RESUMEN
Six unique phage antibodies to human TNF have been selected from a combinatorial library of human single chain fragment variable. ELISA and Western-blotting was used to study selected phage antibodies binding with TNF. The specificity of selected antibodies was determined by binding with interferon alpha and gamma, bovine serum albumin, ovalbumin and ubiquitin. Two antibodies, sA1 and sB3, were converted into a soluble single-chain antibody form and their affinity was 2.5 and 13.7 nM respectively.
Asunto(s)
Anticuerpos de Cadena Única/inmunología , Factores de Necrosis Tumoral/inmunología , Secuencia de Aminoácidos , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Citocinas/inmunología , Humanos , Datos de Secuencia Molecular , Ovalbúmina/inmunología , Biblioteca de Péptidos , Albúmina Sérica Bovina/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificación , Ubiquitina/inmunologíaRESUMEN
A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Ebolavirus/inmunología , Proteínas Recombinantes/biosíntesis , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Línea Celular , Clonación Molecular , Humanos , Nucleoproteínas/inmunología , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transfección , Proteínas del Núcleo Viral/inmunologíaRESUMEN
A combinatorial phage display library of human single-chain antibody fragments (scFv) was constructed on the basis of variable domains of heavy (Vh) and light (VI) genes cloned from the lymphocytes of six healthy donors. The size of the library was 2? 10(8) independent clones. Single-chain antibodies against recombinant human TNF?, vaccinia virus and virus-like particles formed by core protein of hepatitis B virus were selected from the library. Unique scFv sequences were identified using the HaeIII fingerprinting. The specificity of the selected clones was proved by the Western-blot analysis.
Asunto(s)
Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Biblioteca de Péptidos , Especificidad de Anticuerpos , Bacteriófago M13/metabolismo , Antígenos del Núcleo de la Hepatitis B/inmunología , Humanos , Leucocitos Mononucleares , ARN Mensajero , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Donantes de Tejidos , Factor de Necrosis Tumoral alfa/inmunología , Virus Vaccinia/inmunologíaRESUMEN
Human recombinant antibodies against a purified Ebola virus (EV) lysate were selected from a combinatorial library of scFv-antibodies using the phage display technique. Nine unique antibodies were identified after sequencing the Vh- and Vl-genes encoding the selected antibodies. Solid-phase enzyme immunoassay (EIA) indicated that these antibodies were able to bind both inactivated and native EV. Immunoblotting showed that 6 antibodies identified nucleoprotein (NP), one antibody did VP24 and another antibody did VP40. One of the selected antibodies reacted with two EP proteins: VP24 and VP40. Solid-phase EIA demonstrated cross-reactivity with Marburg virus (MAR) and defined VP24 MAR as a target protein for the antibody.
Asunto(s)
Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Ebolavirus/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/genética , Especificidad de Anticuerpos , Regiones Determinantes de Complementariedad/genética , Reacciones Cruzadas , Humanos , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Nucleoproteínas/inmunología , Biblioteca de Péptidos , Estructura Terciaria de Proteína/genética , Proteínas del Núcleo Viral/inmunología , Proteínas Virales/inmunologíaRESUMEN
Eight specific antibodies to live variola virus (VV), Ind-3a strain, and 7 antibodies to VV, Butler strain, were selected from the synthetic combinatorial phage display library on single-chain (scFv) human antibodies. Indirect solid-phase enzyme immunoassay showed the ability of these antibodies to bind the VV strains Ind-3a, Butler, Brazil-131, Kuw-5, and Congo-2. Moreover, earlier selected human scFv antibodies were also tested in the reaction of binding to the above VV strains. The experiments could reveal the antibodies that bound alastrim strains more effectively that did other VV strains. The nucleotide sequences encoding for the selected scFv antibodies were determined.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus de la Viruela/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Técnicas Químicas Combinatorias , Reacciones Cruzadas , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Biblioteca de Péptidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain. Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected. Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced. All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses. Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus. According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.
Asunto(s)
Anticuerpos Antivirales/análisis , Orthopoxvirus/inmunología , Biblioteca de Péptidos , Anticuerpos Monoclonales , Anticuerpos Antivirales/genética , Virus de la Viruela Vacuna/inmunología , Virus de la Ectromelia/inmunología , Electroforesis , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Humanos , Inmunoquímica , Reacción en Cadena de la Polimerasa , Virus Vaccinia/inmunología , Virus de la Viruela/inmunología , Proteínas Virales/análisisRESUMEN
The library of human scFv antibodies displayed on the surface of bacteriophages was panned against Vaccinia virus (VACV), strain Elstree. 75% binding with Vaccinia virus. 5 clones were characterized for their binding with VACV and their ability to neutralize VACV in plaque reduction neutralization test (PRNT). Antibodies from the clones were obtained as soluble individual molecules and their binding activities were confirmed in ELISA.
