J Vet Sci.  2013 Mar;14(1):95-98. 10.4142/jvs.2013.14.1.95.

Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs

• van Wyngaardt W
• Mashau C
• Wright I
• Fehrsen J

• KMID: 1482819
• DOI: http://doi.org/10.4142/jvs.2013.14.1.95

Abstract

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.

Keyword

African horsesickness virus; double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA); phage-displayed scFv; serotype-specific

MeSH Terms

African horse sickness virus/*isolation & purification
Animals
Antibodies, Immobilized
Antibodies, Viral/*immunology
Cercopithecus aethiops
Chickens
Enzyme-Linked Immunosorbent Assay/methods/*veterinary
Immunoglobulin G
*Peptide Library
Serologic Tests/methods/veterinary
Serotyping
Single-Chain Antibodies/*immunology
Vero Cells
Antibodies, Immobilized
Antibodies, Viral
Immunoglobulin G
Peptide Library
Single-Chain Antibodies

Figure

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  Fig. 1 Serotype-specific detection of African horsesickness virus (AHSV)-3 in a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using the phage-displayed single-chain variable fragment (scFv) Lca12. Vero cell homogenate was used as the negative control. Bars represent the mean of two absorbance values.

• 

  Fig. 2 Serogroup-specific detection of the nine AHSV serotypes with a DAS-ELISA using phage-displayed scFv G7 (bars 1~9). Equine encephalosis virus (EEV) Bryanston inoculum (bar C1), Vero cell homogenate (bar C2), and MP/PBS (bar C3) were used as negative controls. Bars depict the mean of two absorbance values. Student's t-test-least significant difference (LSD) = 0.61, p = 0.05. Bars with the same letters are not statistically different at a 5% level of significance.

• 

  Fig. 3 Detection of AHSV-6 amplified in Vero cells inoculated with spleen and lung tissue from 12 infected field horses. Group-specific phage-displayed scFv G7 was used as the detecting antibody in a DAS-ELISA. The average absorbance value for 12 animals was calculated from the mean of two absorbance values for each animal, bar A; EEV Bryanston inoculums, bar B; Vero cell homogenate, bar C; MP/PBS, bar D were used as negative controls. Bars for the controls represent the mean of two absorbance values. Student's t-test-LSD = 0.74319, p = 0.05. Bars with the same letters were not statistically different at a 5% level of significance.

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