Selection and expression of recombinant single domain antibodies from a hyper-immunized library against the hapten azoxystrobin

• Published in: Journal of immunological methods Vol. 373; no. 1-2; pp. 8 - 18
• Main Authors: Makvandi-Nejad, Shokouh, Fjällman, Ted, Arbabi-Ghahroudi, Mehdi, MacKenzie, C. Roger, Hall, J. Christopher
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 28-10-2011; Elsevier
• Subjects: affinity chromatography; Amino Acid Sequence; amino acid sequences; Animals; antibodies; Antibodies - genetics; Antibodies - immunology; Antibodies - metabolism; Antibody Affinity - immunology; Azoxystrobin; Biological and medical sciences; bovine serum albumin; Camelids, New World - genetics; Camelids, New World - immunology; Cattle; clones; enzyme-linked immunosorbent assay; Escherichia coli; Fundamental and applied biological sciences. Psychology; Fundamental immunology; genes; Hapten; haptens; Haptens - immunology; hydrophobicity; Immunization; Immunoblotting; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Heavy Chains - metabolism; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - immunology; Immunoglobulin Variable Region - metabolism; inhibitory concentration 50; Llama; llamas; lymphocytes; Male; Methacrylates - chemistry; Molecular immunology; Molecular Sequence Data; Molecular Structure; ovalbumin; Peptide Library; Pyrimidines - chemistry; Pyrimidines - immunology; Rabbits; Recombinant Proteins - immunology; Recombinant Proteins - metabolism; screening; Sequence Homology, Amino Acid; Serum Albumin, Bovine - chemistry; Serum Albumin, Bovine - immunology; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Single-Chain Antibodies - metabolism; Strobilurins; Techniques; Variable domain of a heavy chain antibody; Azoxystrobin; Variable domain of a heavy chain antibody; Llama; Hapten; Heavy peptide chain; Immunization; Antibody; Pesticides; Strobilurin; Fungicide; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2011.07.006

Three VHHs against the model hapten, azoxystrobin (MW 403), were isolated from a hyper-immunized phage-displayed VHH library. This library was constructed by isolating the VHH-coding genes from the lymphocytes collected from a Llama glama that was immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones containing VHHs specific to the hapten. After screening 95 clones, three VHHs (A27, A72, and A85) with different amino acid sequences were identified, expressed in soluble format in Escherichia coli HB2151, and purified using nickel-immobilized metal affinity chromatography. Competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) showed that A27 and A85 were specific to azoxystrobin while A72 was not. The IC50 values of A27 and A85 VHHs were 7.2 and 2.0μM, respectively. To our knowledge A85 is one of the highest affinity VHHs that has yet been isolated against a hydrophobic hapten such as azoxystrobin.
► A llama was immunized with a model hapten molecule azoxystrobin (MW 403). ► The apparent affinity of the convIgG to azoxystrobin was greater than to the HcIgG. ► A VHH hyper-immunized phage-displayed library was constructed and panned. ► Two VHHs (A27 and A85) were isolated with the IC50 values of 7.2 and 2.0µM, respectively. ► A85 is one of the highest affinity VHHs that has yet been isolated against a hapten.