A simple theoretical treatment of a competitive enzyme-linked immunosorbent assay (ELISA) and its application to the detection of human blood group antigens.

A theory has been developed to explain the behaviour of a competitive enzyme-linked immunosorbent assay (ELISA) in which an immobilized antigen competes with a liquid-phase antigen for a limiting amount of antibody. The binding of antibody to antigen on a solid surface (microtitre well) is described in terms of Langmuirian adsorption with a binding constant kappa. Two equations are presented to describe the behaviour of the ELISA signal as a function of competing antigen concentration; an exact equation and an approximate equation which can be used when the surface coverage of the immobilized antigen is not known. It is shown how curves of ELISA signal vs. competing antigen concentration depend on K/kappa [antibody]. The theory has been tested using several immobilized blood group A antigens competing with ovarian cyst fluid A substance and found to adequately describe these competitive ELISAs which have a detection limit of approximately 1 ng of blood group antigen.

Biochemical characterization of glycoprotein components in human ovarian cyst fluids by lectins.

1. Perchloric acid-soluble glycoprotein fractions (PASFs) were separated, in yields of 180-610 mg per 100 ml of cyst fluid, from the cyst fluids of human ovarian cystadenomas (OCAs) in benign and borderline and ovarian clear cell carcinoma (OCC) in malignant, and then identified as glycoproteins with 19.1-69.5% carbohydrate by their chemical composition analyses. 2. These PASFs reacted with anti-A, -B, -Lea and/or -Leb sera, but did not react with anti-H, -M and -N sera. All of the PASFs reacted with Ricinus communis lectin (RCA-I). Among these PASFs, PASFs from the cyst fluids of OCA in benign did not react with Arachis hypogaea anti-T lectin (PNA) and both of the anti-N lectins of Vicia graminea (VGA) and Vicia unijuga (VUA), and PASFs from OCA in borderline reacted with only PNA and PASFs from OCC in malignant reacted with the three lectins, PNA, VGA and VUA. However, none of PASFs from human normal sera and various normal organ tissues reacted with any of RCA, PNA, VGA and VUA. 3. The above-mentioned results seem to show that the examination of glycoproteins of ovarian cyst fluids by the combined use of RCA-I, PNA and VGA (or VUA) permits biochemical diagnosis of the canceration degree of ovarian cystomas.

Measurement of CA125, carcinoembryonic antigen, and alpha-fetoprotein in ovarian cyst fluid: diagnostic adjunct to cytology.

This study used CA125, carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) to classify ovarian cysts by measuring the levels of the three antigens; this information was useful when fluid obtained through laparoscopic puncture of ovarian cysts was submitted for cytologic examination from patients for whom tissue was unavailable for classification. We studied 136 consecutive cyst fluids (108 benign, 28 malignant) and correlated the findings with the tissue diagnosis. All three antigens were very low (CEA, less than 0.5 ng/ml; CA125, 55-2,143 mu/ml; and AFP, less than 4.8 ng/ml) in follicular and lutein cysts. Markedly elevated CA125 (296-1,950,000 mu/ml) and low CEA (0.5-220 ng/ml) and AFP (less than 4.8 ng/ml) levels were seen in patients with serous neoplasms, both benign and malignant. Elevated CEA (greater than 600 ng/ml) and CA125 (56-65,330 mu/ml) levels were seen in primary mucinous cystadenoma and cystadenocarcinoma. Two patients with colonic carcinoma metastatic to the ovary had an elevated CEA (greater than 600 ng/ml) and a normal CA125. Only one patient, with a malignant teratoma, had an elevated AFP. The adjunctive use of CEA and CA125 is recommended for the classification of ovarian cysts.

The 'simple' ovarian cyst: aspirate or operate?

One hundred ultrasound-guided ovarian cyst punctures were performed in 88 patients. To minimize the risk of unexpected malignancy, only persistent or painful cysts less than 10 cm in diameter were aspirated, cysts with solid areas or multiple locules were excluded. Cytological diagnosis was not possible in 72 of the 100 fluids; of the others 20 contained cells suggestive of follicular or luteal cysts, four samples suggested endometriosis and four benign tumours of epithelial origin. Oestradiol levels were high in 54 cystic fluids, and a combination of oestradiol estimation and cytology facilitated the identification of a follicular origin. Most such patients would normally have undergone surgery, but this was eventually required in only 10 of 60 in whom the cyst fluid was clear or slightly blood-stained and in 16 of the 28 with heavily blood-stained fluid. Ultrasound-guided ovarian cyst puncture would appear to be a valid alternative to surgery for carefully selected benign ovarian cysts especially when the cyst is considered not to contain blood.

[Determination of N-acetylneuraminic acid for the differential diagnosis of cystic ovarian tumors]. / Bestimmung der N-Acetylneuraminsäure zur Differentialdiagnose zystischer Ovarialtumoren.

The concentrations of N-acetylneuraminic acid (NANA) were determined in the fluid of cystic ovarian tumors and in the blood serum from a total of 95 patients. The estimation of NANA was based on the method of Svennerholm. It was found that the mean content of NANA in the cystic fluid is more than 3.0 mmol/l in serous and more than 5.5 mmol/l in mucinous malignant ovarian tumors. The mean value of NANA in the serum of patients is mostly more than 3.5 mmol/l if there is an ovarian cancer. The estimation of NANA is useful for the preoperative diagnosis of cystic ovarian tumors.

Tumour-associated trypsin inhibitor (TATI) in human ovarian cyst fluid. Comparison with CA 125 and CEA.

The levels of tumour-associated trypsin inhibitor (TATI), CA 125 and CEA were measured in ovarian cyst fluids from 21 patients. TATI in cyst fluid was immunologically and physicochemically similar to the peptide originally isolated from the urine of a patient with ovarian cancer. Mucinous cysts contained significantly higher levels of TATI than did serous cysts. Immunohistochemically TATI was localized in the apical parts of cells of mucinous ovarian cysts. These results suggest that this tumour-associated peptide is actually produced by a tumour. Like TATI, CEA occurred at higher concentrations in mucinous than in serous cyst fluids, whereas CA 125 was found in higher concentrations in serous than in mucinous cyst fluids. The concentrations of these tumours markers in cyst fluids did not correlate with circulating levels of the same markers. In spite of the very high levels of all these tumour markers in benign cyst fluids, serum levels were normal or only slightly elevated. Clearly elevated serum levels occurred only in patients with malignant tumours. Cyst fluid levels of these tumour markers could not be used to distinguish between benign and malignant tumours.

Tumor markers in the human ovary and its neoplasms. A comparative immunohistochemical study.

The incidence and histologic characteristics of the expression of placental alkaline phosphatase (PLAP) in ovarian tumors was compared with that of five other tumor antigens. Three monoclonal antibodies were used for the specific localization of PLAP. PLAP was present in some sex cord cells of the 13-16-week fetal ovary, probably germ cells. In normal ovaries, all antigens except carcinoembryonic antigen (CEA) were frequently found in inclusion cysts; the germinal epithelium was positive only for cancer antigen 125 (CA 125). The frequency and extent of PLAP expression in nonmucinous carcinomas was higher than observed for CA 19-9 and CEA, but was lower than for CA 125 and human milk fat globule antigen. Serous tumors had the highest PLAP expression, followed by endometrioid and poorly differentiated adenocarcinomas, and some other tumors. PLAP was predominantly membranous; its histologic distribution was in general heterogeneous. Different antibodies to PLAP gave different staining intensities in some tumors, but the staining patterns were always qualitatively identical.

Placental protein 10: levels in benign and malignant ovarian cyst fluids and pleural exudates.

Placental protein 10 (PP10) was measured in benign (n = 47) and malignant (n = 10) ovarian cyst fluids and in pleural fluid specimens from 19 patients with pleural metastases of various carcinomas (n = 15) or infectious pleurisy (n = 4). PP10 was found in 9 out of 18 follicular or luteal cyst fluids (range 2.0-42.0 micrograms/l) and in 24 out of 29 benign ovarian tumours (range 2.6-38.0 micrograms/l). Nine out of ten malignant ovarian cyst fluids contained detectable PP10 (range 4.0-55.2 micrograms/l). PP10 was found in all the pleural fluids from 15 patients with metastatic bronchial, pulmonary, breast or pancreatic carcinomas (range 5.1-100.2 micrograms/l), as well as from four patients with pleurisy (range 5.0-28.6 micrograms/l). Our results indicate that PP10 is tumour-associated, but not tumour-specific.

Typing of core and backbone domains of mucin-type oligosaccharides from human ovarian-cyst glycoproteins by 500-MHz 1H-NMR spectroscopy.

Human blood-group A active glycoproteins from ovarian-cyst fluid were subjected to Smith degradation and subsequent beta-elimination. The resulting oligosaccharide-alditols represent the core and backbone domains of the O-linked carbohydrate chains. Nine of these, ranging in size from disaccharides to hexasaccharides, were investigated by 1H-NMR spectroscopy. Their primary structures could be adequately characterized. In particular, the core types, i.e. the substitution patterns of N-acetylgalactosaminitol (GalNAc-ol) as well as the types of backbone, i.e. the linkage types of alternating Gal-GlcNAc sequences, were unambiguously identified. The core type GlcNAc beta(1-3)GalNAc-ol is described for the first time as occurring in ovarian-cyst glycoprotein.

Unique finding of prostatic tissue in a benign cystic ovarian teratoma.

Benign cystic ovarian teratomas are neoplasms with a 46,XX karyotype originating from a single abnormal ovarian germ cell after the first meiotic division. Since these tumors lack the Y chromosome (required for production of the H-Y antigen that is presumably obligatory for embryonic male sexual differentiation), identifiable male sex organ structures should not be found in the tumor. We report a case in which prostatic tissue was identified in a benign cystic ovarian teratoma.

[Intracellular distribution of estrogen receptors in the human endometrium]. / Vnutrikletochnoe raspredelenie éstrogennykh retseptorov v éndometrii cheloveka.

Fifty samples of endometrial tissue from healthy women with regular menstrual cycle and from patients with uterine myoma, ovarian cystoma, uterine hypoplasia and endometriosis were studied. The amount of cytoplasmic (total and free) and nuclear estradiol-binding sites as well as estradiol (E2) concentration in the endometrial cells were estimated. Level of E2 in the peripheral blood plasma did not correlate with its content in endometrial cells. At the same time, amount of cytoplasmic estrogen receptors bound with E2 did not correlate with the estradiol content in cytoplasm. The data obtained suggest the leading role of the cytoplasmic estrogen-receptors interaction in development of hormonal effect on the tissue. The formation of hormone-receptor complexes appears to be controlled by some additional factors of unknown nature.

Carbon nuclear magnetic resonance spectra of oligosaccharides isolated from human milk and ovarian cyst mucin.

Natural abundance Carbon-13 nuclear magnetic resonance spectra at 20 MHz were reported for the three common human milk oligosaccharides, lacto-N-tetraose and lacto-N-fucopentaoses I and II, as well as for two related tetra- and hexasaccharide alditols isolated from the alkaline borohydride degradation products of an ovarian cyst glycoprotein. Spectral assignments made with the help of deuterium-induced shift (DIS), attached proton test (APT), and T1 data indicated some very irregular glycosylation shifts which were attributed to effects of steric crowding and non-nearest-neighbor interactions. Samples as small as 10 mumol of oligosaccharide gave acceptable 20-MHz spectra with the use of a 5-mm probe coil.

[Trace element content in the blood and tissues of patients with precancerous and precursor diseases of the female genitalia]. / Soderzhanie mikroélementov v krovi i tkaniakh bol'nykh pri nekotorykh predrakovykh i fonovykh zabolevaniiakh zhenskikh polovykh organov.

Emission spectrum analysis was used for measuring the levels of such trace elements as copper, manganese, zinc, nickel and silver in blood serum and tissues of the diseased organs in female patients with certain precancerous and precursor lesions of genital tract. It was found that such changes as an increase in serum copper, a decrease in zinc concentration in blood cells and a rise in the levels of zinc and silver in the affected tissues were more pronounced in cases of precancerous lesions than in those of precursor pathology.

The combination of normal-phase and reverse-phase high-pressure liquid chromatography with NMR for the isolation and characterization of oligosaccharide alditols from ovarian cyst mucins.

Normal-phase high-pressure liquid chromatography (HPLC) on amino-bonded silica with elution by aqueous acetonitrile is shown to be an especially suitable complement to reverse-phase HPLC on octadecyl silica for the fractionation of oligosaccharide alditols produced by alkaline borohydride degradation of mucin glycoproteins. The former technique separates well on the basis of molecular size, while the latter method shows selectivity for stereoisomers. Stereoisomeric pairs of tetra-, penta- and hexasaccharide alditols show relative retention times ranging from 3 to 12, resulting in excellent preparative separations in reverse-phase chromatography. From a single ovarian cyst glycoprotein, H and Lewis b active, 13 oligosaccharides, representing essentially the entire carbohydrate content, have been isolated. The structures of 12 of the oligosaccharides have been determined by 1H-NMR spectroscopy. For those oligosaccharides which have been isolated from other sources and whose NMR spectra have been previously reported, unambiguous structural identification follows directly. Structures of oligosaccharides differing by only one or two residues from those whose NMR spectra are known may be deduced by a simple algorithm utilizing chemical shift analogies.

Immunochemical studies on blood groups. Purification and characterization of radioactive 3H-reduced di- to hexasaccharides produced by alkaline beta-elimination-borohydride 3H reduction of Smith degraded blood group A active glycoproteins.

Treatment of blood group A active glycoprotein from human ovarian cyst fluid by one stage of Smith degradation followed by alkaline beta-elimination in the presence of NaB[ 3H4 ] (Carlson degradation) liberated tritiated oligosaccharide alditols. The carbohydrate mixture was fractionated by gel filtration, elution from charcoal, paper chromatography, and high pressure liquid chromatography. Structures were established based on sugar composition, periodate oxidation, methylation analysis, and analysis of oligosaccharide alditols as permethylated and N-trifluoroacetylated derivatives by gas-liquid chromatography-mass spectrometry. The following structures have been deduced: Gal beta 1----3GalNAc-ol, GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1---- 3GlcNAc beta 1----6(3-deoxy)GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1----3[GlcNAc beta 1----6]GalNAc-ol, Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3[Gal beta 1----4GlcNAc beta 1----6]Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol. The smaller structures represent pieces of the larger structures. Together they provide direct evidence for the core structure of the carbohydrate side chains in the blood group substances as proposed by K. O. Lloyd and E. A. Kabat [1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1470-1477). Oligosaccharides previously isolated after Carlson degradation of intact human ovarian cyst fluid HLeb , Lea, and B substances and from human and horse B substances contained various alpha-linked L- fucopyranose and alpha-linked Gal substitutions on the composite structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Assay for characterization of human follicular oocyte maturation inhibitor using Xenopus oocytes.

Human follicular fluid from healthy mature Graafian follicles and from pathologic ovarian cyst fluid was found to be inhibitory to progesterone-induced meiotic maturation of oocytes from the South African clawed toad, Xenopus laevis. Human follicular fluid but not human serum, collected from the same individuals, demonstrated a linear dose-response inhibition on the maturation of oocytes in the Xenopus assay system. These findings indicate that the human follicular and cyst fluids contain oocyte maturation inhibitor (OMI). This human OMI was inactivated when subjected to a boiling water bath for 2 min. The OMI action was shown to be reversible in its inhibitory action. The fact that OMI can act directly on the oocyte was demonstrated by its inhibitory action on maturation in defolliculated oocytes. The findings demonstrate that the inhibitory action of human OMI is not species-specific. Xenopus oocytes provide a simple, readily available, year-round bioassay material for testing follicular oocyte maturation inhibitor.

Neonatal ovarian cyst: prenatal diagnosis and analysis of the cyst fluid.

Ovarian cysts of clinical significance are uncommon in the fetus and neonate. The authors diagnosed a large fetal cyst prenatally by ultrasound scan and removed it in the neonatal period. Analysis of the fluid obtained from the cyst demonstrated a capacity of the fetal ovary to synthesize estrogen and progesterone.