RESUMEN
Detection of hepatitis B e antigen (HBeAg) in the sera of individuals infected with hepatitis B virus (HBV) can indicate both a high infectivity of the disease and a poor prognosis of disease treatment. Most of monoclonal antibodies raised against HBV e proteins interact with immuno-dominant epitopes, such as HBeAg-beta. In order to raise antibodies against non-dominant epitopes of HBV e protein, in this study, mice were immunized with both recombinant HBeAg (rHBeAg) and an anti-HBeAg antibody (EWB) recognizing a dominant antigenic epitope of HBeAg (HBeAg-beta epitope). With this strategy, we successfully selected two monoclonal antibodies, S-29-3 and S-72-3. Both S-29-3 and S-72-3 bind to recombinant HBeAg with a high affinity. The epitope mapping assay determined that the S-73-2 recognizes the N-terminal of HBeAg (1-118 aa) and the S-29-3 recognizes the C-terminal of HBeAg (91-149 aa). Further experiment showed that these two antibodies could be formed a pair-Abs that is used in detecting native HBeAg from the sera of HBV patients. The conclusion is that the developed method is useful to raise mAb against non-dominant epitopes in given Ag.
Asunto(s)
Epítopos/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Animales , Anticuerpos Monoclonales , Afinidad de Anticuerpos , Complejo Antígeno-Anticuerpo , Antígenos e de la Hepatitis B/genética , Antígenos e de la Hepatitis B/aislamiento & purificación , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunas ViralesRESUMEN
Severe acute respiratory syndrome-coronavirus (SARS-CoV) causes an infectious disease through respiratory route. Diagnosing the disease effectively and accurately at early stage is essential for preventing the disease transmission and performing antiviral treatment. In this study, we raised monoclonal antibodies (mAbs) against the nucleocapsid (N) protein of SARS-CoV and mapped epitopes by using different truncated N protein fragments. The mapping of those epitopes was valuable for constructing pair-Abs used in serological diagnosis. The results showed that all of the six raised mAbs were divided into two groups recognizing the region of amino acids 249-317 (A group) or 317-395 (B group). This region spanning amino acids 249-395 contains predominant B cell epitopes located at the C-terminus of N protein. One pair-Abs, consisting of N protein-specific rabbit polyclonal antibody and SARS-CoV N protein-specific mAb, was selected to construct a sandwich ELISA-kit. The kit was able to specifically detect SARS-CoV N proteins in serum samples.
Asunto(s)
Anticuerpos Monoclonales/química , Epítopos/química , Proteínas de la Nucleocápside/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Proteínas de la Nucleocápside de Coronavirus , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Conejos , Sensibilidad y Especificidad , Síndrome Respiratorio Agudo Grave/sangreRESUMEN
The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.
Asunto(s)
Péptidos/uso terapéutico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Proteínas Virales/química , Secuencia de Aminoácidos , Western Blotting , Biología Computacional , Humanos , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus. It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection. Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2. In contrast, TGV, FIPV and HCoV-229E are not. Many studies have shown that the cleavage of spike protein seriously affects its function. In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs. Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E.coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis. Furthermore, the anti-S1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation. When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis. The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells.
Asunto(s)
Coronavirus/inmunología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Síndrome Respiratorio Agudo Grave/virología , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Animales , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Chlorocebus aethiops , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Células Procariotas/inmunología , Conejos , Síndrome Respiratorio Agudo Grave/inmunología , Glicoproteína de la Espiga del Coronavirus , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.
Asunto(s)
Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Disulfuros/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Filogenia , Proteómica , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Análisis de Secuencia de Proteína , Glicoproteína de la Espiga del Coronavirus , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas ViroporinasRESUMEN
The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV were predicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodies were isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-induced polyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, it was confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratory syndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified and N protein specific Abs were produced by peptide immunization, which will be usefull for the study of SARS-CoV.
Asunto(s)
Epítopos/inmunología , Proteínas de la Nucleocápside/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/aislamiento & purificación , Vectores Genéticos/genética , Humanos , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Datos de Secuencia Molecular , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión Proteica/inmunología , Conejos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genéticaRESUMEN
Described herein is the synthesis of a hapten of biocyclo[2,2,2]octene 5 designed to mimic the exo transition-state of an aza Diels-Alder reaction. Immunization of rabbits with this hapten provided polyclonal antibodies, Aza-BSA-3, which were used to synthesize adduct 4b in the first reported antibody-catalyzed exo Diels-Alder reaction.
Asunto(s)
Anticuerpos/química , Compuestos Aza/química , Alcadienos/química , Animales , Compuestos Bicíclicos con Puentes/inmunología , Catálisis , Haptenos/inmunología , Inmunización , Cinética , Conejos , Albúmina Sérica BovinaRESUMEN
This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K(M)=6.67 mM and k(cat)/k(uncat)=5.8 x 10(4). This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer.