RESUMO
Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives. However, the lack of the Fc region of the antibody can be difficult in many standard diagnostic platforms. Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated conversion to human IgG with no traces of mouse Fab as determined by Western blot analysis. In addition, the humanized IgG performed better as both a detection and capture reagent in an ELISA format and detected the botulinum toxoid at a lower concentration than the parental murine Fab. This technique offers the ability to convert various species of antibodies or antibody fragments into humanized antibodies with improved characteristics in immunodiagnostic assays, for use as human controls in serological assays, or for possible therapeutic benefit.
Assuntos
Toxinas Botulínicas/análise , Toxinas Botulínicas/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Animais , Biotecnologia/métodos , Botulismo/diagnóstico , Linhagem Celular Tumoral , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/genética , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , TransfecçãoRESUMO
Four serotypes of monovalent live attenuated dengue virus vaccine candidates were tested for reactogenicity and immunogenicity in 49 flavivirus non-immune adult human volunteers. The four monovalent candidates were then combined into a tetravalent formulation and given to another 10 volunteers. Neutralizing antibody seroconversion rates after a single-dose monovalent vaccination ranged from 53% to 100%. Solicited reactogenicity was scored by each volunteer. A composite index, the Reactogenicity Index, was derived by these self-reported scores. Reactogenicity differed among the four serotype candidates with serotype-1 associated with the most vaccine related side effects. A second dose of monovalent vaccines at either 30 days or 90 days was much less reactogenic but did not significantly increase seroconversion rates. Seroconversion rates in the 10 volunteers who received a single dose of tetravalent vaccine ranged from 30% to 70% among the four serotypes. Similar to the monovalent vaccines, a second dose of the tetravalent vaccine at one month was less reactogenic and did not increase seroconversion. A third dose of the tetravalent vaccine at four months resulted in three of four volunteers with trivalent or tetravalent high-titer neutralizing antibody responses.