Jozef De Boever

Expression of Ly49E and CD94/NKG2 on fetal and adult NK cells.

Murine NK cells express inhibitory receptors belonging to the Ly49 and CD94/NKG2 family. Ly49E and CD94 are the only NK cell receptor transcripts detectable in fetal NK cells. Still unproved is the surface expression of Ly49E on NK cells. Here we generated two novel mAbs, a mAb recognizing Ly49E with cross-reactivity to Ly49C, and a mAb against NKG2A/C/E. Ly49E was immunoprecipitated as a disulfide-linked homodimer with 46-kDa subunits. Removal of N-linked carbohydrates revealed a 31-kDa protein backbone. NKG2A was immunoprecipitated as a 38-kDa protein. Although the frequency of fetal NK cells expressing Ly49E was higher than 25%, it decreased drastically from 2 wk after birth. Phenotypic analysis showed that ∼90% of fetal NK cells and ∼50% of adult NK cells express high levels of CD94/NKG2. The remaining 50% of adult NK cells expressed low surface levels of CD94/NKG2. Expression of Ly49E and CD94/NKG2 was not restricted to NK cells, but was also observed on NK T and memory T cells. Functional analysis showed that sorted Ly49E+ and CD94/NKG2+ fetal NK cells could discriminate between MHC class I-positive and MHC class I-negative tumor cells. We also demonstrated that Ly49E becomes phosphorylated following pervanadate stimulation of fetal NK cells. The expression levels of Ly49E and CD94/NKG2 were similar in wild-type compared with β2-microglobulin−/− mice. In conclusion, generation of mAbs against Ly49E and NKG2 extended the phenotypic and functional characterization of NK cells.

Ovulation stigma and concentration of progesterone and estradiol in peritoneal fluid: relation with fertility and endometriosis

The relationship between the presence or absence of an ovulation stigma and (1) the fertility status, (2) the incidence of endometriosis, (3) the concentration of progesterone and estradiol in the peritoneal fluid, and (4) the blood levels of luteinizing hormone, follicle-stimulating hormone, progesterone, and estradiol in 21 fertile and 45 infertile patients who underwent a laparoscopy in the early (n = 48) or late luteal phase (n = 18) was investigated. An ovulation stigma was observed in about half of the patients, irrespective of their fertility status (past and subsequent), the presence of endometriosis, or the time of the luteal phase. Progesterone and estradiol concentrations in the peritoneal fluid were highest in the early luteal phase, but they were not correlated with the presence or absence of an ovulation stigma. No significant differences were observed in peripheral hormone levels between women with and those without an ovulation stigma nor between women with high or low concentrations of progesterone in the peritoneal fluid. From the data, it is concluded that hormone assays are of no aid in the diagnosis of the luteinized unruptured follicle syndrome and that the absence of an ovulation stigma on laparoscopic examination cannot be equated with the luteinized unruptured follicle syndrome.

Antibody binding efficiency of differently labelled steroid hormones

Abstract The binding of estradiol labelled with 3 H or with external labels (isoluminol and horseradish peroxidase) to anti-estradiol—6-carboxymethyloxime—bovine serum albumin antibodies was compared. The external labels were coupled covalently to the steroid via homologous 6-carboxymethyloxime or heterologous 3- and 17-hemisuccinate bridges. Different factors and conditions influencing this binding and the slope and shape of the calibration graph were studied, including the type of label, the liquid- and solid-phase antibody system, the matrix effect and general incubation conditions, e.g., time and temperature. External homologous labels yielded the most sensitive calibration graphs and the lowest detection limits. Large differences in the binding of the label and in the shape of the calibration graph between the different antibodies were observed.

Screening of sera and tumor extracts of cancer patients using a monoclonal antibody directed against human placental alkaline phosphatase

A sensitive endogenous enzyme immunoassay involving an anti-human placental alkaline phosphatase (PLAP) monoclonal antibody was used in the screening of sera and tumor extracts of patients with various types of cancer. In sera of breast cancer patients an incidence of 5.2% was recorded. This value rose to 43% when tumor extracts were analyzed. For lung and bronchial cancers we found 11.2% seropositive patients. On several occasions a good correlation was observed between the PLAP determinations and the histopathological staging of tumor tissue.

Binding of homologous and heterologous isoluminol- and enzyme-labelled progesterone conjugates to monoclonal antibodies

Abstract The binding of three different progesterone-isoluminol and three different progesterone-enzyme (HRP) conjugates to monoclonal antibodies against progesterone-7-carboxy thioether-bovine albumin (clone 2H4) and progesterone-11-hemisuccinate-bovine serum albumin (clone 1E11) was compared. The isoluminol labels were covalently bound to progesterone via hemisuccinate bridges at carbon atoms 3, 7 or 11. The enzyme labels were covalently attached to the steroid using a carboxymethyl-aminocaproic acid bridge at carbon atom 3 or a hemisuccinate bridge at carbon atom 11. The influence of several factors on the binding between antibodies and conjugates and on the slopes of the calibration curves was studied. Considerable differences in the binding of the label and in the shape of the curves between both antibodies was observed. In enzyme immunoassay, using clone 1E11, the binding of the progesterone-enzyme conjugate and the shape of the curves was governed by the presence in the reaction mixture of the antibodies in liquid or solid-phase conditions.

Application of chemiluminescence immunoassays for steroid hormones in clinical endocrinological investigations in women

Abstract Immunoassays based on chemiluminescence for the measurement of serum and plasma steroids (estradiol, estriol, progesterone, testosterone, and cortisol), urinary steroid conjugates (estrone-3-glucuronide, estriol-16α-glucuronide and pregnanediol-3α-glucuronide) and peptide hormones (choriogonadotropin and luteinizing hormone) are surveyed briefly. These immunoassays are simple, robust and valid alternatives to radioimmunoassay. Homogeneous procedures and recent solid-phase assays based on purified specific antibodies, covalently coupled to polymer beads are discussed. Some new results are presented for solid-phase chemiluminescence immunoassays: estradiol is quantified in extracts of serum by using a monoclonal antibody to estradiol with estradiol-6-carboxymethyloxime-aminobutylethyl isoluminol as the marker ligand, and progesterone is quantified in unextracted serum by using a polyclonal antibody to progesterone, progesterone-11-hemisuccinyl-aminobutylethyl isoluminol as the marker ligand, and danazol (17α-pregna-2,4-dien-20-yno[2,3-d]-isoxazol-17-ol) to displace progesterone from serum binding-proteins. Their clinical utility is demonstrated.

Serum estradiol measurement by solid-phase chemiluminescence immunoassay and direct radioimmunoassay

Abstract Estradiol017β is determined in serum extracts by solid-phase chemiluminescence immunoassay. The results are compared with those obtained from unextracted serum in routine conditions with a commercial radioimmunoassay (r.i.a.) kit. For the chemiluminescence procedure, a purified monoclonal antibody to estradiol-6-carboxymethyloxime/bovine serum albumin and the homologous chemiluminescent marker conjugate estradiol-6-carboxymethyloxime aminobutylethylisoluminol are used. Bound and free ligand are separated by washing and simple centrifugation. Results obtained by the chemiluminescence assay ( y ) and by r.i.a. ( x ) on 170 serum specimens from women during ovulation induction showed good correlation ( y = 1.01 x − 16 with r = 0.95). The methods are similar in selectivity, detection limit (ca. 10 ng l −1 ) and precision (interassay relative standard deviation, 8–13%).

Direct chemiluminescence immuoassay of estriol and progresterone and their ratio during pregnancy

Abstract Estriol (E3) and progesterone (P) concentrations in saliva were determined by direct chemiluminescence immunoassays, using solid-phase monoclonal antibodies bound to the wells of microtitre plates, and isoluminol-labelled steroids conjugates. Saliva samples were obtained from women during pregnancy, from 34 to 1 weeks before delivery. The smoothed median and mean salivary E3:P ratios in normal pregnancy rose gradually from 0.45 at -34 weeks to 1.5 at -1 week. In twin pregnancy a similar rise was observed. However, in preterm delivery the smoothed mean ratio rose more slowly, from 0.6 at -30 weeks to 1.1 at -1 week. In pregnancy associated with intra-uterine growth retardation, the smoothed mean ratio did not reach a value of 1 towards the end of pregnancy. In all instances high and low E3:P ratios were observed shortly before spontaneous delivery. This raises the question of whether the E3:P ratio could be used as a predictor providing useful information in relation to the onset of labour.

Correlation between peripheral CA-125 levels and ovarian activity**Supported in part by the Abbott Diagnostics Division, Ottignies, Belgium.

Serum CA-125 concentrations were measured at three different times in normal cycles, pill-suppressed cycles, and cycles stimulated for intrauterine insemination (IUI) or oocyte retrieval, i.e., (1) during the first half of the cycle, (2) at midcycle or at the moment of oocyte retrieval, and (3) the second half of the cycle. Significant variations of serum CA-125 concentrations were not seen during the cycle in normally cycling women or in women taking oral contraceptives: mean +/- SD 28.9 +/- 13.3 U/mL and 26.9 +/- 11.3 U/mL, respectively. In patients stimulated for in vitro fertilization, luteal phase CA-125 levels (60.6 +/- 38 U/mL) were significantly higher than during stimulation (21.5 +/- 5.9 U/mL) or at oocyte retrieval (19.6 +/- 6.4 U/mL). In stimulated cycles for IUI, without laparoscopy or follicular puncture, a comparable rise of CA-125 was observed in the luteal phase (49.6 +/- 37.8 U/mL). However, in patients undergoing laparoscopic sterilization, serum CA-125 concentrations before and after laparoscopy were not significantly different (22.8 +/- 6.3 U/mL and 25 +/- 4.2 U/mL, respectively).

Multiple lutein cysts in an uncomplicated pregnancy

Multiple lutein cysts, pregnancy luteomas and large solitary luteinized follicle cysts are three uncommon luteal formations of the ovary which can arise in pregnancy. They have to be differentiated from the corpus luteum of pregnancy which frequently becomes cystic but usually regresses after the first trimester. In multiple lutein cysts both ovaries are markedly enlarged by multiple follicular cysts which are characterized by luteinization of the theca interna layer. The pregnancy luteoma appears in the form of solid, often bilateral ovarian masses consisting of nodular proliferation of lutein cells. Recently, eight patients with large solitary luteinized follicular cysts occurring during pregnancy or the puerperium have been reported (Clement & Scully 1980). Multiple lutein cysts occur most frequently in pregnancies associated with abnormal trophoblastic growth and excessive human chorionic gonadotrophin (hCG) production such as hydatidiform mole, choriocarcinoma, erythroblastosis fetalis and multiple pregnancy. They can also develop during an apparently normal pregnancy but thorough hormonal investigation of such a case has never been reported.