Inhibition of influenza virus haemolytic and haemagglutination activities by monoclonal antibodies to haemagglutinin glycopolypeptides HA1 and HA2.

Acid treatment of influenza virus enhanced haemagglutination inhibiting (HI) activity of some anti-HA1 monoclonal antibodies (MoAbs). These changes in the HI-activity could be either due to alteration in the mutual orientation of MoAb (e.g.

Changes in the influenza virus haemagglutinin at acid pH detected by monoclonal antibodies to glycopolypeptides HA1 and HA2.

Monoclonal antibodies (Mabs) specific to the HA1 and HA2 subunits of the influenza virus haemagglutinin (HA) were used to show that changes in the antigenicity of the HA molecule at acid pH involve both HA subunits. In solid phase RIA (intact virus adsorbed) the acid-induced change was detected in the form of greatly increased binding of anti-HA 1 Mabs (IVA 1 and IVG 6) and anti-HA2 Mab (IIF 4). This increased binding could be most probably explained by alterations in accessibility of epitopes to the corresponding Mabs.

Monoclonal antibodies to glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin.

Anti-haemagglutinin monoclonal antibodies were prepared and their HA1 or HA2 specificity was determined by solid phase radioimmunoassay (RIA) using purified viral haemagglutinin (HA) and haemagglutinin glycopolypeptides HA1 and HA2, by radioimmunoprecipitation followed with SDS-PAGE, by immunoblotting and by inhibition of virus-induced haemagglutination. The capacity of these methods to estimate HA1 or HA2 specificity of anti-HA monoclonal antibodies (MoAb) was compared. HA1 specificity was demonstrated for all hybridomas originating from lymphocytes of mice immunized with complete influenza virus, except IIF4 hybridoma which was HA2-specific.

Analysis of influenza A virus neuraminidase using lectin test and monoclonal antibodies.

Influenza viruses causing epidemics in the U.S.S.

A simple and rapid characterization of influenza virus isolates by monoclonal antibodies in radioimmunoassay.

Radioimmunoassay (RIA) with infectious allantoic fluid directly bound to solid phase, suitable for detection and further characterization of influenza virus isolates, is described. This simple and rapid method was applied for description of isolates obtained from different regions of Czechoslovakia during influenza epidemic in 1983. The results confirmed that all 13 examined isolates represent influenza A viruses possessing H3 subtype haemagglutinin very similar to haemagglutinin of influenza viruses A/Bangkok/1/79 (H3N2), A/Belgium/2/81 (H3N2) and A/Philippines/2/82 (H3N2), respectively.

Characterization of influenza A-1983 epidemic strains by polyclonal and monoclonal antibodies and detection of two co-circulating antigenic variants.

Influenza virus strains isolated during 1985 epidemic in Czechoslovakia proved to be antigenically closely related to A/Bangkok/79, A/Philippines/2/83 and A/Texas/77 (all H3N2) viruses, if examined in haemagglutination inhibition (HI) tests with standard polyclonal antisera. If examined in HI tests with monoclonal antibody (MAb) IIB4, the virus isolates could be separated into two groups: those reacting to high titres (about two thirds of the isolates) and those negative with IIB4 (titre of less than 20; rest of the strains). A relationship to MAb IIB4 similar to that of freshly isolated A-H3 influenza virus strains was found with prototype strains A/Belgium/2/81 (highly positive with IIB4, HI titre up to 20 000 per 0.

Use of monoclonal antibodies against avian retroviral protein p19 for competitive radioimmunoassay and immunodiffusion.

Monoclonal antibodies were used in competitive binding assays to investigate the arrangement of three epitopes on the protein p19 of avian myeloblastosis virus (AMV). It was reasoned that if the epitopes recognized by two monoclonal antibodies are physically close, the binding of one antibody will sterically block the binding of the second; conversely no blocking will occur if the epitopes are sufficiently distant. The results of these competitive binding assays demonstrated the presence of two distinct antigenic sites on the protein p19.

Radioimmunoassay of influenza A virus haemagglutinin. II. Antigenic cross-reactions of influenza A (H3 subtype) viruses as determined by radioimmunoassay and haemagglutination inhibition tests.

Individual rabbits differed greatly in their antibody response to the "strain-specific" and "cross-reactive" antigenic determinants on the haemagglutinin (HA) subunit of influenza virus recombinant MRC11 (H3N2) and influenza virus Dunedin (H3N2), after immunization with whole virus or bromelain-released haemagglutinin (B-HA). Consequently, diverse cross-reactions between htese viruses and A/Hong Kong/68 virus were found in the haemagglutination inhibition (HI) test as well as in homologous radioimmunoassay (125I-B-HA from MRC11:anti MRC11 serum, and 125I-B-HA from Dunedin: anti Dunedin serum) when sera from different animals were employed. Radioimmunoassay (RIA), over and above to the HI test, was able to differentiate clearly the respective HAs also with antisera reacting to the same HI titre with both corresponding influenza virus strains.

Use of Staphylococcus aureus for rapid radioimmunoassay of influenza A virus haemagglutinin.

In a rapid method for the radioimmunoassay (RIA) of influenza A virus haemagglutinin, Staphylococcus aureus (strain Cowan I, Czechoslovak State Collection No Mau 55/64) was used for separation of bound and free antigens. With rabbit and human immune sera, the binding of antigen-antibody complexes to heat-killed, formalin-fixed staphylocci was comparable to the double antibody technique. The time required for the completion of binding reaction was about 10 min compared to 18--24 hr required for double antibody precipitation.

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. IV. Immunogenic properties of separated haemagglutinin glycopolypeptides.

Highly purified haemagglutinin glycopolypeptides HA1 and HA2 were effective in eliciting an antibody response. HA2 had a markedly greater immunogenic potential than HA1. In gel double immunodiffusion, sera from rabbits immunized with HA2 produced more distinct precipitin lines than sera obtained by immunization with HA1.

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. III. Reactivity with human convalescent sera.

The immune reactivity to both haemagglutinin glycopolypeptides HA1 and HA2 [prepared from bromelain-released haemagglutinin of influenza virus A/Dunedin/4/73 (H3N2)], was demonstrated by both gel double immundiffusion and radioimmunoassay in human convalescent sera obtained after natural infection during influenza epidemics in 1974/75 and 1976/77. In gel double immunodiffusion, the precipitin line(s) corresponding to glycopolypeptide HA1 were always more distinct than precipin line(s) corresponding to glycopolypeptide HA2. In radioimmunoassay, human convalescent sera revealed higher titres for binding of 125-I-labelled HA2 than for 125-I-labelled HA1.

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. II. Reactivity with rabbit sera against intact virus and purified undissociated haemagglutinin.

Rabbit sera produced against either intact virus or purified undissociated haemagglutinin were examined for reactivity with highly purified haemagglutinin glycopolypeptides. Sensitive radioimmunoassay for 125I-labelled glycopolypeptides revealed antibody reactive with either glycopolypeptide HA1, or glycopolypeptide HA2. Antibodies against the carbohydrate moiety were responsible only for a part of the binding activity.

Improved detection by immunodiffusion of type-specific influenza antibody in avian sera.

Various solvents and kinds of agar and agarose as well as various ribonucleoprotein (RNP) antigen preparations were tested in a search for optimal conditions for the detection of low levels of type-specific influenza antibody in avian sera by gel double diffusion. The best results were obtained with one kind of agarose in a solvent with lowered ionic strength (approx. 0.

Antigenic glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. I. Gel filtration in 6 M guanidine hydrochloride.

Highly purified glycopolypeptides HA1 and HA2 were separated from bromelain-released haemagglutinin of influenza virus A/Dunedin/4/73 (H3N2) by gel filtration in 6 M guanidine hydrochloride under reducing conditions. The purity of both glycopolypeptides was proved by extensive studies. Despite the lack of C-terminal end, the isolated HA2 glycopolypeptide displayed some hydrophobic properties.

Radioimmunoassay of influenza A virus haemagglutinin. I. Preparation and properties of radioactive 125I-labelled bromelain-released haemagglutinin.

Haemagglutinin released from influenza A virus recombinant MRC11 [antigenically identical to the strain A/Port Chalmers/1/73 (H3N2)] by bromelain treatment and purified by rate zonal centrifugation (further on B-HA) was examined for eventual contamination by neuraminidase. According to specific enzymatic activities corresponding to MRC11 virus and B-HA alone respectively, B-HA contained less than 0.1% of enzymatically active neuraminidase orginally present in the virus.

Evaluation of the effectiveness of receptor destroying enzyme preparations.

A procedure has been developed for testing receptor destroying enzyme (RDE) preparations used to remove nonspecific inhibitors before carrying out haemagglutination inhibition tests with influenza virus. Four criteria should be taken into account: (1) titre of RDE, usually used to indicate the activity of the preparation; (2) neuraminidase activity of the preparation, determined biochemically, which partially corresponds to the RDE titre; (3) direct demonstration of the complete removal of nonspecific inhibitors; and (4) determination that the RDE preparation does not affect specific antibody.

Some biological activites of rabbit anti-interferon serum.

After prolonged immunization of rabbits with a semipurified mouse interferon preparation in Freund-incomplete and/or Al-Span-Oil-adjuvant a specific interferon neutralizing immunoglobulin was obtained from antiserum. The specific activity of the antiserum and immunoglobulin was confirmed in tests in which the interaction of antibodies with the cell-surface was ruled out. The antiserum (and the immunoglobulin) neutralized both the anti-viral and the cell-growth inhibitory activity of interferon.

A competitive-inhibition radioimmunoassay for influenza virus envelope antigens.

A double-antibody competitive-inhibition radioimmunoassay for influenza virus envelope antigens is described. A viral antigen preparation from influenza A virus recombinant MRC11 [antigenically identical to A/Port Chalmers/1/73 (H3N2)] consisting of haemagglutinin and neuraminidase was labelled with radioiodine. Rabbit antisera were allowed to react with the labelled antigen and the resultant antigen-antibody complexes were precipitated with the appropriate antiglobulin.

Some biological activities of rabbit anti-interferon serum.

After prolonged immunization of rabbits with a semipurified mouse interferon preparation in Freund's incomplete or Al-Span-Oil adjuvant, a specific interferon-neutralizing immunoglobulin was obtained from antiserum with a capacity of neutralizing about 49000 mouse interferon units per ml. The specific activity of the antiserum and immunoglobulin was confirmed in tests in which the interaction of antibodies with the cell surface was ruled out. The antiserum (and the immunoglobulin) neutralized both the antiviral and the cell-growth inhibitory activities of interferon.

[Preparation of porcine IgA and its detection using the fluorescent antibody technic].

The preparation of immunoglobulin A (IgA) from porcine colostrum, intestinal content and serum is described. The best results were achieved with colostrum, from which an antigen of satisfactory purity was prepared by purification on Sephadex G-200, on DEAE cellulose and subsequent filtration on Sephadex G-200. The serum to this antigen raised in rabbits was adsorbed to an immunoadsorbent from porcine serum (PS) or porcine IgG.

[Study of anti-T-agglutinins in active and inactivated human sera (author's transl)].

Anti-T-agglutinins were found in 252 of 283 human sera investigated applying the two-step neuraminidase test and using an RDE-preparation of czechoslovakian origin. Storage and heating of the sera was followed by a reduction or loss of T-agglutination dependent on the time of incubation at 4 degrees C and 56 degrees C. This was proved to be caused by the inactivation of the anti-T-agglutinins and not by the loss of complement.