Sheep monoclonal antibody fragments generated using a phage display system.
Monoclonal sheep antibodies have great potential for biomedical, veterinary and agricultural purpose. Although conventional sheep monoclonal antibodies can be generated by a modified hybridoma technology, the procedures are not routine. Here, we describe a method to generate recombinant sheep antibody fragments from immunised animals using a modified phage display system. Total RNA from pooled spleens of sheep immunised with the model antigens human serum albumin and conalbumin were used to amplify immunoglobulin V gene repertoires and an efficient two-step cloning method was employed to rapidly construct a phage display single-chain Fv (scFv) library. A total of 14 different scFvs were isolated and characterised. Sequence analysis indicated typical ovine immunoglobulin characteristics. Thirteen Vlambda and 11 VH genes were identified that could be grouped into the sheep Vlambda families I, II, VI and a single VH family. Soluble monomeric scFvs, produced in the periplasm of Escherichia coli, were subjected to affinity measurement via surface plasmon resonance analysis and affinities typical of the secondary immune response were observed. The method described here should be of value for the study of sheep immunology as well as for biorecognition in general.
Li
Y
Y
Department of Biology, University of Leicester, University Road, Leicester, UK. YL4@leicester.ac.uk
Kilpatrick
J
J
Whitelam
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification; D000915:Antibody Affinity; D001483:Base Sequence; D003207:Conalbumin / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000276:immunology; D012756:Sheep; D012995:Solubility
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D012709:Serum Albumin; C055966:immunoglobulin Fv; D003207:Conalbumin