Single-Chain Antibody Fragments from a Display Library Derived from Chickens Immunized with a Mixture of Parasite and Viral Antigens

Abstract

Phage-displayed chicken single-chain antibody fragment libraries can provide useful diagnostic and research reagents. Using avian immunoglobulin genes simplifies the construction of such repertoires since far fewer primer sets are required to access the avian antibody repertoire than is the case with mice or humans. Libraries constructed using mRNA from an immune source are enriched in affinity-matured sequences and consequently need not be as large as "universal" non-immune repertoires to have a reasonable probability of yielding high-affinity binders. Repertoires focused on a number of defined targets can be constructed using lymphocyte mRNA from chickens immunized with a mixture of several different antigens. This approach was evaluated with the aim of economically and rapidly deriving immunodiagnostic reagents for malaria, trypanosomiasis, and malignant catarrhal fever, all of which are important to health or food security in Africa. Two chickens were each immunized with a mixture comprised of recombinantly expressed histidine-rich protein, the aldolase and the lactate dehydrogenase of Plasmodium falciparum, the variant surface glycoprotein of Trypanosoma sp., and purified malignant catarrhal fever virus, a herpesvirus that causes an economically important disease of cattle and other ruminants. Immune responses to each of the individual antigens were determined by extracting egg-yolk IgY and testing for antigen-specific antibodies in ELISA. The chicken splenocytes were then recovered, RNA was extracted, and after reverse transcription, the immunoglobulin VH and VL regions were amplified by PCR and joined via a single glycyl residue for surface expression on a collection of filamentous bacteriophages. The resulting display library was then screened by panning to isolate binders. The immunized chickens did not, however, respond equally well to all the different antigens, nor was it possible to derive antibody fragments against all the targets. These limitations notwithstanding, several useful binders with the potential to be used in malaria diagnosis were obtained.

Serological investigation of highly pathogenic avian influenza H5N2 in ostriches (Struthio camelus)

“…After the hen was euthanized the spleen was collected, sliced into small fragments and stored in RNAlater (Qiagen, Hilden, Germany) at (708C. A phage-displayed single-chain fragment variable (scFv) library was constructed using the repertoire of the immunized chicken as described before (van Wyngaardt et al, 2004;Chiliza et al, 2008). Phages were rescued from the library of 3.82 )10 7 primary clones and 5)10 12 phages were subjected to selection on rH5 HA1.…”

IgY-Technology Applied to Studies of Toxoplasma gondii Infection

“…Libraries constructed, using mRNA from an immune source, are enriched in affinity-matured sequences. Repertoires focused on a number of defined targets can be constructed using lymphocyte mRNA from chickens immunized with a mixture of several different antigens from Plasmodium falciparum, Trypanosoma sp., or human viruses [47,48].…”

The complementarity-determining region sequences in IgY antivenom hypervariable regions

“…Punctual interferences of horse immunization schedules with lower immunizing venom doses and an increased number of immunizations interspaced with longer periods and meticulous antibody quality evaluations [1], [2], [3] were transferred to immunize hens [1]. More specific antibodies endowed with higher affinity and toxin neutralizing potency were developed.…”