Production of a functional chicken single-chain variable fragment antibody derived from caecal tonsils B lymphocytes against macrogamonts of Eimeria tenella

• Published in: Veterinary immunology and immunopathology 2004-02, Vol.97 (3), p.219-230
• Main Authors: Réfega, Susana, Cluzeaud, Marianne, Péry, Pierre, Labbé, Marie, Girard-Misguich, Fabienne
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; B-Lymphocytes - immunology; Base Sequence; Caecal tonsils; Cecum - immunology; Chicken single-chain variable fragment antibody; Chickens - immunology; Cloning, Molecular; Coccidiosis - immunology; Coccidiosis - parasitology; Coccidiosis - veterinary; Eimeria; Eimeria tenella; Eimeria tenella - immunology; Enzyme-Linked Immunosorbent Assay - veterinary; Fluorescent Antibody Technique - veterinary; Hybridomas - immunology; Immunoglobulin Fragments - biosynthesis; Immunoglobulin Fragments - immunology; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Light Chains - immunology; Immunoglobulin Variable Region - biosynthesis; Immunoglobulin Variable Region - immunology; Life Sciences; Lymphoid Tissue - immunology; Macrogamonts; Molecular Sequence Data; Poultry Diseases - immunology; Poultry Diseases - parasitology; Sequence Alignment
• ISSN: 0165-2427; 1873-2534
• DOI: 10.1016/j.vetimm.2003.09.008

Avian coccidiosis is due to a protozoan intracellular parasite belonging to the genus Eimeria which multiplies in the intestine of the host. In order to identify Eimeria antigens which reflect the natural avian humoral immune response, chicken hybridomas were produced by fusion of myeloma MuH1 with B lymphocytes from Eimeria tenella infected chicken. B lymphocytes used for fusions were isolated from tonsils at the basis of caeca where the parasite develops. One of the clones (G1F5) recognised oocyst antigens and the macrogamont stage of the parasite in ELISA and immunofluorescence assay. A single-chain variable fragment (scFv) antibody was cloned from the light chain variant region (V L) and heavy chain variant region (V H) genes of the hybridoma. This recombinant antibody (scFv G1F5) exhibited antigen binding specificity to oocysts and macrogamonts of E. tenella equivalent to the mAb produced by the clone G1F5. Nucleotide sequence analysis of V L genes from scFv G1F5 compared to the germ-line revealed vestiges of gene conversion. scFv derived from chicken B lymphocytes isolated from the gut-associated lymphoid tissue following experimental infection can reveal specific antigens recognised by the avian immune response.