RESUMO
Solid phase enzyme-immunoassay (EIA) was employed to assess the antigenic reactivity of matrix protein (M) and nucleoprotein (NP) of influenza A virus adsorbed to polystyrene in the presence of different detergents such as beta-octaglucoside (OG), Triton X-100, Tween-20, sodium dodecylsulphate (SDS), sodium deoxycholate (Doch-Na), Nonidet P-40 (NP-40), and sarcosyl at concentrations ranging from 0 to 2%. The antigenic reactivity of NP was the highest in the absence of detergents. For M protein, Doch-Na, SDS, NP-40 and sarcosyl of 0.05-0.1% enhanced the chromatophoric response in EIA 1.5-2 times. In contrast, the antigenic reactivity of M protein remained unchanged after OG or Triton X-100 treatments, and it decreased in the presence of Tween-20.
Assuntos
Antígenos Virais/imunologia , Detergentes , Vírus da Influenza A/imunologia , Nucleoproteínas/análise , Nucleoproteínas/imunologia , Proteínas de Ligação a RNA , Tensoativos , Proteínas do Core Viral , Proteínas da Matriz Viral/imunologia , Proteínas Virais/imunologia , Adsorção , Epitopos/análise , Epitopos/imunologia , Técnicas Imunoenzimáticas , Vírus da Influenza A/efeitos dos fármacos , Proteínas do Nucleocapsídeo , Nucleoproteínas/isolamento & purificaçãoRESUMO
Enzyme-linked immunosorbent assay (ELISA) has been adopted for simultaneous determination of the levels of antibodies to different influenza virus proteins in human sera with known haemagglutination-inhibition (HI) titre. Whole virus of serotypes H1N1 and H3N2, haemagglutinin (HA), matrix (M) and nucleoprotein (NP) proteins have been used as antigens. For detection of antibodies bound to the antigen, peroxidase labelled Staphylococcus protein A conjugate has been used. Correlation of the ELISA and HI titres of anti-HA antibody has been demonstrated. The use of isolated HA as antigen increased the specificity of ELISA. The analysis of human reconvalescent sera has shown that increase in the titre of antibodies to internal proteins does not always coincide with the increase of antibody level to HA. Out of 8 sera with significant increase of the HI titre to the H3 subtype 5 specimens showed 4-fold increase of antibody titre to NP protein. The antibody titre to M protein was elevated in 2 sera only, while 1 serum showed no rise of antibody response to the tested viral proteins.
Assuntos
Anticorpos Antivirais/análise , Hemaglutininas Virais/imunologia , Vírus da Influenza A/imunologia , Proteínas do Core Viral/imunologia , Proteínas da Matriz Viral/imunologia , Adolescente , Adulto , Animais , Embrião de Galinha , Ensaio de Imunoadsorção Enzimática , Testes de Inibição da Hemaglutinação , Humanos , Vírus da Influenza A/classificação , Influenza Humana/diagnóstico , Masculino , Testes Sorológicos , Sorotipagem , Fatores de TempoRESUMO
ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.
Assuntos
Antígenos Virais/isolamento & purificação , Vírus da Influenza A/imunologia , Nucleoproteínas/isolamento & purificação , Proteínas de Ligação a RNA , Ribonucleoproteínas/isolamento & purificação , Proteínas do Core Viral , Proteínas Virais/isolamento & purificação , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Centrifugação com Gradiente de Concentração , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Proteínas do Nucleocapsídeo , Nucleoproteínas/imunologia , Coelhos , Ribonucleoproteínas/imunologia , Proteínas Virais/imunologia , Vírion/imunologiaRESUMO
A test-system was developed on the basis of solid-phase enzyme-immunoassay using protein A/peroxidase conjugate for the determination of antibody levels to influenza virus in sera of humans who had experienced a natural infection or received a live influenza vaccine. The accurate observation of the test conditions was demonstrated to give the results well correlating with those of the HI test. The use of isolated hemagglutinin as the antigen considerably increased the specificity of the enzyme-immunoassay and in a number of cases detected a 4-fold or higher rise of antibody titres to hemagglutinin in paired sera of the vaccinees where the HI test showed no rise in antibody titres.
