In order to obviate the drawbacks of plasma immunoglobulins, the whole molecular recombinant human anti-HAV (hepatitis A virus) monoclonal antibody (anti-HAV IgG) produced and secreted by rCHO cells was purified and its physicochemical properties were extensively characterized. The rCHO cells were cultured in serum-free medium and the supernatants were collected. The recombinant human IgG molecules were sequentially purified by ultrafiltration, rProtein A Sepharose Fast Flow affinity chromatography, ion exchange chromatography and diafiltration. In affinity chromatography, prior to the target protein elution, an intermediate high salt wash step was inserted, different pH and salt concentrations were evaluated for the capacity of removing host cell DNA. The yield of the downstream purification process was approximately 40%. The purity of anti-HAV IgG thus generated was assayed with SEC-HPLC method, integration result showed that the monomeric IgG content was more than 99%. Western-blot was carried out with AP-antiHuman IgG (Fab specific) and AP-antiHuman IgG (Fc specific) respectively, the blot result demonstrated that the anti-HAV IgG is human antibody with Fab and Fc structure. The specific anti-HAV activity determined by ELISA was 100 IU/mg, with anti-HAV immunoglobulin as the working standard reference. Ligand leakage in the eluate of the affinity column was approximately 32 ng/mg IgG, while after further purification steps, it was decreased to less than 2 ng/mg IgG. Residual host cell DNA was monitored with solid dot blot assay, DNA can be removed effectively with intermediate high salt wash step in the affinity chromatography. Free sulfhydryl content of anti-HAV IgG was assayed with fluorescent spectrophotometer, the low molecular weight bands appeared in non-reducing SDS-PAGE may be caused by the presence of free sulfhydryl. The endotoxin content was less than 1EU/ mg examined by standard LAL test procedures. Anti-HAV IgG prepared with this process is able to fulfill the regulatory requirements of State Food and Drug Administration for recombinant products.
Antibodies, Monoclonal ; biosynthesis ; immunology ; isolation & purification ; Chromatography, Affinity ; methods ; Hepatitis A Antibodies ; biosynthesis ; immunology ; isolation & purification ; Hepatitis A virus ; immunology ; Humans ; Immunoglobulin G ; biosynthesis ; immunology ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification

Interferon is an important cytokine that plays a critical role in the initial host defense against viral infection. Recombinant human adenoviruses expressing human interferon-alpha (Ad-HIFNalpha) or pig interferon-beta fused with interleukin-18 (Ad-PIFNbeta-IL18) were constructed and used to induce an early protective response against foot and mouth disease (FMD). To analyze the antiviral effect, bovine thyroid and porcine kidney IBRS-2 cells and ICR mice were treated with Ad-HIFNalpha, Ad-PIFNbeta-IL18, and cocktail of Ad-HIFNalpha and Ad-PIFNbeta-IL18. The survival rate of suckling mice was monitored after foot and mouth disease virus (FMDV) challenge following intra-peritoneal (IP) administration of appropriate adenovirus. Indirect antigen ELISA was performed to evaluate inhibition of FMDV replication following challenge with the FMDV O, A, or Asia 1 serotypes in vitro. These recombinant adenoviruses reduced the replication of FMDV in susceptible cells, thereby decreasing the fatality in mice, suggesting that they can be a useful control method for the early protection against FMD infection in livestock after field trial.
Adenoviridae ; Adenoviruses, Human ; Animals ; Asia ; Enzyme-Linked Immunosorbent Assay ; Foot ; Foot-and-Mouth Disease ; Foot-and-Mouth Disease Virus ; Humans ; Interferon-alpha ; Interferon-beta ; Interferons ; Interleukin-18 ; Kidney ; Livestock ; Mice ; Mice, Inbred ICR ; Survival Rate ; Thyroid Gland

Congenital Microtia/surgery* ; Ear, External ; Humans ; Reconstructive Surgical Procedures ; Surgical Flaps ; Tissue Expansion Devices ; Treatment Outcome