RESUMO
We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein. The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen. Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity. The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift.
Assuntos
Anticorpos Monoclonais/imunologia , Vírus da Encefalite Equina Venezuelana/imunologia , Região Variável de Imunoglobulina/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Clonagem Molecular , Escherichia coli , Região Variável de Imunoglobulina/genética , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We have generated a single-chain variable fragment (ScFv) antibody, from a previously well-characterized monoclonal antibody (MAb) to Venezuelan equine encephalitis (VEE) virus, 5B4D-6. The variable regions of the heavy (V(H)) and light (V(L)) chain antibody genes, were connected by a DNA linker and cloned in the phagemid vector pCANTAB5E. The ScFv clone in Escherichia coli strain TG-1, 5B4D-6-6, was expressed as a approximately 30 kDa ScFv protein and higher molecular weight fusion products which were functional in recognizing VEE virus by enzyme-linked immunosorbent assay (ELISA). Results were reproduced in Escherichia coli strain HB2151, where clone D66 was expressed mainly as soluble periplasmic protein. The D66 ScFv antibody bound VEE virus strongly as determined by ELISA. Nucleotide sequence analysis of 5B4D-6-6 ScFv indicated that the Vkappa gene belonged to family XVI, subgroup V, while the V(H) gene was unique in its sequence, though its amino acid sequence could be subgrouped as IA. The deduced protein sequence of D66 was highly homologous to published murine ScFv protein sequences. This work demonstrates, for the first time, cloning of a functional ScFv antibody against VEE virus.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Vírus da Encefalite Equina Venezuelana/imunologia , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Expressão Gênica , Genes de Imunoglobulinas , Hibridomas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/imunologiaRESUMO
Nasopharyngeal cells of 24 patients diagnosed as being in the prodromal phase or with established measles were examined by indirect immunofluorescence microscopy for the presence of measles antigen. Eighteen patients were positive for measles antigen by this technique. In ten of the 18 patients it was necessary to remove masking globulins from the antigen-containing cells prior to staining. Results obtained by immunofluorescence were supported by cytologic and serologic findings. No virus isolations were achieved. Immunofluorescence microscopy with elution of adherent globulins offers the most rapid and sensitive method for the laboratory diagnosis of measles.