Construction of recombinant extended single-chain antibody peptide conjugates for use in the diagnosis of HIV-1 and HIV-2.
The construction, expression and evaluation of recombinant scFv based HIV diagnostic reagents are described. In a whole-blood, erythrocyte agglutination assay format, recombinant scFv antibodies (expressed in Escherichia coli), linked to a spacer domain and HIV-gp36 or -gp41 peptides, were shown to be able to detect efficiently natural antibodies against HIV in human serum. Performance in trials suggests that these single chain reagents have potential as alternatives to existing Fab-peptide chemical conjugates. We also report the construction of an inducible expression vector, pGC, which can be used both in laboratory experiments and in large-scale fed-batch fermentations. It was found that while the base scFv reagent (lacking a spacer) functioned as well as the Fab peptide conjugate in assays where whole (negative) blood was spiked with mouse monoclonal anti-HIV antibodies (IgG or IgM), clinical assays using human sera showed lower sensitivities and increased false negatives. This deficiency was overcome by inclusion of the natural 1C3 kappa (light) chain domain as a spacer arm between the scFv and HIV peptide tags. This spacer was thought to overcome steric constraints which would otherwise prevent efficient interaction between the reagent (once bound to the surface of red blood cells) and the various serum antibodies against the respective C-terminal peptide epitopes. As a result of this important modification, performance of the extended scFv reagent (for both HIV-1 and HIV-2) equalled that of the current commercial technology in limited trials.
Coia
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G
CSIRO Division of Biomolecular Engineering, Parkville, Australia.
Hudson
P J
PJ
Lilley
G G
GG
D000595:Amino Acid Sequence; D001483:Base Sequence; D015686:Gene Products, env / Q000096:biosynthesis / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis / Q000138:chemical synthesis; D015488:HIV Antigens / Q000096:biosynthesis / Q000235:genetics; D015700:HIV Envelope Protein gp41 / Q000096:biosynthesis / Q000235:genetics; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology; D015498:HIV-2 / Q000235:genetics* / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics*; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000276:immunology*; D054299:env Gene Products, Human Immunodeficiency Virus
D015686:Gene Products, env; D015488:HIV Antigens; D015700:HIV Envelope Protein gp41; D007128:Immunoglobulin Fragments; D007202:Indicators and Reagents; D010455:Peptides; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C059763:env protein gp36, Human immunodeficiency virus; C055966:immunoglobulin Fv