特異抗体を用いた乾燥濾紙血中17-Hydroxyprogesteroneの酵素免疫測定法

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Specific antiserum for 17-hydroxyprogesterone (17-OH-P) was prepared by immunizing 7α- (2-carboxyethylthio) -17-OH-P conjugated bovine serum albumin (BSA) in rabbits. Using this antiserum, 17-OH-P enzyme immunoassay for dried blood spots on filter paper was established. As a label, alkaline phosphatase was coupled covalently with 7a-carboxymethylthio-17-OH-P by carbodiimide method. B/F separation was carried out by the addition of anti-rabbit IgG goat antiserum. All specimens used were punched out with a paper puncher of 3mm diameter. The assay sensitivity was 2pg/tube, which was estimated by two standard deviation at zero concentrations of the calibration curve. Cross reactivities of this antibody were as follows : 11-deoxycortisol (8.21%), 17-0H-pregnenolone (3.33%), progesterone (1.67%), 11-deoxycorticosterone (0.31%), cortisol (0.16%), pregnenolone-3-sulfate Na salt (0.03%), dehydroepiandrosterone (DHEA) (<0.03%), 16a-OH-DHEA (<0.03%), DHEA-3-glucuronide (<0.03%), DHEA-3-sulfate Na salt (<0.03%), pregnenolone (<0.02%). Intra-and inter-assay coefficient of variations were 4 14% and 9-18%, respectively.<BR>In normal babies, 17-OH-P concentrations measured directly (without sample extraction) were below 23pg/disk (n=204). The histogram of 17-OH-P level in normal babies obtained by the direct method was distributed lower than that obtained by the enzyme immunoassay system (range : 4-79pg/disk, n=268) which used antibody raised against 17-OH-P-3-O-carboxymethyloxime conjugated BSA (Enzaplate, Sapporo Diagnostic Laboratory). Correlation of 17-OH-P concentrations in dried blood spots of normal babies obtained by direct method (x) and ether extracted method (y) was y = 0.67x - 0.11 (n=45). The values of 17-OH-P concentrations by the direct method were about 1.5 times higher than those obtained by the ether extracted method. However, this difference was smaller than that reported by other investigators (-10 times higher) who used antibody raised against 17-OH-P antigen with bridge site at 3 or 4 position. In immature babies, 17-OH-P concentrations on dried blood spots obtained by the direct method ranged from 6 to 189 pg/disk (n=18). Although these values were higher than the 17-OH-P values in normal babies, the values were distributed in the same range as those in normal babies determined directly by the other investigators who used antibody raised against 17-OH-P antigen with bridge site at 3 or 4 position. These results suggest that the present enzyme immunoassay using anti 7a- (2-carboxyethylthio) -17-OH-P antiserum is more specific than those so far reported. Meanwhile, in the present study, large differences in 17-OH-P values between direct method and extracted method (n=18, range : 1-29pg/disk) were observed in the dried blood spots of immature babies (max. 19 times). This may possibly be due to cross reactant substances in dried blood spots in immature babies.
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特異抗体を用いた乾燥濾紙血中17-Hydroxyprogesteroneの酵素免疫測定法

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