Radioimmunoassay for hypocretin-2.
OBJECTIVE
To develop radioimmunoassay for hypocretin-2 (Hcrt-2). And search for its presence in certain rat tissues.


METHODS
Anti-Hcrt-2 serum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic Hcrt-2 with bovine serum albumin. Radioiodination of Hcrt-2 was performed by chloramine T method, followed by purification of radoiodinated material on Sephadex G-25 column.


RESULTS
The obtained antibody did not cross react with hypocretin-2, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. The assay was performed with a double antibody system. Hcrt-2 was extracted from the tissues with acid acetone. The dilution curve of acid acetone extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue Hcrt-2 was about 85 % and the intra-assay and inter-assay variation were 5.6 % and 8.0 %, respectively. Hcrt-2 was found in the hypothalamus, cerebrum, brain stem and testes.


CONCLUSIONS
The obtained data suggest that the assay system developed is suitable to measure Hcrt-2 in tissues and that Hcrt-2 is mainly found in the hypothalamus.
Mitsuma
T
T
Fourth Department of Internal Medicine Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
Hirooka
Y
Y
Kayma
M
M
Mori
Y
Y
Yokoi
Y
Y
Izumi
M
M
Rhue
N
N
Ping
J
J
Adachi
K
K
Ikai
R
R
Kawai
N
N
Nakayashiki
A
A
Nogimori
T
T
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D001923:Brain Chemistry; D001933:Brain Stem / Q000737:chemistry; D007031:Hypothalamus / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000032:analysis* / Q000276:immunology; D000068797:Orexins; D009928:Organ Specificity; D011786:Quality Control; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D013737:Testis / Q000737:chemistry
D000906:Antibodies; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D000068797:Orexins