A newly developed enzyme-immunoassay for measuring the tissue contents of PACAP in fish.
We have developed a novel and easy enzyme-immunoassay (EIA) for pituitary adenylate cyclase-activating polypeptide (PACAP). We used it to determine immunoreactive PACAP levels in the central nervous system (CNS) and peripheral tissues of two fishes, a teleost (the stargazer) and an elasmobranch (a stingray). An antiserum was raised in a white rabbit immunized with a conjugate of synthetic stargazer PACAP27 plus keyhole limpet hemocyanin. The EIA system used an antiserum/biotin-labeled PACAP/avidin/biotin-conjugated enzyme complex, and a double antibody method was used to precipitate the immune complexes. We call the system the avidin-biotin complex detectable EIA (ABCDEIA) for PACAP. ABCDEIA with biotin-labeled PACAP27 detected only PACAP27, recognizing neither the longer forms of PACAP nor any other peptides. PACAPs with 27, 38, and 44 residues cross-reacted in another ABCDEIA with biotin-labeled PACAP38 or PACAP44. Whole brains of both fishes contained much higher levels of PACAP, 6-30 times as high as the levels in the mammalian brain, but unexpectedly, no immunoreactive PACAP27 was found in any CNS or peripheral tissue in either fish. The gastrointestinal tracts of fish also contained lower, but significant amounts of PACAP.
Matsuda
Kouhei
K
Department of Biology, Faculty of Science, Toyama University, 3190-Gofuku, Toyama, Toyama 930-8555, Japan. kmatsuda@sci.toyama-u.ac.jp
Onoue
Satomi
S
Kashimoto
Kazuhisa
K
Hamakawa
Aya
A
Kikuchi
Minori
M
Uchiyama
Minoru
M
Mochizuki
Tohru
T
Arimura
Akira
A
D000595:Amino Acid Sequence; D000818:Animals; D004064:Digestive System / Q000276:immunology; D005260:Female; D005399:Fishes / Q000378:metabolism*; D007124:Immunoenzyme Techniques / Q000379:methods*; D008297:Male; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000032:analysis* / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
C495403:Adcyap1 protein, rat; D009479:Neuropeptides; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide