RESUMO
A test kit as an immunochip designed for the diagnosis of hepatic C virus (HCV) has a high sensitivity and specificity. Recombinant HCV antigens were separately immobilized on the activated slides together with internal controls. Serum test results were red by ScanArray Express. K-factor and corresponding value of cut-off were calculated for each antigen and internal controls. Comparative evaluation of the sensitivity and specificity of the immunochip was carried out by commercial ELISA test kits and linear blotting analyses on 448 blood samples containing and free from NCV antibodies.
Assuntos
Anticorpos Anti-Hepatite C/sangue , Hepatite C/diagnóstico , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Análise em Microsséries , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
AIM: Development of test-system on the basis of immunochip for detection of IgG to Treponema pallidum. MATERIALS AND METHODS: Recombinant T. pallidum antigens Tp47, Tp17, Tp15, TmpA were separately immobilized on activated slides as individual spots. The specific antibodies to T. pallidum in human serum were detected by indirect immunofluorescent assay on the immunochip using antispecies antibodies to human IgG. Fluorescent scanner was used to read results of testing. For each antigen threshold level of fluorescent signal and positivity coefficient were calculated. Assessment of specificity and sensitivity of the immunochip was performed on 400 human serum samples containing or not-containing antibodies to T. pallidum. RESULTS: From 200 positive serum samples 5 were interpreted as inconclusive because positive results on antigen Tp17 only were registered that was comparable with the results of immunoblotting. For each other 195 serum samples, positive result on 2 or more antigens was obtained. Specificity of the immunochip-based test-system was 100%. CONCLUSION: Experimental immunochip-based test-system for differential serologic diagnostics of the syphilis was developed. Its sensitivity and specificity are comparable with that of ELISA-based test-system.
Assuntos
Anticorpos Antibacterianos/sangue , Ensaio de Imunoadsorção Enzimática , Análise Serial de Proteínas/métodos , Sorodiagnóstico da Sífilis/métodos , Sífilis/diagnóstico , Treponema pallidum/isolamento & purificação , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/imunologia , Diagnóstico Diferencial , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Sífilis/sangue , Treponema pallidum/imunologiaRESUMO
Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.
Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Aldeídos/química , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , DNA/química , DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Eletroforese em Gel de Poliacrilamida , Modelos Químicos , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
A rapid and effective method of testing of a repair enzyme, uracil-DNA-glycosylase, was proposed. As a substrate, a deoxyuridine-containing 5'-32P-labeled deoxyoligonucleotide covalently attached to a polystyrene support (Tenta Gel S-NH2) was used. The ammonia cleavage of the apyrimidine site formed in the enzymic reaction followed by the transition of the labeled oligonucleotide fragment from the solid phase into solution allowed the detection of the enzymic activity.