RESUMEN
Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of the layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1 x 10(10) pfu/mL. Each variation of layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1 x 10(10) approximately 2.5 x 10(10) pfu/mL, with the sensitivity of 10(9) pfu/mL. Compared with other methods, the optical protein-chip requires only short measurement time, is label free, is a quantitative test, and can be visualized. This study could be significant on the investigation of interactions between the antibody and virus, and shows potential in the early diagnosis of virosis.
Asunto(s)
Animales , Ratones , Anticuerpos Antivirales , Alergia e Inmunología , Bacteriófago M13 , Alergia e Inmunología , Análisis por Matrices de Proteínas , MétodosRESUMEN
A novel human ScFv H12 against SARS-CoV has been selected from a SARS immune library. In order to produce a large amount of ScFv H12, pET28a-H12 expression vector was constructed and ScFv H12 was expressed at yield about 30% of total proteins in E. coli . Here two different refolding procedures were used to refold ScFv H12 from inclusion body: gel filtration chromatography and dilution. The results showed that ScFv H12 could be efficiently refolded by both procedures. However, the refolding via gel filtration was 1.5 time more effective than that of dilution. The affinity of ScFv H12 to SARS-CoV virion was detected as Kd = 73.5 nmol/mL.
Asunto(s)
Humanos , Anticuerpos Monoclonales , Genética , Anticuerpos Antivirales , Alergia e Inmunología , Escherichia coli , Genética , Metabolismo , Fragmentos de Inmunoglobulinas , Genética , Alergia e Inmunología , Región Variable de Inmunoglobulina , Genética , Alergia e Inmunología , Cuerpos de Inclusión , Genética , Alergia e Inmunología , Renaturación de Proteína , Proteínas Recombinantes , Química , Alergia e Inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Alergia e InmunologíaRESUMEN
After its advent, monoclonal antibody has gone an uneven way to its present wide applications in clinical practices, during which the humanized antibody set an important milestone accompanying a series of technique renovations, such as PCR technique, phage display and transgenic animals. Humanized antibody has developed from chimeric antibody and reshaped antibody to the present fully human antibody. Humanization of murine antibodies has been the future direction of therapeutic antibodies and this can be reflected from the fact that humanized antibodies or even human antibodies have made up majority of the therapeutic antibodies both in clinical test and in the market. The present techniques have enabled the production of fully human antibodies and given chances to the arising of antibody derivatives. They not only overcome the deficiency in application of murine antibody with different strategies, but also provide more weapons for human therapeutics. However, modifications of monoclonal antibody aiming at clinical applications need more research work in the mechanisms of antibody effector system, as well as comprehensive understanding in regulation of human immune system.