KUWERT, E.K.; WIKTOR, T.J. & KOPROWSKI, H., ed. Cell culture rabies vaccines and their protective effect in man. Geneva, International Green Cross, 1981. p. 60-63.

[5] 5. DIAZ, A.M.O. - Rabies neutralizating antibodies determination by the modified counterimmunoelectrophoresis test and the rapid fluorescent focus inhibition test. Zbl. Bakt. Hyg. A., 256: 1-6, 1983.

[6] 6. DIAZ, A.M.O. - The counterimmunoelectrophoresis techniques for "in vitro" detection of antigens in rabies vaccines. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. 4. ed. Geneva, World Health Organization, 1992. (In press).

[7] 7. DIAZ, A.M.O.; ASSAD MILLER, R.; CORTES, M.A.; DELLEPIANE, N.; ELBERGER, D.; FABREGA, F.; MICELI, G.S.; MENDOZA, G.; OBARRIO, M.E.; OCHOA, M.T.; BECCO, O. & PERDOMO, G. - Rabies reference vaccine for use as regional standard for Latin America and the Caribbean Countries. Biologicals, 18: 281-287, 1990.

[8] 8. DIAZ, A.M.O.; NEBEL, A.E. & MICELI, G.S. - A counterimmunoelectrophoresis technique for in vitro detection of antigens in rabies vaccines. In: THRAENHART, O.; KOPROWSKI, H.; BOGEL, K. & SUREAU, P., ed. Progress in rabies control. England, Wells Medical, 1989. p. 305-310.

[9] 9. DIAZ, A.M.O.; PERDOMO, G. & BECCO, O. - Estabilidad de la vacuna antirrábica de cerebro de ratón lactante almacenada a distintas temperaturas. Bol. Ofic.sanit. panamer., 104: 261-271, 1988.

[10] 10. FERGUSON, M.; SEAGROATT, V. & SCHILD, G.C. - Single radial immunodiffusion assays for the standardization of the antigenic content of rabies vaccines. Develop. biol. Stand., 64: 81-86,1986.

[11] 11. FERGUSON, M. & SCHILD, G.C. - A single-radial-immunodiffusion technique for the assay of rabies glycoprotein antigen: application for potency tests of vaccines against rabies. J. gen. Virol., 59: 197-201, 1982.

[12] 12. FERGUSON, M.; WAXHMANN, B.; NEEDY, C.F. & FITZGERALD, E.A. - The effect of strain difference on the assay of rabies virus glycoprotein by single radial immunodiffusion. J. biol. Stand., 5: 73-77, 1987.

[13] 13. FITZGERALD, E.A.; GALLAGHER, M.; HUNTER, W.S. & SELIGMAN Jr., E.B. - Use of the antibody assay in immunized mice for the determination of rabies vaccine potency. In: HENESSEN, W. & REGAMEY, R.H., ed. Developments in biological standardization. Basel, Karger, 1978. v.40, p. 183-186.

[14] 14. FITZGERALD, E.A.; GREEN, O.L. & SELIGMAN Jr., E.B. - Rabies vaccine potency testing: a comparison between the antibody binding test and the NHI test. Symp. Ser. Immunobiol. Stand., 21: 300-307, 1974.

[15] 15. FITZGERALD, E.A. & NEEDY, C.F. - Use of the single-radial-immunodiffusion test as a replacement for the NIH mouse potency test for rabies vaccines. In: IABS SYMPOSIUM ON REDUCTION OF ANIMAL USAGE IN THE DEVELOPMENT AND CONTROL OF BIOLOGICAL PRODUCTS. London, 24-26 April 1985. Develop. biol. Stand., 64: 73-97, 1986.

[16] 16. KUWERT, E.K.; WERNER, J.; MARCUS, I. & SCHEIERMANN, N. - Introductory remarks: stumbles and heaps in rabies vaccination of man with special regard to the F.R. Germany. In: KUWERT, E.K.; WIKTOR, T.J. & KOPROWSKI, H., ed. Cell culture rabies vaccines and their protective effect in man. Geneva, International Green Cross, 1981. p. 31-51.

[17] 17. LAFON, M.; PERRIN, P. & SUREAU, P. - Enzyme-immunoassays (EIA) for potency control of rabies vaccines. In: THRAENHART, G.; KOPROWSKI, H.; BOGEL, K. & SUREAU, P, ed. Progress in rabies control. England, Wells Medical, 1989. p. 315-319.

[18] 18. LARGHI, O.P. & NEBEL, A.E. - Rabies virus inactivation by binary ethylenimine: new method for inactivated vaccine production. J. clin. Microbiol., 11: 120-122, 1980.

[19] 19. MAYNER, R.E. & NEEDY, C.F. - Evaluation of the single radial immunodiffusion assay for measuring the glycoprotein content of rabies vaccines. J. biol. Stand., 15: 1-10, 1987.

[20] 20. ORGANIZACIÓN MUNDIAL DE LA SALUD. Comité de Expertos de la OMS en Rabia. 7º Informe. Ginebra, 1984. Org. Mund. Salud Ser. Inf. Técn., (709), 1984.

[21] 21. ROUMIANTZEFF, M.; AJJAN, N.; BRANCHE, R.; FOURNIER, P.; MONTAGNON, B.; TROTEMANN, P. & VINCENT-FALQUET, J.C. - Rabies vaccine produced in cell culture: production, control and clinical results. In: KURSTAK, E., ed. Applied virology. New York, Academic Press, 1984. p. 241-296.

[22] 22. SELIGMAN, E.B. - The NIH test for potency. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. Geneva, World Health Organization, 1973. p. 279-286.

[23] 23. SCHNEIDER, L. - Aluminium phosphate method for rabies virus purification. In: KAPLAN, M.M. & KOPROWSKI, H., ed. Laboratory techniques in rabies. 3. ed. Geneva, World Health Organization, 1973. v. 1b, p. 179-181.

[24] 24. SPIEGEL, M.R. - Teoría de la correlación. In: SPIEGEL, M.R., ed. Estadística. 5. ed. México, Mc Graw-Hill, 1969. p. 247-264.

[25] 25. THRAENIIART, O. & RAMAKRISHNAN, K. - Standardization of an enzymed immunoassay for the in vitro potency assay of inactivated tissue cultures rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera. J. biol. Stand., 17: 291-309, 1989.

[26] 26. WORLD HEALTH ORGANIZATION - Expert Committee on Biological Standardization. Thirty-first Report, 1981. Wld. Hlth. Org. techn. Rep. Ser., (658), 1981.

[27] 27. WORLD HEALTH ORGANIZATION - Report on the inventory of techniques for potency testing of rabies vaccines. Geneva, 1991.

Brasil

Brasil

Evaluacion de la tecnica de contraimmunoelectroforesis para determinar la potencia antigena de las vacunas antirrabicas

Evaluation ot the counterimmunoelectrophoresis technique to determine the antigenic potency of antirrabic vaccines

Resúmenes

Fechas de Publicación

Histórico