Heikki Mikola

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Preparation of europium-labeled derivatives of cortisol for time-resolved fluoroimmunoassays

Cortisol 3-(O-carboxymethyl)oxime, 6- and 21-hemisuccinoxycortisol, and cortisol 7-carboxyethyl thio-ether were synthesized. These carboxyl derivatives were labeled using a described general labeling method with a europium chelate. The labeled steroids were tested in a competitive time-resolved fluoroimmunoassay using antibodies raised against cortisol. Only the site-homologous antigen-antibody pairs underwent immunoreaction and gave satisfactory calibration curves.

Polar lipids and net photosynthesis potential of subarctic Diapensia lapponica

Abstract The overall net photosynthesis potential of Diapensia lapponica was very low. It was highest at the end of August (11.1 μmol CO 2 (μmol chlorophyll) −1 hr −1 ) and decreased during the autumn and winter, reaching a minimum in February (0.6 μmol CO 2 (μmol chlorophyll) −1 hr −1 ). In March, with increased light in the subarticum, the potential rose temporarily, and after another minimum at the beginning of May, the potential slowly rose to the summer level (2.3–6.5 μmol CO 2 (μmol chlorophyll) −1 hr −1 ). Ihe seasonal fluctuation pattern of the potential of net photosynthesis was the same when calculated on a dry weight basis. Seasonal changes also occurred in the chlorophyll content and in the contents of polar lipids, particularly DGDG (digalactosyl diacygglycerols) and PC (phosphatidyl choline) and less clearly in MGDG (monoglactosyl diacylglycerols), PE (phosphatidyl ethanolamine) and PG (phosphatidyl glycerols). The contents of chlorophylls, DGDG and PC increased during autumn and early winter during the hardening process and decreasing light and temperature. Their contents decreased in late winter and spring in response to dehardening and increased light and temperature in the subarcticum. Thus, the molar ratios of MGDG:DGDG and PE:PC varied throughout the year, being lowest in winter. In addition, fatty acids of individual lipid classes showed seasonal fluctuation. In both MGDG and DGDG, the proportion of linolenic acid was higher in summer than in winter and that of linoleic acid was vice versa . In both PC and PE, the proportion of palmitic acid was highest in summer and lowest in winter,and, particularly in PC, this variation was compensated for by changes in linoleic acid and less clearly in linolenic acid, and, in PE, by the long-chain behenic acid. In PG, the proportions of trans -16:1 and oleic acid were higher in summer than in winter, whereas the proportions of palmitic and linoleic acids were higher in winter than in summer. Thus, there was not a clear increase in the degree of unsaturation of fatty acids during winter-time.

Syntheses of europium-labeled digoxin derivatives and their use in time-resolved fluoroimmunoassay.

A simple and efficient method of derivatizing and labeling the terminal sugar ring of digoxin (and other steroid glycosides) is described. The terminal sugar ring was oxidized by periodate to dialdehyde, followed by a reaction with the aminooxy group of heterobifunctional spacer reagent. Usually, reductive amination is needed to stabilize the Schiff base-type compounds produced in reactions between amino and aldehyde groups. Here the oxime compounds produced are stable wherefore the reductive amination step can be avoided and the reaction gives high yields without significant side reactions. After characterization the digoxin dioxime derivatives formed were labeled with europium chelates. These labeled digoxin derivatives bearing one or two europium-containing chelates, coupled via different spacers, were purified and tested in a competitive time-resolved fluoroimmunoassay. Several digoxin-specific monoclonal antibodies were also tested to determine the most suitable antibody-tracer combination, which was then employed to develop a simple competitive time-resolved fluoroimmunoassay for digoxin.