| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1991: ¥100,000 (Direct Cost: ¥100,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
The concentration of many peptides in the circulation has been determined by competitive radioimmunoassay only after extraction and concentration. This is due to insufficient sensitivities of competitive immunoassays. The purpose of this research was to develop noncompetitive immunoassay methods for peptides. [Methods]Two noncompetitive enzyme immunoassay methods (hetero - two - site enzyme immunoassy methods) were developed. <Method I>Peptide was biotinylated and trapped onto an anti - peptide IgG - coated polystyrene ball. After washing the polystyrene ball to eliminate other biotinylated substances, biotinylated peptide was eluted from the polystyrene ball with HCl and reacted with anti -peptide Fab' -peroxidase conjugate. The complex formed was trapped onto streptavidin - coated polystyrefie balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. <Method II>Bjotinylated peptide, eluted from anti - peptide IgG - coated polystyrene ball as described abov
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e, was reacted with 2, 4 - dinitrophenyl - fluorescein disulfide - bovine serum albumin - rabbit antipeptide IgG conjugate. The complex formed was trapped onto[anti - 2, 4 - dinitrophenyl group]IgG - coated polystyrene balls and, after washing, reacted with avidin - beta - D -galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N - 2, 4 - dinitrophenyl - L - lysine and transferred to anti - fluorescein IgG - coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2 - mercaptoethylamine and transferred to[anti - rabbit IgG]IgG - coated polystyrene balls. beta - D - Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. [Results]The detection limits by Methods I and 11 for peptides such as[Arg^8] - vasopressin, alpha - atrial natriuretic peptide and angiotensin I were 10 amol and I amol, respectively. The principle of the two methods may be applicable to the measurement of hastens including peptides that can be derivatized so as to be bound simultaneously by both anti - hapten antibody and avidin(streptavidin)molecules. Less
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