Escherichia coli expression of a bifunctional Fab-peptide epitope reagent for the rapid diagnosis of HIV-1 and HIV-2.
BACKGROUND
The current format of a rapid whole-blood agglutination assay for HIV relies on a bifunctional molecule which comprises a 1C3 Fab fragment, with specificity for the human red blood cell surface marker (glycophorin A), chemically conjugated to a synthetic peptide that corresponds to a single immunodominant region of HIV envelope glycoprotein. In this assay erythrocyte agglutination occurs if the blood sample contains anti-HIV antibodies.


OBJECTIVE
To establish whether a bacterially synthesised Fab fragment encoding several C-terminal immunodominant peptide tails can be produced in sufficient purity and yield to function in whole-blood agglutination assays.


METHODS
An E. coli dicistronic Fab expression cassette was constructed comprising of light and heavy chain gene fragments derived from a glycophorin specific monoclonal antibody (1C3), genetically linked with C-terminal immunoreactive peptide epitopes. Expression and purification procedures were established to enable the rapid production of 1C3 Fab-peptide epitope conjugates.


RESULTS
A recombinant 1C3 Fab fragment was expressed with two different immunological epitope markers, Glu-Glu-Phe (EEF) and FLAG, at the C-terminus of the Fd heavy and kappa light chain, respectively. This model Fab-EEF/FLAG conjugate was detected in culture supernatant by SDS-PAGE gels and Western blots, and could be successfully used in erythrocyte agglutination assays. Furthermore, an HIV specific 1C3 Fab reagent, containing immunoreactive peptide epitopes from the surface glycoproteins of HIV-1 and HIV-2, was also expressed but at lower levels and with increased sensitivity to proteolytic degradation. Nevertheless, this recombinant Fab reagent with dual diagnostic specificity performed very effectively in whole-blood diagnosis of patients infected with either HIV-1 or HIV-2.


CONCLUSIONS
A recombinant 1C3 Fab fragment terminated by immunoreactive peptide epitopes can be expressed in E. coli in a soluble, antigen-binding form, and it can successfully mimic the commercial Fab-HIV reagents in whole-blood agglutination assays.
Dolezal
O
O
CSIRO, Division of Biomolecular Engineering, Parkville, Victoria, Australia.
Coia
G
G
Guthrie
R E
RE
Lilley
G G
GG
Hudson
P J
PJ
D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000276:immunology*; D018604:Epitope Mapping; D004926:Escherichia coli; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1; D015498:HIV-2; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology*; D007202:Indicators and Reagents; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology
D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007202:Indicators and Reagents; D011994:Recombinant Proteins