RESUMO
By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.
Assuntos
Antígenos HIV/imunologia , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Sequência de Bases , Western Blotting , Clonagem Molecular , Mapeamento de Epitopos , Biblioteca Gênica , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Humanos , Epitopos Imunodominantes , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Recombinantes/química , Análise de Sequência de DNARESUMO
The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.
