Limits...
Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV).

Kirsch MI, Hülseweh B, Nacke C, Rülker T, Schirrmann T, Marschall HJ, Hust M, Dübel S - BMC Biotechnol. (2008)

Bottom Line: The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved.The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated.The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

View Article: PubMed Central - HTML - PubMed

Affiliation: Abteilung Biotechnologie, Institut für Biochemie und Biotechnologie, Technische Universität Braunschweig, Spielmannstrabetae 7, 38106, Braunschweig, Germany. martina.kirsch@tu-bs.de

ABSTRACT

Background: Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.

Results: In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.

Conclusion: For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

Show MeSH

Related in: MedlinePlus

Cross-reactivity of the anti-VEEV scFv clones and different anti-Alphavirus specific mAbs analyzed by ELISA. Antigens: VEEV strains TC83, 230 and H12/93 were captured by using anti-VEEV mAb VEE-WIS1 (3 μg/mL); Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) and Chikungunya (CHIK) were captured by using an anti-Alphavirus mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (3 μg/mL); Culture supernatant of non-infected Vero cells was captured once by anti-VEEV mAb VEE-WIS1 (VERO VEEWIS1) or by a mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (VERO mAb mix). A. Staining with biotinylated anti-VEEV mAb 8/6 (1:10000) and streptavidin conjugated with HRP (1:4000). B. Staining with a biotinylated mixture of antibodies consisting of mAb 8/6 (1:10000), mAb VEE-WIS1 (1:10000), mAb 12/2 (1:5000) and mAb 42/2 (1:2000) followed by a streptavidin-HRP (1:4000) incubation. C. Staining with 1 × 109 (cfu) scFv phage per well was followed by an incubation with mAb anti-M13 conjugated with HRP (1:5000). The IIB6 scFv phage was used as negative control. The mean values of two ELISAs from two independent scFv phage productions are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2543005&req=5

Figure 6: Cross-reactivity of the anti-VEEV scFv clones and different anti-Alphavirus specific mAbs analyzed by ELISA. Antigens: VEEV strains TC83, 230 and H12/93 were captured by using anti-VEEV mAb VEE-WIS1 (3 μg/mL); Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) and Chikungunya (CHIK) were captured by using an anti-Alphavirus mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (3 μg/mL); Culture supernatant of non-infected Vero cells was captured once by anti-VEEV mAb VEE-WIS1 (VERO VEEWIS1) or by a mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (VERO mAb mix). A. Staining with biotinylated anti-VEEV mAb 8/6 (1:10000) and streptavidin conjugated with HRP (1:4000). B. Staining with a biotinylated mixture of antibodies consisting of mAb 8/6 (1:10000), mAb VEE-WIS1 (1:10000), mAb 12/2 (1:5000) and mAb 42/2 (1:2000) followed by a streptavidin-HRP (1:4000) incubation. C. Staining with 1 × 109 (cfu) scFv phage per well was followed by an incubation with mAb anti-M13 conjugated with HRP (1:5000). The IIB6 scFv phage was used as negative control. The mean values of two ELISAs from two independent scFv phage productions are shown.

Mentions: An established VEEV-specific (figure 6A) and Alphavirus genus-specific sandwich ELISA (figure 6B) served as positive control. As negative control, cell culture of non-infected Vero cells was used. As marker antibody the biotinylated anti-VEEV mAb 8/6 was used for the detection of all VEEV strains (figure 6A) and a biotinylated mixture of antibodies consisting of mAb 8/6, mAb VEE-WIS1, mAb 12/2 and mAb 42/2 was used for the group specific detection of Alphaviruses (figure 4B). All viral antigens were captured by either the VEEV-specific mAb VEEV-WIS1 or a mAb mixture of anti-Alphavirus antibodies, consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (WIS, Munster, Germany). Some virus strains (VEE-230) were captured better than others (VEE-H12/93).


Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV).

Kirsch MI, Hülseweh B, Nacke C, Rülker T, Schirrmann T, Marschall HJ, Hust M, Dübel S - BMC Biotechnol. (2008)

Cross-reactivity of the anti-VEEV scFv clones and different anti-Alphavirus specific mAbs analyzed by ELISA. Antigens: VEEV strains TC83, 230 and H12/93 were captured by using anti-VEEV mAb VEE-WIS1 (3 μg/mL); Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) and Chikungunya (CHIK) were captured by using an anti-Alphavirus mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (3 μg/mL); Culture supernatant of non-infected Vero cells was captured once by anti-VEEV mAb VEE-WIS1 (VERO VEEWIS1) or by a mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (VERO mAb mix). A. Staining with biotinylated anti-VEEV mAb 8/6 (1:10000) and streptavidin conjugated with HRP (1:4000). B. Staining with a biotinylated mixture of antibodies consisting of mAb 8/6 (1:10000), mAb VEE-WIS1 (1:10000), mAb 12/2 (1:5000) and mAb 42/2 (1:2000) followed by a streptavidin-HRP (1:4000) incubation. C. Staining with 1 × 109 (cfu) scFv phage per well was followed by an incubation with mAb anti-M13 conjugated with HRP (1:5000). The IIB6 scFv phage was used as negative control. The mean values of two ELISAs from two independent scFv phage productions are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543005&req=5

Figure 6: Cross-reactivity of the anti-VEEV scFv clones and different anti-Alphavirus specific mAbs analyzed by ELISA. Antigens: VEEV strains TC83, 230 and H12/93 were captured by using anti-VEEV mAb VEE-WIS1 (3 μg/mL); Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE) and Chikungunya (CHIK) were captured by using an anti-Alphavirus mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (3 μg/mL); Culture supernatant of non-infected Vero cells was captured once by anti-VEEV mAb VEE-WIS1 (VERO VEEWIS1) or by a mAb mix consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (VERO mAb mix). A. Staining with biotinylated anti-VEEV mAb 8/6 (1:10000) and streptavidin conjugated with HRP (1:4000). B. Staining with a biotinylated mixture of antibodies consisting of mAb 8/6 (1:10000), mAb VEE-WIS1 (1:10000), mAb 12/2 (1:5000) and mAb 42/2 (1:2000) followed by a streptavidin-HRP (1:4000) incubation. C. Staining with 1 × 109 (cfu) scFv phage per well was followed by an incubation with mAb anti-M13 conjugated with HRP (1:5000). The IIB6 scFv phage was used as negative control. The mean values of two ELISAs from two independent scFv phage productions are shown.
Mentions: An established VEEV-specific (figure 6A) and Alphavirus genus-specific sandwich ELISA (figure 6B) served as positive control. As negative control, cell culture of non-infected Vero cells was used. As marker antibody the biotinylated anti-VEEV mAb 8/6 was used for the detection of all VEEV strains (figure 6A) and a biotinylated mixture of antibodies consisting of mAb 8/6, mAb VEE-WIS1, mAb 12/2 and mAb 42/2 was used for the group specific detection of Alphaviruses (figure 4B). All viral antigens were captured by either the VEEV-specific mAb VEEV-WIS1 or a mAb mixture of anti-Alphavirus antibodies, consisting of mAb 3/4, mAb 12/2 and mAb VEE-WIS1 (WIS, Munster, Germany). Some virus strains (VEE-230) were captured better than others (VEE-H12/93).

Bottom Line: The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved.The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated.The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

View Article: PubMed Central - HTML - PubMed

Affiliation: Abteilung Biotechnologie, Institut für Biochemie und Biotechnologie, Technische Universität Braunschweig, Spielmannstrabetae 7, 38106, Braunschweig, Germany. martina.kirsch@tu-bs.de

ABSTRACT

Background: Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.

Results: In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.

Conclusion: For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.

Show MeSH
Related in: MedlinePlus