Establishment of a reference ELISA for measurement of universal thresholds of pregnanediol glucuronide excretion rates using urine samples diluted to a constant volume per unit time.

An enzyme-linked immunosorbent assay (ELISA) for measurement of pregnanediol-3α-glucuronide (PdG) excretion rates in urine samples diluted to 150 mL/h before analysis is described. The sensitivity of the 9 optimized standard curves was 0.093 ± 0.070 µmol PdG/24 hr, with the multiple combined standard curves having a mean mid-point (EC50) of 6.88 µmol PdG/24 hr. The PdG threshold excretion rate of 7.0 µmol/24 hr, which is used as a marker for the end of fertility, was situated in the most accurate region of the standard curve. The specificity of the ELISA was determined using normal variate transformation to compare seven menstrual cycle profiles obtained with the ELISA method with the profiles obtained previously using a validated radioimmunoassay (RIA) method. The cycle profiles all agreed within experimental error, and a high degree of correlation using Deming regression was obtained. The correlation equation was Y = 1.57X-0.11 µmol PdG/24 hr (n = 200; r = 0.932). The PdG excretion rates determined by the ELISA were 50% higher than given by RIA, but the normal ranges were similar to those given by the original reference gas liquid chromatographic method. The ELISA assay was therefore suitable as a reference method for measurement of thresholds of PdG excretion rates.

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh.

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990bp) and MD-ACO3 (966bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3' untranslated regions (3'-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3'-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K(m)) of 89.39 microM (MD-ACO1), 401.03 microM (MD-ACO2) and 244.5 microM (MD-ACO3) for the substrate ACC, catalytic constants (K(cat)) of 6.6x10(-2) (MD-ACO1), 3.44x10(-2) (Md-ACO2) and 9.14x10(-2) (MD-ACO3) and K(cat)/K(m) (microMs(-1)) values of 7.38x10(-4) microMs(-1) (MD-ACO1), 0.86x10(-4)Ms(-1) (MD-ACO2) and 3.8x10(-4) microMs(-1) (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.

Validation of a reference ELISA for estrone glucuronide using urine samples normalized by dilution to a constant rate of urine production.

A direct enzyme linked immunosorbent assay (ELISA) system has been optimized as a reference method for the measurement of first statistically significant rises in estrone glucuronide excretion rates in human urine by analysing samples pre-diluted at the time of the collection by the women subjects to a constant urine production rate of 150 mL/h. Validation was achieved by correlation of the individual menstrual cycle profiles with the corresponding estrone glucuronide excretion rates determined by radioimmunoassay (RIA) on the same urine samples for a total of 221 samples from nine cycles. The pre-dilution procedure removed random variations due to fluctuations in the daily rate of urine excretion and minimized between sample matrix effects. When the ELISA data were correlated with the RIA data, Deming regression gave a slope of 1.20+/-0.03 and an intercept of 4.6+/-1.8 nmol/24h (r=0.944) and a random experimental error of 14.2 nmol/24h. The major difference in the measurements was a proportional error of 20%, which was present in either the ELISA or RIA methods or in both. Comparison of the standard normal variate transformation of the ELISA and RIA data gave hormonal profiles of the individual menstrual cycles (N=9) that overlapped almost perfectly. Statistically significant rises or falls in the magnitude of the excretion rate in one profile were mirrored faithfully in the other.