Haemagglutinin of measles virus: purification and storage with preservation of biological and immunological properties.

Measles virus envelope haemagglutinin (H) was purified rapidly with Triton X-100-solubilized virions by a two-step anion-exchange chromatography using fast protein liquid chromatography. The purity of the glycoprotein in its dimeric form was demonstrated by SDS-PAGE followed by silver staining or autoradiography. The purified H glycoprotein was further freed from contaminating detergent by dialysis of octylglucoside detergent. This purification procedure, together with subsequent lyophilization and storage at -70 degrees C of the H glycoprotein which was incorporated into phospholipid vesicles allowed the full preservation of its haemagglutinating activity, its reactivity with a monoclonal anti-H antibody that recognized a conformational epitope and its capacity to elicit anti-H antibodies with haemagglutination-inhibiting and neutralizing activities.

The British reference preparation for influenza virus haemagglutinin.

The two methods for measuring the haemagglutinin content of an influenza virus suspension are the haemagglutinating (HA) and chick cell agglutinating (CCA) techniques and both measure the same biological activity. With the establishment of an international reference preparation for influenza virus haemagglutinin (type A), however, it seems logical to express the haemagglutinin content of influenza vaccines in international units. Accordingly a collaborative study was arranged in order to obtain agreement on the number of units to be assigned to a British reference preparation for influenza haemagglutinin. It was agreed that the preparation contains 190 i.u. per ampoule and 1 i.u. is contained in 0.0622 mg. of the dried material.

The international reference preparation of influenza virus haemagglutinin (type A).

This paper describes the international collaborative assay that led to the establishment in 1967 of the International Reference Preparation of Influenza Virus Haemagglutinin (Type A) and the studies completed during the following years on the use of the preparation for evaluating the haemagglutinin content of 46 influenza virus vaccines in terms of international units. The WHO Expert Committee on Biological Standardization (1967) defined the International Unit as 0,09361 mg of the International Reference Preparation.Altogether 14 laboratories in 12 countries took part in one or both studies, using a total of 24 methods (HA titrations and, in a few cases CCA titrations). Major differences in the HA titres were found between laboratories, while the potencies (the haemagglutinin content values) relative to the International Reference Preparation were free from most of these differences. Haemagglutination titres varied over a range factor up to 50, while the corresponding relative "potencies" varied with a factor of only 2. The CCA method used in a few laboratories gave results close to the lowest haemagglutination titres and showed relatively small variations between laboratories. The analyses of variance disclosed differences in the variation within laboratories, but for the majority of the laboratories the variation allowed an overall estimate of a standard error.The calculation of haemagglutinin content (in IU) from relative potencies is described. Advice is given on the selection, preparation, and titration of a local reference vaccine with a view to expressing its haemagglutinin content in international units.The test results with 46 local vaccines are also given. The deviations of the relative potencies from the average per vaccine showed a distribution with eight major discrepancies instead of the expected one. The background for these cases is discussed.