Preparation of a Monoclonal Antibody to Common Amino Acid Sequence of LHRH and Its Application

Abstract

In order to prepare an antibody directed at the common amino acid se-quence of mammalian, avian, and fish luteinizing hormone-releasing hormones (LHRHs), C-terminal free LHRH was conjugated with bovine thyroglobulin, and was used as the antigen. A monoclonal antibody (LRH13) was obtained an ascitic fluid by fusing the spleen cells of a BALB/c donor mouse im-munized with the antigen to X63. Ag8.653 mouse myeloma cells followed by limiting dilution cloning and transplanting a positive clone to BALB/c mice. This monoclonal antibody seems to belong to Ig G2b as it was eluted from protein A-Sepharose CL-4B with citrate buffer pH 3.5. Competitive binding experiment using fragment peptides of LHRH indicated the binding site LRH13 was a region around serine and tyrosine, and modification mammalian LHRH by radioiodination caused a marked decrease in the binding activity. LRH13 has an affinity constant of 0.134×109 M-1to native mammalian LHRH, and binds C-terminal free LHRH with a similar affinity (1.6×), however, it binds with higher affinities to N- and C-terminal free LHRH (12.9×), N-terminal free LHRH (10.4×), salmon LHRH (8.3×) and chicken LHRH-I (6.0×). Chicken LHRH-II, where tyrosine is replaced histidine, has a lower affinity (0.3×) than that of mammalian LHRH. From its high affinity to N-, C-terminal free LHRH, LRH13 is also expected bind possible precursor peptides of LHRH. Immunohistochemical staining the brain sections obtained from rats, mice, chickens, Japanese quail, and rainbow trout successfully visualized cell bodies and fibers distributed from the olfactory bulb to the median eminence, indicating high LHRH specificity and wide crossreactivity in animal classes of this monoclonal antibody. With this antibody, LHRH-like immunoreactive substance in the pineal gland was also stained with fixation at neutral pH.