Investigation of diethylstilbestrol residue level in human urine samples by a specific monoclonal antibody.

Diethylstilbestrol (DES) is used as a kind of animal feed additive and affects people's health through the food chain. The purpose of this study is to detect the residue level of DES in 576 human urine samples directly. DES-BSA was used to immunize Balb/c mice. The monoclonal antibody was produced by hybridoma that was screened through cell fusion techniques. Finally, we developed the indirect competitive ELISA method to analyze 576 human urine samples from Zhejiang Province, China. The IC50 of this method was 3.33 ng/mL. The LOD and LOQ were 0.16 and 0.54 ng/mL. Linear range of the standard curve was from LOD to 12.50 ng/mL. There was no cross-reactivity with two kinds of estrogens and two structural analogs with DES. Five hundred seventy-six urine samples were analyzed by the indirect competitive ELISA method, and the detection rate was 98.78%. The mean concentration and geometric mean were 4.70 and 3.50 ng/mL. The indirect competitive ELISA method based on monoclonal antibody was sensitive and reliable for the detection of DES in human urine samples. The results warned us to pay more attention to human health and food safety.

[Synthesis of artificial diethylstilbestrol antigen for preparation of polyclonal antibodies].

OBJECTIVE: To synthesize artificial diethylstilbestrol (DES) antigen and to prepare DES polyclonal antibody with high titer and sensitivity. METHODS: The derivative of DES (DES-HS) was synthesized from diethylstilbestrol, ethyl bromoacetate,bovine serum albumin (BSA) and chicken ovalbumin (OVA) with the nucleophilic substitution reaction; the compound was identified by electrospray ionization mass spectrometry(ESI-MS). The DES-HS and the carrier proteins (BSA, OVA) were cross-linked to prepare the artificial antigen; the UV absorption spectrophotometry and high performance liquid chromatography (HPLC) were used to identify the prepared artificial antigen. The rabbits were immunized with the DES artificial antigen to prepare the DES polyclonal antibodies. RESULTS: The DES-HS was synthesized. The DES artificial antigen was prepared successfully with a coupling rate of 22:1. The DES polyclonal antibodies with a titer of 1:25 600 and IC50 of 10.81 ng/ml were prepared with DES artificial antigen. CONCLUSION: A set of methods to synthesize DES artificial antigen and to prepare the DES polyclonal antibodies has been developed successfully.

Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured K(D) was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

Colloidal gold-based immunochromatographic assay for detection of diethylstilbestrol residues.

One-step membrane-based competitive colloidal gold-based immunoassays in immunochromatographic formats for the rapid detection of diethylstilbestrol (DES) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and DES hapten-ovalubumin conjugate (test line). Anti-DES polyclonal antibody labeled with colloidal gold particles was first incubated with DES. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for immunochromatographic of 0.5 microg/kg for detecting DES standard solution, and the limit of detection was 5 microg/kg for detecting the DES spiked in swine pork and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.

Comparison of the efficiences of enzymatic and chemical hydrolysis of (nortestosterone and diethylstilboestrol) glucuronides in bovine urine.

Residues of 19-nortestosterone (19-NT) and diethylstilboestrol (DES) are excreted in bovine urine, mainly conjugated to glucuronic acid. Prior to quantification, urine must be deconjugated, which is commonly performed by enzymatic or chemical hydrolysis. The efficiencies of two enzymatic and two chemical deconjugation methods were studied. The range of efficiencies obtained for DES were 51.8% (beta-glucuronidase, incubation at 37 degrees C overnight) and 2.7% (methanolic HCl), respectively. Similarly, efficiencies for NT ranged from 43.1% (beta-glucuronidase, incubation at 55 degrees C for 2 h) to 12.7% (methanolic HCl). The results highlight that within control laboratories significant underestimation of drug residue content in samples may occur, due to poor deconjugation.

Differential effect of estradiol on antibody secretion of murine hybridomas.

The need for increased antibody production by hybridomas has been approached by the addition to cell cultures of different growth factors; in vitro addition of estradiol-17beta (E2) to human blood lymphocytes increases the accumulation of plasma-blasts and Ig-secreting cells. Four different murine-murine hybridomas secreting different monoclonal antibodies (MAbs) were treated with E2. Specific antibody concentration was measured by enzyme-linked immunoadsorbent assay (ELISA) in culture supernatants whereas expression of E2-receptor in the hybridoma cells was determined by polymerase chain reaction (PCR). When E2 was added as a growth supplement to alpha-estrogen receptor positive murine-murine hybridomas it enhanced MAb secretion by as much as 255%, in a dose-dependant manner. This effect lasted for as long as the alpha-estrogen receptor was detected in the hybridoma cells, was inhibited by tamoxifen and was not observed in alpha-estrogen receptor negative hybridomas. The synthetic estrogen analogue diethylstilbestrol had no effect. Estradiol-17beta should be added to the list of hybridoma-inducing growth factors.

Immune alterations in geriatric mice dosed subacutely with diethylstilbestrol (DES).

Exposure to both physiological and pharmacological doses of estrogenic compounds has been reported to alter immunologic responses in humans as well as in developmental and adult murine models. Despite the current therapeutic use of potent estrogens, including diethylstilbestrol (DES), in geriatric human disorders, elucidation of the effects of these agents on the aged immune system is limited. The present report describes highly significant alterations in the thymus and bone marrow of aged mice (21 +/- 1 months) exposed subacutely to DES. Severe thymic hypocellularity developed in treated mice following five consecutive days of intraperitoneal injection with 1.5 or 6.0 mg kg(-1) DES. Cell maturation within the thymus was also affected, as indicated by a significant decrease in CD4+8+ cells and a concomitant increase in CD4-8- cells. Cellularity of the bone marow was unchanged by DES. However, significant changes were observed in percentages of bone marrow cells expressing surface antigens CD45 (common leukocyte lineage), Mac-1 (macrophage lineage) and CD45R (B-lineage lymphocytes). Both percentages and total numbers of cells in the spleen expressing Thy 1.2 (T-lineage lymphocytes) were also reduced. These immune changes in geriatric mice exposed to DES were similar in direction but more severe than those reported in either young adult or perinatal models. These data may suggest a need for considering the geriatric immune system separately from other age groups when determining effects of immunosuppressive or immunotoxic compound exposure.

A 125I-radioimmunoassay for diethylstilbestrol in serum of patients with prostatic cancer treated with stilphostrol.

A new radioimmunoassay for determining diethylstilbestrol in serum using N-(4'-OH-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)-hex anamide as a radiotracer and a double antibody as a separation reagent is described. The radiotracer is prepared by synthesizing 6-(4-O-diethylstilbestryl)-hexanoic acid and coupling its succinimidyl ester with mono-[125I]tyramine in tetrahydrofuran (16 h, 20-22 degrees C). The standard curve is linear (semi-log transformation) and the assay is sensitive (< 0.022 pmol/tube), reproducible (intra- and interassay coefficient of variation values, 5.3 and 8.1%, respectively), and accurate (recovery values, 95-101%), with a non-specific binding less than 3.2%. Diethylstilbestrol concentrations measured in sera of nine patients with prostatic cancer by the proposed assay ranged from 0.170 to 2.517 mumol/l, which corresponded to an only three-fold dosage variation. In all cases tested, dosing was adequate to retain markers of prostatic cancer in serum within accepted limits; nevertheless, individualization of dosing may be necessary to minimize toxicity.

Neutrophil involvement in effects of diethylstilbestrol and strontium 89 on macrophage activation by Propionibacterium acnes.

We have recently demonstrated that diethylstilbestrol (DES) significantly suppresses macrophage (M phi) activation by Propionibacterium acnes. Because the initial activation of M phi by P. acnes appears to involve the close interaction of the killed bacteria with inflammatory neutrophils (PMN) and resident M phi in the peritoneal cavity, we investigated whether the DES inhibition of M phi activation was associated with inhibition of the PMN response. Our data demonstrate that treatment of mice with DES did not interfere with the acute inflammatory peritoneal PMN influx 5 h after P. acnes injection. DES treatment also did not affect development of the early (day 4) tumor cytotoxic activity of P. acnes activated M phi; this M phi activity has been shown to be mediated by the acute PMN influx. DES treatment, however, did reduce M phi activation as evidenced by alterations in other markers typically associated with M phi activation by P. acnes, including the characteristic reductions in alkaline phosphodiesterase (APD) ectoenzyme activity and the total RNA synthesis, as well as the characteristic persistence of the peritoneal PMN response seen on days 4 and 7 after P. acnes injection. In addition, M phi activity 7 days after P. acnes injection was inhibited in DES treated mice, as evidenced by reduced antitumor activity, and alteration of the markers mentioned above. As a second approach to elucidate the involvement of the acute and persistent PMN response in the M phi activation process, we depleted mice of circulating PMN by treatment of mice with 89Sr before administration of P. acnes.(ABSTRACT TRUNCATED AT 250 WORDS)

[Demonstration of specific antibodies against diethylstilbestrol during treatment of prostatic cancer]. / Mise en évidence d'anticorps spécifiques anti-diéthylstilboestrol au cours du traitement du cancer prostatique.

Antibodies against diethylstilboestrol were detected in prostatic cancer patients treated with diethylstilboestrol (DES). Direct radioimmunoassay (DRIA) was performed in 109 patients divided into three groups: a control group of 33 patients (group I), a group of 38 patients treated by DES and free of cardiovascular complications (group II), and a group of 38 patients treated by DES with cardiovascular complications (group III). Antibody count was significantly higher in group III than in the two other groups (p less than 0.05). These results suggest that DES antibodies may play a role in estrogen-associated cardiovascular toxicity. For this reason, DES should not be used in patients with a positive assay.

An improved radioimmunoassay for the anabolic agent hexoestrol, using a monoclonal antibody.

A monoclonal antibody was raised against hexoestrol coupled to bovine serum albumin. The antibody cross-reacted with the stilbenes, diethylstilboestrol (10%) and dienoestrol (4%), but had no cross-reaction (less than 0.01%) with other anabolic agents. A radioimmunoassay method using the monoclonal antibody has been validated and used to measure residues of hexoestrol in the urine of treated cattle. The limit of detection was 0.6 pg/ml urine at the 95% confidence limit. The results were compared with those obtained using polyclonal antibodies. Although there was a good correlation between the results, the use of monoclonal antibody gave more reliable results than those obtained with available polyclonal antibodies. The monoclonal antibody, because of its quality and theoretically limitless supply, is very suitable for use in large scale screening or monitoring programmes for regulating the use of hexoestrol.

[Immunogenicity of synthetic sex hormones and thrombogenesis]. / Immunogénicité des hormones sexuelles synthétiques et thrombogenèse.

The ingestion of synthetic steroidal and non-steroidal estrogens may induce antiestrogen antibodies in women on oral contraceptives, and in prostatic patients treated with diethylstilbestrol (DES). Natural sex hormones have no such effect. A radioimmunoassay with tritiated ethinylestradiol or DES was applied to study the prevalence of synthetic sex hormone antibodies in 2 populations: 100 women on estroprogestative hormones and 93 cases of DES treated prostatic cancers. Homologous non-treated controls were compared. Results allowed to identify among treated and asymptomatic subjects an immunoreactive population of 30% women and 47% men. Furthermore, the antibodies were found with a much higher frequency (p less than 0.001) in patients who had experienced a thromboembolic disease while on treatment: 90% of women and 74% of men. The importance of these antibodies as a risk factor, their possible role in promoting vascular lesions, the interest of their detection for the prevention of the vascular risk induced by synthetic sex hormones, are considered.

Estrogen immunosuppression is regulated through estrogenic responses in the thymus.

Previous studies have clearly established that physiologic and pharmacologic levels of estrogens modulate immunologic responses that result in simultaneous activation of the reticuloendothelial system and depression of cell-mediated immunity. The mechanisms of estrogen immunoregulation were examined in adult female mice administered pharmacologic levels of exogenous estrogens. Evaluation of steroidal and nonsteroidal compounds with varying degrees of estrogenicity (i.e., uterotrophic activity) provided evidence that their immunotoxicity, for the most part, correlates with estrogenicity. The mechanisms responsible for these effects appear to be complex, mediated through a direct chemical interaction with lymphoid target cells, as well as with nonlymphoid tissue, resulting in the release of soluble immunoregulatory factors. The latter phenomenon was examined in detail and it appears to constitute a regulatory factor(s) produced by thymic epithelium in response to an estrogen stimulus. This response is not only estrogen specific but may involve specific binding to estrogen receptors or receptor-like structures present in cytosol preparations from thymic epithelial cells.

Ethinylestradiol and diethylstilbestrol induced antibodies and vascular thrombosis.

Antiethinylestradiol antibodies were demonstrated in several oral contraceptive users. The antibodies could be precipitated from serum immune complexes by 25% saturated ammonium sulfate. This test of serum precipitation was applied to a comparative study of 116 women on O.C and 65 men treated with diethylstilbestrol for a prostatic cancer. Controls without hormones were included in each group. The test was shown to discriminate among the estrogen users reactive and unreactive persons. 34% of women and 41% of men had high levels of precipitated immunoglobulins in 25% saturated ammonium sulphate. Study of the specificity of the Igs purified from the precipitates in reactive cases, showed that they were binding ethinylestradiol with an affinity constant consistent with an antigen antibody reaction. It is concluded that the oral ingestion of two different synthetic estrogen compounds may induce antiestrogen antibodies. The relation of these antibodies with the increased incidence of vascular thrombosis is considered.

PIP: This work investigated the effects of ethinylestradiol (EE) and diethylstilbestrol (DES) on serum immunoglobulin levels, and these findings were compared; in addition, the correlation with vascular thrombosis was investigated. 2 populations were studied: Population 1 consisted of 166 women who were divided into 3 groups--A) 50 healthy women, mean age 27 years, who had never used the pill or other hormones (controls); B) 100 women, mean age 29 years, who were taking the pill without thrombotic complications; and C) 16 women, mean age 33 years, with a vascular thrombosis during oral contraceptive use. Population 2 consisted of 108 men who were divided in 4 groups and were aged 58-87 years: 1) 24 men with no evidence of cancer (controls); 2) 19 men with prostatic cancer never treated with DES; 3) 47 men with prostatic cancer treated with DES; and 4) 18 men with a thrombosis during DES treatment for prostatic cancer. Anti-EE antibodies could be precipitated from serum immune complexes by 25% saturated ammonium sulfate, and this test was applied to the comparative study. The test discriminated among the estrogen users reactive and unreactive; 34% of women and 41% of men had high levels of precipitated immunoglobulins in the 25% saturated ammonium sulfate. When the specifity of the immunoglobulins was studied in reactive cases, it was found that they were binding to EE with an affinity constant consistent with an antigen-antibody reaction. Hence, the study concludes that oral ingestion of 2 different synthetic estrogen compounds may induce anti-estrogen antibodies. These increased anti-estrogen antibodies may be associated with the incidence of vascular thrombosis.

Methods for controlling the application of anabolics in farm animals.

In the use of anabolic agents, the most pronounced effect on growth is caused by estrogens. For this reason primarily attention will be given to the methods of detection of estrogen administration to fattening animals. The detection methods can mainly be divided in histological, biological, chemical, and immunological determinations and these will be briefly discussed in the light of the present situation in many countries, where the use of anabolic agents is prohibited. From the point of view of control, this prohibition is much easier to handle than a situation in which the application of some specified products is permitted. The possibilities and limitations of control, when certain anabolic agents are permitted for use, will be discussed and evaluated. The conclusion is drawn that in this latter case a sufficient control is very difficult if at all possible considering the methods of control available at the time.