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Archive: 1999 Session - Appendix 17

1999 Session of the Research Group of the Standing Technical Committee of EuFMD



Need for further Standardization of the 146-S test as the Basis for final Foot-and-Mouth Disease (FMD) Vaccine Formulation
(discussion topic)


S.J. Barteling, ID-Lelystad, P.O. Box 65, AB, Lelystad

For more than 20 years FMD vaccine producers and international vaccine banks use the 146 S test (quantitative sucrose density gradient analyses; ref. 1, 2, 3) to quantify the virus antigen concentrations and, on that basis, formulate their vaccines. Therefore, it is very important that the results of this test refer to international standards. This is not the case.

Between 1978 and 1982 phase III, IV and V of the FAO International Collaborative Study for the Standardization of Methods used in FMD-laboratories were dedicated to this problem.

The first effort (phase III) showed up to 10-fold differences between the laboratories. After an analysis of the equipment and of the way the surface of the 146 S peaks were used to calculate the antigen concentration, the results of the next phase (phase IV) were a lot better. Most results were pretty well comparable, however, in some cases still a twofold difference was observed.

In phase V a series of adenine preparations were used to assess the UV monitors. MS2 ribophage standards were used to assess the sucrose gradient analysis. It was demonstrated by close cooperation between 2 laboratories that meaningful results could be obtained (6). However, the efforts did not really result in a system of international test evaluation using international standards.

Integration of the 146 S peak by a computer (3), as is nowadays general practice in most production laboratories, certainly will reduce the variation further, however, will not overcome the problem of 146 S aggregates that are spinned down to the bottom of the centrifuge tube.

Lei (2) reported considerable "masking" of 146 S antigen in harvest from BHK roller bottle monolayers. Yields in terms of 146 S increased considerably - in some cases more than a two-fold - if samples were pre-treated with 10 mM EDTA, with 0.5 % of chloroform, or with 0.5 % of one of the detergents TEGO 51 or Empigen 88.

Very likely aggregates are also the cause of the variation observed for the 146 S antigen content of antigen stocks of the International Vaccine Bank at Pirbright (5).

In Onderstepoort (S. Africa) they face similar problems with their antigen stocks (table 1). Therefore, a new effort of international standardization must also encompass a method that disaggregates complexes of 146 S particles and thus works for all FMD antigen preparations.



146 S before storage

146 S after storage























68 (calculated)

55 (observed), 75 calc.

Mixture after chloroform




  • Barteling, S.J. and Meloen, R.H., 1974. A simple method for the quantification of 140 S particles of foot-and-mouth disease virus. Arch. Ges. Vrusforsch., 45, 362.
  • Lei, J.C., 1979. Comparative investigation of virus content in samples of foot-and-mouth disease virus from BHK 21 suspension cells by sedimentation rate and density equilibrium ultra-centrifugation. Rep. Meeting Research Group Standing Techn. Comm. Eur. Comm. Contr. Foot-and-mouth Disease. Uccle, Belgium, FAO Rome, pp 67-78.
  • Doel, T.R., Fletton, B., and Staple, R.F., 1982. Further developments in the quantification of small RNA viruses by UV photometry of sucrose density gradients. Dev. Biol. Stand. 50, 209.
  • FAO international Collaborative study for the standardisation of laboratory methods used for foot-and-mouth disease vaccine preparation and control. Preliminary report of the results of Phases III and IV.
  • Rep. Meeting Research Group Standing Techn. Comm. Eur. Comm. Contr. Foot-and-mouth Disease. Vienna, Austria, FAO Rome, pp 49-52.
  • Barnett, P.V. and Statham, R.J. 1998. Long term stability and potency of antigen concentrates held by the International Vaccine Bank. Rep. Meeting Research Group Standing Techn. Comm. Eur. Comm. Contr. Foot-and-mouth Disease. Aldershot, U.K., FAO Rome, pp 272-275.