Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

Serum IgG-like glycoferroprotein: identification of its final dissociation form of thermostable protein coupled with albumin.

Human serum IgG-like glycoferroprotein identical to ascitic IgG-like glycoferroprotein that binds labeled monoclonal antibodies to CA125 is a complex consisting of three proteins: IgG, human serum albumin, and unidentified thermostable protein. Final dissociation form of serum IgG-like glycoferroprotein also appears as a complex of three nonidentical polypeptides with a molecular weight of 55 kDa (PC55) migrating in the albumin zone of thermostable protein coupled with albumin and structures chemically identical to human serum albumin and IgG heavy chains. Under denaturing conditions of electrophoresis in polyacrylamide gel, IgG-like glycoferroprotein and PC55 have the same molecular weight (about 55 kDa), while under reducing conditions their weight is about 75 kDa. Transition form (form the lower to the higher molecular weight) appears as an oblique (at about ≈ 30°) protein band creating a ladder string effect. Ladder string effect was reproduced with thermostable protein coupled with albumin, PC55, IgG-like glycoferroprotein, with all commercially available human and bovine albumins, rat albumin as well as with heated and renatured albumins and can serve as electrophoretic identification sign for thermostable protein coupled with albumin. Renatured after boiling (100°C for 15 min) bovine albumin under reducing conditions appeared as bow string twisted in helix, that raises molecule in 2.5 turns from ≈ 2 to ≈ 75 kDa. These data attest to the existence of an albumin double and to its possible double structure.

Identification and characterization of serum protein in patients with ovarian cancer.

A 36 kDa protein was isolated from the sera of patients with ovarian cancer and rabbit antisera to this protein were prepared. Precipitation test with these antisera detected an antigen with electrophoretic mobility corresponding to alpha-1-globulins and molecular weight of 36 kDa. Direct comparison of precipitating test systems showed that this antigen is not identical to the known carcinoembryonic, placental, and reactive proteins. Serum alpha-1-globulin was not detected in the sera of healthy humans, pregnant women, newborns, and in human adult and fetal visceral tissues at the level of precipitating test system sensitivity 1 mg/liter. It was detected in the sera of patients with ovarian cancer, in ovarian tumor (cancer) tissues, in the contents of ovarian tumor cavities, and in concentrated specimens of amniotic fluid. The antigen was not detected in ascitic fluid of patients with ovarian cancer, but it was present in 75% serum samples from these patients. The antigen was called serum oncoovarian alpha-1-globulin. SDS-PAAG electrophoresis showed that this antigen is an oligomer consisting of subunits (monomers) with molecular weight of 36 kDa. Under denaturing conditions in the presence of 2-mercaptoethanol these monomers dissociate into polypeptide chains with a molecular weight of 18 kDa. The protein is liable to oligomerization. Comparative characteristics of serum oncoovarian alpha-1-globulin and CA-125 antigen are presented.

Hyperfine interactions in the iron cores from various pharmaceutically important iron-dextran complexes and human ferritin: a comparative study by Mössbauer spectroscopy.

Mössbauer spectroscopy has been used to study the hyperfine interactions in the iron cores of pharmaceutically important industrial and elaborated iron-dextran complexes (ferritin models) and human ferritin. Mössbauer spectra of frozen solutions and lyophilized samples of iron-dextran complexes at 87 K demonstrated magnetic, superparamagnetic and paramagnetic states of iron in various complexes. Mössbauer spectra of human ferritin in frozen solution and lyophilized form showed paramagnetic state of iron at 87 K. Small variations of Mössbauer hyperfine parameters were observed for different samples at 87 and 295 K, respectively, supposing the homogenous iron cores. The values of quadrupole splitting for iron-dextran complexes and ferritin in frozen solutions at 87 K varied from 0.639 to 0.744 mm/s while those of lyophilized samples at 87 K varied from 0.714 to 0.788 mm/s. The values of quadrupole splitting for iron-dextran complexes and ferritin in lyophilized form at 295 K varied from 0.687 to 0.741 mm/s. The values of hyperfine magnetic fields on the 57Fe nuclei in several iron-dextran complexes at 87 K varied from approximately 231 to approximately 485 kOe. These small variations of the hyperfine parameters were related to several types of the hydrous iron oxide microstructural modifications in the core and variations of the iron core size. The influence of lyophilization on the iron core structure was also assumed. In addition, Mössbauer spectra were evaluated in supposition of heterogeneous iron core in all samples.

Antigenic structure of ovarian cancer metastases.

Twenty highly specific (organ-specific and tumor-associated) antigens named ovarian metastatic antigens were revealed using polyclonal antibodies isolated from rabbits after immunization with soluble antigens from ovarian cancer metastases. These protein antigens were not detected by precipitating test system (sensitivity 1 mg/liter) in tissues of adult humans (except for organ-specific antigens) and blood plasma from healthy donors. Ovarian metastatic antigens included organ-specific, placenta-specific, tumor embryonic, tumor-associated, and reactive proteins. The precipitating test system identified 15 antigens in the amniotic fluid from pregnant women and 7 antigens in the plasma from patients with ovarian cancer (incidence 16-75%).

[Immunochemical identification of onco-ovarian acid-soluble alpha-2-globulin]. / Immunokhimicheskaia identifikatsiia onkoovarial'nogo kislotorastvorimogo al'fa-2-globulina.

Antisera were obtained by rabbits immunization with pooled extract from ovarian carcinoma and its metastases into the ome tum. Using standard test-system antigen, precipitating in agar, was identified in the tissue of 12 out of 24 primary ovarian carcinomas and in 8 out of 20 metastases. It was not revealed in adult healthy internal organs tissues and in fetal tissues, except embryonal large intestine, where it was determined in trace amounts in isolated samples. Using immunodiffusion method it wasn't determined in blood serum of healthy people and patients, pregnant women and neonates. Immunochemical identification using standard test-systems showed that this antigen is not identical with already known carcino-embryonic antigen, placental, reactive and onco-ovarian proteins. It presents acid-soluble alpha-2-globulin (ASAG-2) with MW 55 kD determined by gel-filtration and 55 kD determined by electrophoresis in 10% polyacrylamide gel with dodecylsulfate under reducing conditions. Physico-chemical and antigenic properties of ASAG-2, as we think, give the opportunity to present it as a new onco-ovarian antigen, which differs from the known proteins.

[Immunoenzyme analysis of ovarian metastatic antigen-8]. / Immunofermentnyi analiz ovarial'no-metastaticheskogo antigena-8.

Immunoenzymatic method for the determination of new ovarian-metastatic antigen-8 in human blood serum was worked out. OMA-8 level was studied in blood serum of 40 healthy women, 40 healthy men, 16 neonates, 33 pregnant women, 103 patients with genital tumours. It was found out that OMA-8 level in blood serum of healthy women changed between 2 to 15.4 micrograms/l, in men between 5-17.0 microgramS/l. OMA-8 concentration higher than 15.4 micrograms/ was considered elevated. The elevated OMA-8 level was marked in neonates (100%) and in pregnant (39.3%). High level was discovered in the amniotic fluid.

[Detection of trophoblastic beta 1-glycoprotein in tumor tissue and blood in ovarian neoplasms]. / Opredelenie trofoblasticheskogo beta 1-glikoproteina v opukholevoi tkani i syvorotke krovi pri rake iaichnika.

Using immunochemical analysis methods (the reaction of precipitation in agar, immunoenzymatic method, immunofluorescence), trophoblastic beta 1-glycoprotein (TBG) concentration in tumour tissue and in the blood serum of patients with ovarian cancer was studied. By rabbits immunization with glycoprotein fraction of ovarian adenocarcinoma, dissolved in 0.6 M sulfosalicylic acid, the authors obtained antibodies to TBG. Immunoenzymatic method showed, that TBG level is raised during ovarian cancer (more than 3 micrograms/l): in 18% of tumour extracts, in 12.5% of blood sera samples and in 41.6% of cases in ascites fluid. Utilizing indirect immunofluorescence method morphological structures of trophoblastic type were identified in paraffin sections of ovarian adenocarcinoma. The authors suppose, that such structures may be responsible for TBG biosynthesis in ovarian tumours.

[Ovarian metastatic antigen in the blood serum of patients with ovarian tumors]. / Antigen metastazov ovarial'nogo raka v syvorotke krovi bol'nykh s opukholiami iaichnikov.

Enzyme immunoassays of ovarian metastatic antigen 8 (OMA-8) in sera of patients with ovarian cancer have shown its elevation in 74.5% of samples. OMA-8 levels did not correlate with histology, differentiation, clinical stage of the tumour or the presence of ascites. Increased OMA-8 levels in sera of 70% of patients with stage I-II ovarian cancer suggest that it may be a marker of this disease.

[Serum ferritin and trophoblastic globulin in genital tumors in women]. / Syvorortochnyi ferritin i trofoblasticheskii globulin pri opukholiakh polovykh organov u zhenshchin.

Blood serum levels of ferritin and trophoblastic globulin were measured by immunoassay in 389 and 114 cases, respectively, suffering malignant or benign tumors of the uterus and ovary as well as in controls. Hyperferritinemia identified at serum ferritin levels in excess of 200 micrograms/l was established in 94% of cases of ovarian cancer, 57%--benign ovarian tumors, 60%--endometrial carcinoma and in 16% of patients with uterine myoma. Patients with ovarian and uterine malignancies were shown to have the highest serum ferritin levels. The study failed to establish an increase in trophoblastic globulin concentration in cases of nontrophoblastic tumors of the genitals. It is suggested that serum ferritin level be measured in patients presenting with ovarian and uterine tumors and in subjects at high risk for ovarian cancer to assure early diagnosis of disease.

[Immunochemical identification of beta2-globulin in metastases of ovarian tumors]. / Immunokhimicheskaia identifikatsiia beta2-globulina v metastazakh ovarial'nykh opukholei.

Antisera were obtained upon immunization of rabbits with the extracts obtained from metastatic tissues of primary ovarian carcinoma into the omentum. Eight antigens were found, which were termed "ovarian-metastatic antigens" (OMA). OMA 1-7 showed cross-reactions with the antigens of one of the normal organs of the adult man: kidneys, spleen, brain. OMA-8 was not identified in the internal organ tissues of the adults and fetuses. Using immunodiffusion method, OMA-8 was revealed in the tissues of metastases of primary ovarian cancer into the omentum in 55% of cases (3-180 mg/l), in 50% of cases it was detected in primary ovarian carcinomas (3-50 mg/l) and in mature placenta (38-40 weeks) (6-12 mg/l). OMA-8 was detected in the chorion (8-20 weeks) and in the amniotic fluid at all periods at the maximum sensitivity of immunodiffusion method (1-2 mg/l). OMA-8 is a beta 2-globulin of protein nature with NW 35 kD, containing alpha- and beta-subunits with MW 18 and 19.5 kD. No carbohydrates, lipids and ferrum were determined in OMA-8. Its physico-chemical and antigenic properties differ from those of the described carcinoembryonic and placental proteins.

[Distribution of cytoplasmic ferroproteins and iron in human kidney tumors]. / Osobennosti raspredeleniia tsitoplazmaticheskikh ferroproteinov i zheleza v opukholiakh pochki cheloveka.

Using immuno- and histochemical techniques, it has been shown that the primary tumoural node in kidney cancer is a locus with an extraordinarily high concentration of cytoplasmic ferroproteins and ferrum (Fe3+). Fe3+ is located in the cytoplasm of nephrothelium of tumour-affected kidney tubules, intensively incrustates the cell cytoplasm in the primary tumoural node and is revealed in separate nuclei of the primary node cells. Possible consequences of high local ferrum concentration in cells is discussed. It is suggested that ferroprotein accumulations and the peculiarities of their distribution in kidney tumours result from the disturbances in ferrum metabolism that antecede the onset of kidney tumours.

[Physico-chemical characteristics of human neurospecific beta 2 globulin]. / Fiziko-khimicheskaia kharakteristika neirospetsificheskogo beta2-globulina cheloveka.

Neurospecific beta 2-globulin was found to be a protein with molecular mass 20,000 +/- 2,000 daltons, exhibiting electrophoretic mobility of beta 2-globulins (0.29 +/- 0,05). Contrary to the S-100 protein the globulin was readily precipitated by ammonium sulfate at 30-60% of saturation. No carbohydrate and lipid components were found in the globulin. The beta 2-globulin appears to be a typical polypeptide.

[Isolation and physico-chemical characteristics of human cancerocerebral antigen]. / Vydelenie i fiziko-khimicheskaia kharakteristika kantserotserebral'nogo antigena cheloveka.

During gel filtration on Sephadex G-200 human cancerocerebral antigen (CCA) was eluted as two protein fractions with molecular mass of 135,000 and 270.000 daltons. Only one band of protein with molecular mass of about 15,000 daltons was noted after electrophoresis in 10% polyacrylamide gel containing SDS. As characteristic properties of CCA were recognized an electrophoretic polymorphism and a distinct trend to polymerization and isomeria. The antigen was not stained with dyes designed for staining base proteins, lipo-,glyco- and ferroproteins; CCA was thermostable (5 min at 80 degrees), it was inactivated by trypsin and protease but was resistant to pronase, hexokinase, alpha-amylase and beta-glucuronidase. A procedure was developed for isolation of CCA from brain, including fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephadex A-50. The procedure enabled to obtain the CCA preparations suitable for radioimmunological, immunobiological assays and amino acid analyses.

[Ferritin study in tumors of the female genitalia]. / Izuchenie ferritina pri opukholiakh zhenskikh polovykh organov.

Ferritin levels in extracts of tumor tissue and serum of patients with gynecological malignancies were assayed by immunodiffusion methods using a standard system of testing for ferritin. A high blood-ferritin level (500-1,000 ng/ml) was found in 8 out 21 patients with ovarian cancer, 8 out of 24 patients with endometrial carcinoma. 2 out of 8 cases of mucinous cystadenoma and in 2 out of 4 cases of pyoovarium. It was lower in other malignancies of the female genital tract. There was no correlation between the ferritin level of blood serum and that of tumor tissue extracts. Although blood serum ferritin was found in very few healthy subjects (2.5%), it is suggested that blood-ferritin level test should be carried out in the course of examination of slowly-progressing purulent adnexitis and mass screening for tumors of female genitals.

[Immunochemical identification of ferritin and its immunological analogs beta-fetoprotein and alpha 2H-globulin]. / Immunokhimicheskaia identifikatsiia ferritina i ego immunologicheskikh analogov beta-ferritina i al'fa 2H-globulina.

Immunochemistry techniques were made use of to identify the immunological analogs of ferritin - beta-fetoprotein and alpha 2H-globulin. Direct correlation of the test systems for ferritin, beta-fetoprotein and alpha-globulin demonstrated their complete identity. Electrophoretic mobility of beta-fetoprotein was found to be identical to that of ferritin, corresponding with blood serum alpha 2-globulins. alpha 2H-Globulin was obtained from human kidney tumors according to the method devised by the author. The glycoprotein component of alpha 2H-globulin isolated from the preparation alpha 2H-globulin did not prove identical to kidney tumor ferritin and was thus signed to serum alpha 2-macroglobulins of healthy subjects. Since there are no differences between ferritin and its immunological analogs, it is suggested that beta-fetoprotein and alpha 2H-globulin be termed ferritin.