Production of monoclonal antibodies against avian LHRH-I

• Published in: In Vitro Cellular & Developmental Biology 1989-10, Vol.25 (10), p.934-938
• Main Authors: Alston-Mills, B. (University of Maryland, College Park, MD), Li, Q.C, Ottinger, M.A
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antibodies, immunoglobulins; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - immunology; Antibody Formation; ANTICORPS MONOCLONAL; ANTICUERPOS MONOCLONALES; Antigens; Biological and medical sciences; Biotechnology; CHICKENS; EXPERIMENTACION IN VITRO; EXPERIMENTACION IN VIVO; EXPERIMENTATION IN VITRO; EXPERIMENTATION IN VIVO; Feeder cells; Fundamental and applied biological sciences. Psychology; Fundamental immunology; GONADOLIBERINE; GONADOTROPIN RELEASING HORMONE; Gonadotropin-Releasing Hormone - immunology; Health. Pharmaceutical industry; HORMONA LIBERADORA DE GONADOTROPINA; Hormones; Hypothalamus; Hypothalamus - analysis; Immunization; Immunoenzyme Techniques; IN VITRO EXPERIMENTATION; IN VIVO EXPERIMENTATION; Industrial applications and implications. Economical aspects; Luteinization; Mice; Mice, Inbred BALB C; Miscellaneous; Molecular immunology; MONOCLONAL ANTIBODIES; POLLO; POULET; Production of active biomolecules; Spleen - immunology; Spleen - metabolism; Spleen cells
• ISSN: 0883-8364; 0073-5655; 2327-431X; 1475-2689
• DOI: 10.1007/BF02624006

Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1-to 3-d posthatched chicks, mature quail, and mature mouse.