Cryopreservation of lateral buds of in vitro-grown innala plants (Solemostemon rotundifolius) by vitrification.

We succeeded in cryopreserving of innala (Solenostemon rotundifolius) in vitro-grown young lateral buds by vitrification. Nodal segments from in vitro-grown shoots (2-4 mm in length) were cultured on MS medium containing 0.1M sucrose in Petri dishes for 3 weeks under 16-h photoperiod at 25 degree C. This pre-growth induced a large number of uniform young lateral buds. Nodal segments (0.5 to 1.0 mm in length) with two lateral buds were dissected from the shoots and precultured with 0.3 M sucrose for 2 days at 25 degree C. They were then treated with loading solution containing 2 M glycerol and 0.4 M sucrose (LS solution) for 20 min at 25 degree C and dehydrated with the PVS2 vitrification solution for 18 min at 25(C prior to either rapid immersion in liquid nitrogen. Surviving lateral buds resumed growth within 3 days and developed shoots without intermediary callus formation. The average growth recovery after cryopreservation amounted to 85%.

Production and utilisation of antibodies directed against oestrone sulphate using specific hapten synthesis.

Oestrone-3-sulphate (E13S) is an important metabolite of oestrone. Studies in cattle had previously shown that it is synthesised in the gravid uterus by the foetus, but not by the corpus luteum. Progesterone measurement in milk by radioimmunoassay (RIA) is routinely carried out in some laboratories as a pregnancy test for cattle. The major drawback of progesterone measurement in milk, by RIA, as a pregnancy test was the failure to detect the lack of conceptus in those cows where early embryonic death had occurred but the corpus luteum still persisted thereby giving false positive results. We have developed a direct RIA for E13S by raising antibodies to an immunogen prepared from a specific hapten synthesised by an unambiguous chemical synthesis. The sensitivity of the RIA in milk was found to be 0.368 nmol/l. The levels of E13S in non-pregnant cows are undetectable but during a viable pregnancy, the levels are elevated to greater than 0.40 nmol/l by day 100. There was no cross-reactions in the assay with any free oestrogens. The measurement of this metabolite of oestrone promises to provide an accurate marker for the detection of a viable conceptus in cattle.

Prediction and detection of ovulation by the measurement of urinary pregnanetriol-3 alpha-glucuronide. A multicentre study.

The urine excretion pattern of pregnanetriol 3 alpha-glucuronide (PT-3G) throughout the menstrual cycle in 26 normal ovulating women was evaluated in a multicentre study. The concentration of PT-3G was measured by radioimmunoassay in daily samples of early morning urine (EMU) from 20 women for three consecutive cycles and from 6 women who conceived during the period of study. PT-3G was also measured in 24-h urine samples from 5 additional women. The peak of urine LH was used as a reference point for ovulation (Day 0). The EMU concentration of PT-3G in the follicular phase of 60 normal ovulatory cycles was 5.10 mumol/l +/- 0.11 (arithmetic mean +/- SE). The first PT-3G defined rise (CUSUM analysis) occurred during the late follicular phase (Days -3 to 0) with a PT-3G maximum excretion (9.69 mumol/1 +/- 0.55) on Day 0, whereas a PT-3G excretion peak occurred during mid-luteal phase (Days +5 to +9). The overall PT-3G excretion during the luteal phase (8.06 mumol/1 +/- 0.17) was significantly higher than that of the follicular phase (P less than 0.001). A further sustained increase in PT-3G excretion was noted after day +11 in the conceptional cycles. The 24-h excretion profile of PT-3G was similar to that obtained in EMU samples. No inter-centre significant variation was noticed in terms of PT-3G concentration values. The results were interpreted as demonstrating that the PT-3G excretion profile throughout the cycle exhibits a close resemblance to that of serum 17-OH progesterone. The data also indicates that although the immunoanalytical measurement of this urine steroid metabolite does not give an early sign for the occurrence of ovulation, it can be used for both the immediate prediction and the detection of ovulation.

The radioimmunoassay of pregnanetriol 3 alpha-glucuronide.

The hapten (5 beta-pregnane-17,20 alpha-diol-3 alpha-yl glucuronide) required to develop the radioimmunoassay was synthesized by an unambiguous chemical synthesis. An immunogenic complex was synthesized by coupling bovine serum albumin (BSA) to the glucuronide by mixed acid anhydride reaction. The immunogenic complex was used to induce the formation of specific antibodies in rabbits. In addition, the required radioligand [6,7-3H]5 beta-pregnanetriol 3 alpha-glucuronide was prepared with appropriate specific radioactivity. The characteristics of the antibody were determined with regard to specificity and sensitivity and the precision and accuracy of the assay method established. As applied to urine samples this method is superior to existing procedures in that they avoid the hydrolysis step and can be used directly on diluted urine. Examples have been given of useful application of this technique in clinical practice.

Specificity studies of monoclonal and rabbit antibodies raised against steroid glucuronides.

Monoclonal and rabbit antibodies raised against estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide have been studied with respect to their ability to bind free estrone and its conjugates or free pregnanediol and its conjugates, respectively. High titre and high specificity were observed with monoclonal antibodies produced against pregnanediol-3 alpha-glucuronide, whereas the monoclonal antibodies produced against estrone-3-glucuronide were not so specific when compared with the corresponding rabbit antibodies. Both monoclonal and rabbit antibodies had affinity constants in the range of 10(9)--10(10) liter/mole.

The isolation of estriol-16 alpha-glucuronide and pregnanediol-3 alpha-glucuronide from late pregnancy urine.

A simple procedure is described, whereby estriol-16 alpha-glucuronide and pregnanediol-3 alpha-glucuronide have been isolated from late pregnancy urine. The conjugates isolated were used as haptens for raising antisera for radioimmunoassay.

The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.

Antisera were prepared against three related oestrogen ring-A glucuronides, oestrone 3-glucuronide, oestradiol 3-glucuronide and oestriol 3-glucuronide. The corresponding 6,7-3H-labelled conjugates were synthesized as radioligands and the cross-reactions of the antisera against ring-A oestrogen glucuronides and other steroid conjugates were examined. The specificity of the antiserum against oestriol 3-glucuronide was compared with that raised against oestriol 16alpha-glucuronide, and the measurement of the former conjugate in late-pregnancy urine is discussed.