Effects of a monoclonal antibody against progesterone, on embryo transport, development and implantation in laboratory mice.

A monoclonal antibody against progesterone (11P27) given on Day 2 of pregnancy interrupted pregnancy in BALB/c mice but not in BCF1 mice. The reason for this strain difference remains unclear, although it may involve discrimination by the recipient's immune system. The effects in BALB/c mice were reversed by progestin treatment. A dose of 5 nmol, which completely blocked implantation, had no significant effects on embryo transport and development on Day 4. A dose of 10 nmol did not increase the proportion of abnormal embryos, even though it accelerated tubal transport and increased embryo loss. No tubal retention was evident; most remaining embryos reached the uterus at the normal time. The transformation of morulae to blastocysts was only slightly delayed in 11P27-treated mice, and transfer experiments showed no decrease in embryo viability. The antibody appeared to act by blocking actions of endogenous progesterone on the uterus: uteri of 11P27-treated mice failed to develop a decidual cell reaction to intrauterine oil, and embryos from untreated donors failed to implant in 11P27-treated recipients. Antagonism of progesterone by antibody treatment prevented implantation in BALB/c mice apparently by actions on the uterus rather than on the embryo.

Measurement of human corticosteroid binding globulin by enzyme-linked immunoassay.

Thirteen monoclonal antibodies have been raised against corticosteroid binding globulin (CBG). From four of those with highest affinity for the antigen, two were selected for development of a sandwich enzyme-linked immunoassay (ELISA). The sensitivity of the assay was such that 0.7 fmol CBG could be detected. Levels of the binding protein in men (740 +/- 67 nmol/l) and women (690 +/- 103 nmol/l) were not significantly different, while those found during the third trimester of pregnancy (1500 +/- 423 nmol/l) were approximately twice these levels. CBG denatured by heating to 60 degrees C could not be detected by the ELISA.

Urinary epidermal growth factor excretion and breast cancer risk.

The amount of urinary epidermal growth factor (EGF) excreted was determined in 350 normal women of whom 37 subsequently developed breast cancer. These were a group of women selected on a case-control basis from 5000 volunteers who had participated in a prospective epidemiological study. Urinary EGF excretion was not correlated with known risk factors such as age at menarche or menopause, age at first or last full-term child or parity. Neither was it associated with day or length of menstrual cycle, breast mammographic parenchymal pattern or the blood concentration of prolactin, dehydroepiandrosterone or its sulphate ester. Univariate analysis indicated that the amount of urinary EGF was significantly correlated with urinary creatinine (P less than 0.001), age (P less than 0.001), urinary androsterone (P less than 0.02) or aetiocholanolone (P less than 0.02), height (P less than 0.05) and weight (P less than 0.05). However, multivariate analysis showed that the amount of urinary EGF was correlated only with creatinine excretion (P less than 0.001) and age (P less than 0.001) and that the significance of the other correlations were probably due to the confounding influence of creatinine.

Salivary oestradiol and progesterone levels in premenopausal women with breast cancer.

The concentrations of oestradiol and progesterone have been measured in salivary specimens collected daily over a complete menstrual cycle in 12 patients with operable breast cancer and 12 normal control volunteers. There was no significant difference (P greater than 0.05) for either hormone between these two groups. Both showed a mid-cycle rise in oestradiol levels followed by a smaller but sustained increase during the luteal phase. The progesterone concentration increased markedly during the luteal phase of the cycle. Total or non-protein bound oestradiol levels measured in blood samples from 19 normal women were both linearly correlated (P less than 0.001) with the concentration of oestradiol in matched saliva samples. The amount of free oestradiol in blood was about twice that found in saliva.

A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody.

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was covalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 microliter serum. The method is precise, accurate and specific and can detect 19.5 pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.

Simultaneous production of monoclonal antibodies to dehydroepiandrosterone, oestradiol, progesterone and testosterone.

Mice were immunized with a mixture of four steroid antigens: dehydroepiandrosterone (DHA), oestradiol, progesterone and testosterone linked to bovine serum albumin through the 7, 6, 11 alpha and 17 beta positions respectively. The response to immunization varied widely with no one mouse producing an optimal response to all four steroids. In the two fusion experiments performed, antibodies to all four antigens were developed. Data are presented which show that the immune response of the spleen donor is related to the relative numbers and quality of the antibodies produced to each steroid. Despite the structural identity of the progesterone and testosterone haptens, antibodies elicited in response to their respective antigens could readily be distinguished from each other. From the large number of monoclonal antibodies obtained those most useful for radioimmunoassay were three high affinity antibodies to oestradiol and two antibodies raised against DHA but with high affinity for DHA sulphate.

Characterisation of monoclonal antibodies raised against testosterone.

Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics.

The production of high affinity monoclonal antibodies to progesterone.

Using different immunisation regimens and the antigens 6 beta-hydroxyprogesterone hemisuccinate and 11 alpha-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin, we have produced 35 monoclonal antibodies against progesterone with a wide range of specificities and affinities (Ka = 8 x 10(7)-3 x 10(10) M-1). The 10 antibodies raised against the 6 beta-antigen showed no advantage in terms of specificity when compared with those raised against the 11 alpha-antigen. The four antibodies with the best binding characteristics were all derived from fusion experiments performed after long (5 booster injections) immunisation with the 11 alpha-antigen. Although the specificities of these monoclonal antibodies were similar to those of a high affinity rabbit antiserum raised against the same antigen, a measurable improvement in sensitivity was obtained when one of these four antibodies was used for radioimmunoassay.