Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV.
Recombinant single chain Fv (scFv) antibody fragments can form the basis of a rapid, whole-blood diagnostic assay. The scFv described in this study is derived from a monoclonal antibody which has a high affinity for glycophorin A, an abundant glycoprotein on the human red blood cell membrane surface. The prototype reagent built around the scFv was designed to detect, in whole blood samples, the presence of antibodies that have arisen through infection with a foreign organism such as human immunodeficiency virus. The scFv was composed of the antibody heavy-chain variable domain (Vh) joined by a 15 residue linker -(GGGGS)3- to the light-chain variable domain (V1) terminated by either a C-terminal octapeptide tail (FLAG) or a 35 amino acid segment from the gp41 surface glycoprotein of HIV-1. Constructs were cloned into a Escherichia coli expression vector, pHFA, and expressed in a soluble form into culture supernatant. The product retained anti-glycophorin activity which could be detected directly in culture supernatants by ELISA. Furthermore, the scFv-epitope fusion functioned efficiently in the whole blood agglutination assay and was able to distinguish between HIV-1 positive and negative sera.
Lilley
G G
GG
CSIRO Division of Biomolecular Engineering, Victoria, Australia.
Dolezal
O
O
Hillyard
C J
CJ
Bernard
C
C
Hudson
P J
PJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics*; D000936:Antigen-Antibody Complex / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis*; D015497:HIV-1; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007128:Immunoglobulin Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins