RESUMO
The OOO "Research-and-Production Association "Diagnostic Systems" has developed a "DC-EIA-HIV-AT/AG-SPECTRUM" screening enzyme immunoassay system designed to detect separately antibodies to certain HIV-1 and HIV-2 proteins, as well as antigen p24. The determination of antibodies of all classes and the marker of early-stage infection antigen p24 with a high (5 pg/ml) sensitivity substantially reduces the number of void results obtained in the use of immunoblots. The developed "DC-EIA-HIV-AT/AG-SPECTRUM" plate system is an effective tool to support positive screening results and may be used at the final stage of laboratory diagnosis of HIV infection.
Assuntos
Anticorpos Anti-HIV/sangue , Infecções por HIV/sangue , HIV-1 , HIV-2 , Antígenos Virais/química , Antígenos Virais/imunologia , Biomarcadores/sangue , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/química , HIV-1/imunologia , HIV-2/química , HIV-2/imunologia , Humanos , Técnicas Imunoenzimáticas/métodos , Técnicas Imunoenzimáticas/normas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.
Assuntos
Epitopos/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/sangue , HIV-1 , Peptídeos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/diagnóstico , Infecções por HIV/genética , Infecções por HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Humanos , Técnicas Imunoenzimáticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Analysis of the sequence of a number of proteins of the virus causing severe acute respiratory syndrome (SARS-CoV) was used to theoretically predict antigenic epitopes. Nine sequences, encoding for the predicted epitopes in spike, nucleocapside, and membranous SARS CoV proteins, were synthesized by polymyrase chain reaction and cloned into the pGEX 4-T2 vector. The resultant plasmids were expressed in the E. coil cells and purified by glutathione-Sepharose affinity chromatography.
