Assay buffer

Serum testosterone, estradiol and progesterone concentrations were measured by time-resolved immunofluorometric assay, DELFIA (PerkinElmer Life and Analytical Sciences, Turku, Finland) after diethyl ether (Merck KGaA, Darmstadt, Germany) extraction. Ether-extracted serum samples were reconstituted to DELFIA Diluent II buffer (PerkinElmer Life and Analytical Sciences, Turku, Finland) and used for analysis. The sensitivity of the assay was 100 pg/ml for testosterone, 13.6 pg/ml for estradiol and 250 pg/ml for progesterone. The intra- and interassay coefficients of variation (CV) were below 6 and 12%, respectively. To enhance the sensitivity, commercial tracer and antiserum were additionally diluted 5∶8 with assay buffer (PerkinElmer Life and Analytical Sciences, Turku, Finland) in testosterone assay. For estradiol and progesterone, dilution rate was 1∶2. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels were determined from unextracted samples by DELFIA as described previously [30] (link), [31] (link). The sensitivity of the rat FSH assay was 0.1 µg/l, intra-assay CV 4.3% and inter-assay CV 10.4% at 4.8 µg/l, and the sensitivity of the rat LH assay was 0.03 µg/l, intra-assay CV 19% at 0.04 µg/l, >5% at >1 µg/l and inter-assay CV 12.5% at 0.24 µg/l and 7.8% at 0.78 µg/l.

The CDI for human PrP was performed as described previously^{39 (link),79 (link)}, with following modifications: white Lumitrac 600 High Binding Plates (E&K Scientific, Santa Clara, CA) were coated with mAb 8H4 (epitope 175–185)^{80 (link)} and aliquots of each sample containing 0.007% (v/v) of Patent Blue V (Sigma) were dilutedinto plate wells filled with 200 µl of Assay Buffer (Perkin Elmer, Waltham, MA); he captured PrP was detected by a europium-conjugated^{81 (link)} anti-PrP mAb 3F4 (epitope 108–112)^{74 (link)} and the time-resolved fluorescence (TRF) signals were measured by the multi-mode microplate reader PHERAstar Plus (BMG LabTech, Durham, NC).