NISHIDA Toshinobu The University of Tokushima, School of Dentistry Instructor, 医学部附属病院, 助手 (50156080)
SHUNTANI Yasumi The University of Tokushima, School of Medicine Instructor, 医学部附属病院, 助手 (10235773)
YOKOGOSAHI Yutaka The University of Tokushima, School of Medicine Instructor, 医学部, 助手 (00253203)
|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1992 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Sensitive noncompetitive enzyme immunoassay (EIA) for hypothalamic hormone (growth hormone-releasing hormone, GHRH ; somatostation, SRIF) was developed using sandwich method.
Specific antibody was raised in rabbits by immunizing them with an antigen-carrier protein complex. We first tried to obtain two kinds od antibody, which recognize the C-terminal or the N-terminal portion of the peptides, to establish sandwich EIA.However, only antibodies that C-terminal portion but not the N-terminal portion of GHRH(1-44), and the middle portion of SRIF(1-14) were obtained. Thus these antisera were purified by affinity chromatography, and their IgG fractions were used to EIA.
EIA was conducted by sandwich method utilizing high affinity of avidin (streptavidin) to biotin and antigen-antibody reaction. The peptides were biotinylated using biotinamidocaproate N-hydroxysccinimide ester and reacted with avidin, which had been immobilized to a plate, and the purified specific antibody described above. Th
is complex formed was further reacted with goat anti-rabbit IgG-peroxidase conjugate and o-phenylenediamine (OPD), and the color development at 420 nm was measured. By means of this assay method, the peptides could be quantified in a range of 5.0-1,000 pg/tube (GHRH) and 0.61-100 pg/tube (SRIF). The assay sensitivity was almost the same as that by the conventional competitive radioimmunoassay. In normal subjects, the Plasma GHRH and SRIF levels were 27 (〕SY.+-.〔) 9.7 pg/ml and 15 (〕SY.+-.〔) 6.4 pg/ml(mean (〕SY.+-.〔) SD), respectively.
The problem to be solved was a relatively strong color development due to nonspecific reaction irrespective of the good dose-response curves. However, the color development of OPD due to non-specigic reaction was decreased to a negligible level by adding normal goat serum (10%) to the assay system, and by the use of antiserum with low non-specific reaction. In the future, Fluorometry at 320 nm, 405 nm, instead of colorimetry using OPD will be considered to increase the assay sensitivity. To reduce non-specific reaction, the method to trap biotinylated peptide to specific anti-peptide IgG-coated polystirene balls is now under investigation. Less