A monoclonal antibody to tryptophan hydroxylase: applications and identification of the epitope.
Recombinant rabbit tryptophan hydroxylase (TPH) was expressed in Escherichia coli and purified from inclusion bodies by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A mouse monoclonal antibody and rabbit and sheep polyclonal antibodies were generated. In immunohistochemical studies of formaldehyde-fixed primate brain, the monoclonal strongly labeled not only cell bodies in the raphe nuclei but also fibers in the cerebral cortex. Truncation mutants and peptide pre-competition were used to localize the epitope to E103SVPWFP109. Although the primary sequences of TPH encoded by mRNAs from brain and pineal gland are identical, differences in the immunoreactivity of TPH protein from these two sources were observed in blot immunolabeling studies. TPH immunoreactivity migrated as an M(r) approximately equal 56000 band in each of the tissues except human pineal glands, in which the TPH reactivity was approximately 3 kDa lower. In addition, the relative intensities of TPH immunolabeling across the four tissues differed among these antibodies and a previously described monoclonal antibody against phenylalanine hydroxylase (PH8), which cross-reacts with TPH. Whereas PH8 exhibited roughly equivalent TPH reactivity per protein in both tissues from both species, TPH from human and rat raphe nuclei was preferentially recognized by the present monoclonal. By contrast, the affinity-purified sheep polyclonal antibody reacted preferentially with TPH from human and rat pineal gland, and the affinity-purified rabbit polyclonal antibody appeared to selectively recognize TPH from human pineal gland.
Haycock
John W
JW
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, LSUHSC-BIOCHEM, 1100 Florida Avenue, New Orleans, LA 70119, USA. jhayco@lsuhsc.edu
Kumer
Sean C
SC
Lewis
David A
DA
Vrana
Kent E
KE
Stockmeier
Craig A
CA
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008252:Macaca fascicularis; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry; D010870:Pineal Gland / Q000737:chemistry; D011817:Rabbits; D011903:Raphe Nuclei / Q000737:chemistry; D051381:Rats; D012756:Sheep; D013045:Species Specificity; D014365:Tryptophan Hydroxylase / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D014365:Tryptophan Hydroxylase