[Development of an immunoenzyme test system for the quantitative demonstration of influenza virus hemagglutinin in biological specimens]. / Razrabotka immunofermentnoi test-sistemy dlia kolichestvennoi indikatsii gemaggliutinina virusa grippa v biologicheskikh obraztsakh.

A test system for the detection and quantitation of influenza virus hemagglutinin (HA) in biological specimens by solid-phase enzyme immunoassay (SPEIA) is described. The test system implies sequential adsorption on polystyrene base of anti-HA guinea pig gammaglobulins, detectable HA-containing antigen, monospecific anti-HA rabbit serum, and peroxidase antirabbit conjugate. Adsorption of the HA-containing antigen is run in the presence of a high concentration of nonionic detergent. The developed method is highly sensitive (0.3-0.5 ng in a specimen) and permits the detection and quantitation of HA in whole influenza virions, preparations of surface glycoproteins and in preparations of hemagglutinin recovered from virus particles. The analysis (after pre-sensitization of the polystyrene adsorbent) takes from 2 to 21/2 hours. The possibility of using the developed test system for control and standardization of inactivated influenza vaccines (whole-virion, split, and subunit) is discussed.

[Isolation of preparative amounts of influenza virus hemagglutinin by an affinity chromatographic method]. / Vydeelnie preparativnykh kolichestv gemaggliutinina virusa grippa s ispol'zovaniem metoda affinnoi khromatografii.

The use of affinity column sepharose-tyrosine-sulfanilic acid permits one to obtain preparative amounts of pure hemagglutinin of the types H1, H2, H3 from the total amount of surface glycoproteins solubilized by octylglycoside from antigenically different strains of influenza A virus. The yield of hemagglutinin ranges from 30% to 84% depending on the virus strain.

[Characteristics of isolating influenza virus hemagglutinin using purified bromelin]. / Osobennosti vydeleniia gemaggliutinina virusa grippa s pomoshch'iu ochishchennogo bromelina.

Gel filtration and ion-exchange chromatography were used to isolate a fraction with the highest proteolytic activity and virtually lacking glycosidases from a commercial bromeline preparation ("Serva", grade B) showing, in addition to proteolytic, also glycosidase activity. The utilization of purified bromeline allows isolation of hemagglutinin without residual neuraminidase activity from influenza virus of various serotypes (H1, H1, and H3). The results are discussed on the basis of an assumption of analogous localization but different conformation accessibility of bromeline-sensitive parts of the light chains of influenza viruses of different subtypes, and a possible role of the carbohydrate component of surface glycoproteins, hemagglutinin and neuraminidase of influenza virus virions.

Influenza virus haemagglutinin: estimation of tryptophan and tyrosine content and localization of tryptophan residues.

The tryptophan and tyrosine content of the bromelain-released subtype H3 haemagglutinin (H3 BHA) of influenza virus were measured by u.v. absorption and fluorescence techniques. The values obtained (8 and 18 residuces, respectively) are in close agreement with those derived from amino acid analysis. Essentially all of th tryptophan residues are demonstrated to be localized on the surface of the BHA molecule.

[Hemagglutinin of influenza virus type H3: conformation in solution]. / Gemaggliutinin tipa H3 virusa grippa: konformatsiia v rastvore.

The method of circular dichroism was used to evaluate the conformation parameters of the secondary structure of the molecule of isolated influenza virus hemagglutinin H3. Comparison of the data obtained with the similar known parameters for hemagglutinin H2 has shown significant differences in the secondary structure, which are, probably, responsible, (at the level of a higher structural organization) for the variations in the antigenic specificity of hemagglutinins H2 and H3. Temperature and pH stability of alpha-spiral areas of the hemagglutinin H3 molecule was studied, and the direct participation of aromatic amino acid residues in formation of the alpha-spiral areas was shown. The native state of the hemagglutinin alpha-spiral areas may serve as a factor determining potential hemagglutination.

[Influenza virus hemagglutinin: hydrodynamic properties and molecular weight]. / Gemaggliutinin virusa grippa: gidrodinamicheskie svoistva i molekuliarnaia massa.

Hydrodynamic properties of hemagglutinin isolated with bromelain from influenza virus were studied. The sedimentation constant determined by the method of analytical ultracentrifugation was 9.17 +/- 0.17 S. The translation diffusion coefficient estimated by the method of optic mixing spectroscopy comprised (3.6 +/- 0.1) X 10(+7) cm2/s. A comination of both the constants in Svedberg's equation allowed calculation of the hemagglutinin molecular weight (approximately 230,000) which suggests a three-subunit structure of the molecule. The hydrodynamic diameter and friction ratio of an isolated molecule were calculated.

Structure of bromelain-released influenza virus haemagglutinin as revealed by electrophoresis, sedimentation and electron microscopy.

Sigma bromelain (EC 3.4.22.4) was used to isolate the haemagglutinin (HA) from the MRC-11 (H3N2) and A/U.S.S.R./90/77 (H1N1) influenza A virus strains. Sedimentation analysis of bromelain-solubilized preparations revealed 9.5S and 5.5S protein components, the former being identified as the bromelain-released haemagglutinin (BHA). No residual neuraminidase (NA) activity was detected in the BHA isolated from the MRC-11 strain whereas up to 80 per cent of the enzymatically active NA was found to be preserved in the electrophoretically pure BHA isolated from the A/U.S.S.R./90/77 strain. Increased electrophoretic mobilities were exhibited by both the light and heavy chains of the BHA subunit. The difference observed in the molecular weights of the polypeptide fragments removed by bromelain from the light chains is interpreted in terms of the different depth of penetration of antigenically distinct HAs through the influenza virus lipid membrane. Splitting off of approximately 15 and 26 per cent of the sugars from the carbohydrate portions of the light and heavy chains respectively, was demonstrated. This suggested involvement of glycosidase impurities present in the bromelain preparation employed. The rod-shaped BHA molecules proved to be 110 +/- 5 Angstrom long and 40 +/- 5 Angstrom wide as measured by electron microscopy. It is proposed that the 45,000-molecular-weight polypeptide observed constantly in egg-grown influenza viruses is host actin.