Generation of chicken single chain antibody variable fragments (scFv) that differentiate and neutralize infectious bursal disease virus (IBDV).
Phage-displayed recombinant antibody libraries derived from splenic mRNA of chickens immunized with an Australian strain of infectious bursal disease virus (IBDV) were constructed as single chain variable fragments (scFv) by either overlap extension polymerase chain reaction (PCR) or sequential ligation of the individual heavy (V(H)) and light (V(L)) chain variable gene segments. Sequential cloning of the individual V(H) and V(L) genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly(4)Ser)(3) was more efficient than cloning by overlap extension PCR, increasing the library size 500 fold. Eighteen IBDV specific antibodies with unique scFv sequences were identified after panning the library against the immunizing antigen. Eight of the clones contained an identical V(H) gene but unique V(L) genes. In ELISA analysis using a panel of Australian and overseas IBDV strains, one scFv antibody was able to detect all strains, whilst 3 others could discriminate between Australian and overseas strains, classical and variant strains and Australian field strains and vaccine strains. In addition, some scFvs showed significant neutralization titres in vitro. This report shows that generation of chicken antibodies in vitro by recombinant means has considerable potential for producing antibodies of diverse specificity and neutralizing capacity.
Sapats
S I
SI
CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, VIC, Australia. sandra.sapats@csiro.au
Heine
H G
HG
Trinidad
L
L
Gould
G J
GJ
Foord
A J
AJ
Doolan
S G
SG
Prowse
S
S
Ignjatovic
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D018175:Birnaviridae Infections / Q000276:immunology / Q000517:prevention & control; D002645:Chickens / Q000276:immunology*; D005818:Genetic Engineering / Q000379:methods; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology*; D007243:Infectious bursal disease virus / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000276:immunology
D000914:Antibodies, Viral; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library