Escherichia coli expression of a bifunctional Fab-peptide epitope reagent for the rapid diagnosis of HIV-1 and HIV-2

• Published in: Immunotechnology (Amsterdam, Netherlands) Vol. 1; no. 3-4; pp. 197 - 209
• Main Authors: Dolezal, Olan, Coia, Gregory, Guthrie, Robin E., Lilley, Glenn G., Hudson, Peter J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.1995
• Subjects: AIDS/HIV; Antibodies, Bispecific - biosynthesis; Antibodies, Bispecific - immunology; Blood; Diagnosis; E. coli; Epitope Mapping; Escherichia coli; Fab; HIV; HIV Infections - diagnosis; HIV-1; HIV-2; Humans; Immunoglobulin Fab Fragments - biosynthesis; Immunoglobulin Fab Fragments - immunology; Indicators and Reagents; Models, Molecular; Peptide epitope; Recombinant Proteins - biosynthesis; Recombinant Proteins - immunology; anti-FLAG antibody; M2; antigen-binding fragment; culture medium; antibody(ies); 10 anti-glycophorin antibody; human immuno-deficiency virus; HIV-2 surface glycoprotein; Blood; constant domain of the immunoglobulin light chain; anti-EEF antibody; gp41; VH and VL; anti-gp41 antibody (IgM); Diagnosis; PBS; Peptide epitope; EEF; isopropyl-β-d-thiogalactopyranoside; SDS-PAGE; HIV-1 surface glycoprotein; single letter amino acid code; gp36; 2 x YT; ELISA; tripeptide a-tubulin epitope; Ab; IgG; antigen-binding fragment (VH and VL domains); enzyme linked immunosorbent assay; FLAG; A-Z; immunoglobulin type G; CH1 first constant domain of the immunoglobulin heavy chain; singlechain Fv fragment; sodium dodecyl sulphate-polyacrylamide gel electrophoresis; variable domains of antibody heavy and light chains, respectively; anti-gp41 antibody (IgG); 1B1/114; E. coli; Fd; 2A6/105; Fab; truncated heavy chain consisting of VH and CH1; YL1/2; CL; octapeptide epitope; Fv; HIV; scFv; phosphate buffered saline; IPTG
• ISSN: 1380-2933; 1879-162X
• DOI: 10.1016/1380-2933(95)00021-6

Background: The current format of a rapid whole-blood agglutination assay for HIV relies on a bifunctional molecule which comprises a 10 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. Objectives: 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. Study design: 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 I C3 Fabpeptide epitope conjugates. Results: A recombinant I C3 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 κ 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 I C3 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 dial diagnostic specificity performed very effectively in whole-blood diagnosis of patients infected with either HIV-1 or HIV-2. Conclusion: A recombinant 10 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.