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Biomedical subjects

E R Karamova

Publications and source records attributed to E R Karamova.

18 recordsLinked to original sources
PubMed1998

Inducible protein in rat hepatomas with expression alternative to alpha-fetoprotein.

The rat hepatoma cell line McA RH7777 was cloned into alpha-fetoprotein-producing (AFP+) and non-producing (AFP-) sublines. A monoclonal antibody (MAb A2/3) reacting with an antigen (Ag A2/3) present only in AFP- clones or AFP- cells in mixed clones was obtained. Ag A2/3 was absent from the liver of embryonic, fetal, newborn and adult rats, but it was present in gastric and intestinal mucosa of adult rats. Ag A2/3 was found to be a heavy metal-inducible protein: Cd2+ and Pb2+ strongly induced the expression of Ag A2/3 in vivo in the liver of adult rats, while xenobiotics and CCl4 were not active in this respect. In vitro Cd2+ and Pb2+ induced Ag A2/3 expression in several AFP+ clones, leading to a simultaneous marked decrease of AFP+ cells from such clones. The effect of Cd2+ in the induction of Ag A2/3 and suppression of AFP was reversible. SDS PAGE revealed one protein band with an m.w. close to 45,000, which was not sensitive to mercaptoethanol. Despite its inducible properties, Ag A2/3 was shown not to belong to metallothioneins, cytochrome P-450, glutathion-transferase or heat shock proteins families, well-known as being inducible cell stress proteins. Expression of Ag A2/3 could be one of the factors determining the high amplitude of AFP production by individual liver tumors. The nature of Ag A2/3 and its alternative expression with respect to AFP remain to be studied.

Animals↗
PubMed1998

Epitope mapping of human alpha-fetoprotein.

Thirty monoclonal antibodies (MoAbs) to human alpha-fetoprotein (AFP) were compared with one another by two methods: Immunoaffinity electrochromatography or additive ELISA. The first method permitted to analyse the epitopes of native AFP in solution [Abelev et al., Immunol Lett 1994;40:133-138] while the other approach also detects the epitopes of conformationally modified (partly denatured) AFP fixed on the plastic [Yazova et al., Immunol Lett 1990;25:325-330]. Competitive analysis of all MoAbs revealed 10 epitopes, 9 expressed on native AFP and 1 only on the partly denatured molecule. The cross-reactions between separate MoAbs allowed to include them into 6 distinct epitope clusters, or immunodominant groups with the characteristic patterns of reactivity. The obtained epitope map of AFP is necessary for the construction of AFP detection kits as well as for the identification of its antigenic and functional subfractions.

Antibodies, Monoclonal↗
PubMed1998

Conformational variants of human alpha-fetoprotein.

The immunological heterogeneity of human alpha-fetoprotein (AFP) was demonstrated using immunoaffinity electrochromatography on monoclonal antibodies (MoAbs) to 3 non-cross-reacting epitopes of this protein. At least 4 subfractions expressing different epitopes were found in the native AFP. These subfractions demonstrated molecular weights similar to the major component of the original AFP. The difference between epitope F5-positive and F5-negative subfractions disappeared when epitope-negative subfraction was conformationally changed after fixation onto nitrocellulose membrane (NCM). Thus, the epitope under study exists in two forms on the native human AFP molecule: an open and a cryptic form. The cryptic form could be revealed after partial denaturation by fixation on NCM. The epitope variants of AFP could possess different functions in multifunctional AFP. The AFP epitope heterogeneity found in this work should be taken into account when constructing diagnostic AFP kits and when isolating purified AFP using anti-AFP MoAbs.

Antibodies, Monoclonal↗
PubMed1996

[Automation on membranes: countercurrent isotachophoresis and immunoelectrochromatography].

The review discusses two related methods--counterflow isotachophoresis and immunoaffinity electrochromatography on porous membranes developed by the authors. The methods exploit the peculiarity of electroendosmosis in porous membranes at the conditions of discontinues electrophoresis. There are described the principles and application of the above methods for automatic performance of electrophoretic analysis of low-protein biological fluids, of western blotting, epitope analysis of monoclonal antibodies and multiantigen assays. The possibility of DNA-binding protein detection is also considered.

Automation↗
PubMed1994

Electrochromatography: a method for automatic immunoaffinity chromatography on porous membranes.

Electrochromatography (ECHR) exploits a very high electro-osmotic counterflow developed in porous membranes at discontinuous electrophoresis. This counterflow exceeds considerably the anodic migration of any negatively charged protein and is used as a 'conveyer belt' for sequential transfer of immunoreagents to the specific adsorbents (antigens or antibodies) fixed on the nitrocellulose membrane. This approach was applied for simultaneous detection of two antigens (alpha-fetoprotein and carcino-embryonic antigen) in one sample, for determination of subfractions of alpha-fetoprotein, different in their epitope specificity, and for detection of L chains with certain idiotype on the background of heterogeneous L fraction. ECHR was used also for the partition of different antibodies to DNA adducts, demonstrating the possibility of applying this method to the study of DNA-binding proteins.

Antibodies, Monoclonal↗
PubMed1991

Counterflow affinity isotachophoresis on cellulose acetate membranes.

Counterflow isotachophoresis on cellulose acetate membranes of human alpha-fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.

Ricinus communis↗
PubMed1990

Immuno-isotachophoretic determination of monoclonal immunoglobulin light chains produced by neoplastic B-cells: use in diagnosis, monitoring and detection of residual disease.

We suggest that countercurrent isotachophoresis performed on cellulose acetate membranes (ITP-CAM) should be used for detecting trace amounts of Bence-Jones protein (BJP) in urine of patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL) and related diseases. ITP-CAM allows simultaneous concentration and electrophoretic separation of proteins present in highly diluted solutions, as well as easy immunological detection of separated substances. BJP was found in 24 out of 42 patients with CLL, 33 of 56 with NHL and 3 of 3 with Waldenström macroglobulinemia. Twenty-three patients were followed during the course of chemo- or radiotherapy. In 19 cases the BJP findings correlated well with clinical status. In no case of partial or complete clinical response did BJP completely disappear from the urine.

B-Lymphocytes↗
PubMed1989

Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes--I. Immunoblotting.

Urine with trace amounts of different proteins from healthy people or B-lymphoma patients was concentrated and separated simultaneously by counterflow isotachophoresis on cellulose acetate membranes (CAM). The protein zones were blotted onto nitrocellulose membrane (NCM) by direct contact of CAM and NCM. NCM-blots were exposed to second isotachophoresis with the leading electrolyte 0.06 M Tris-HCl and the terminating one, 0.012 M Tris-beta-alanine. Under these conditions the moving boundary formed by Cl-/beta-alanine- migrated towards the anode with decreasing velocity. At a certain point the rate of migration of the moving boundary became completely compensated by the electroendosmotic counterflow. In this steady state position the boundary stopped on the NCM support, while the electroendosmotic rate in the area before the boundary was much higher than the rate of the opposite migration of any protein to the anode. Under these conditions electroendosmosis served as a "conveyer belt" which transferred consecutively the immunoreagents, antibodies, immunoconjugates, or antiperoxidase-peroxidase system through the protein blots "printed" on NCM. The immunoblots obtained in this way were developed by the substrate for the immunoenzyme complex used in the experiment. The technique could be used to characterize light chains present in the urine of normal donors and monoclonal light chains in the urine of patients with B-cell malignancies.

B-Lymphocytes↗
PubMed1989

Performance of multistep immunochemical reactions by counterflow isotachophoresis on nitrocellulose membranes--II. Epitope analysis of monoclonal antibodies.

The method for comparison of epitope specificity of different monoclonal antibodies to one antigen, using a panel of monoclonal antibodies to mouse and human alpha-fetoprotein is described. The method used exploits the special properties of electroendosmotic flow in nitrocellulose membranes under the conditions of anionic isotachophoresis. Electroendosmosis allows successive transfer of several immunoreagents to the dots of monoclonals previously bound to the nitrocellulose membrane. The inhibition of antigen binding to monoclonal dot, if the antigen is mixed with excess of another monoclonal antibody, demonstrates that both monoclonals are directed to the same epitope, and vice versa. The method requires neither purified monoclonals nor antigens, or radio labelling, and is performed semi-automatically. It was shown that each monoclonal antibody to mouse and human alpha-fetoprotein had its own epitope specificity.

Animals↗
PubMed1988

[Epitopic analysis of monoclonal antibodies based on countercurrent isotachophoresis on nitrocellulose membranes].

A variant of the counterflow isotachophoresis on nitrocellulose membranes (NCM) was applied for the analysis of the epitope specificity of monoclonal antibodies (Mabs). Different Mabs against mouse and human alpha-fetoprotein were fixed on the membrane and the immunoreagents were transferred consequently to these dots by the electroendo-osmotic flow. The epitope specificity of the Mabs was estimated by the competition for the homologous antigen between the Mab added to the antigen and the one fixed on the membrane.

Animals↗
PubMed1988

[Countercurrent immunoblotting].

Urinary proteins, concentrated and separated on cellulose-acetate membranes by counterflow isotachophoresis (ITP), were transferred by direct contact onto a strip of nitrocellulose membrane (NCM). ITP in the system of electrolytes: tris-HCl, pH 6.7 (the leading one) and tris-beta-alanine, pH 8.6 (the terminal one) gives rise to a strong electroendo-osmotic flow (EEF) in NCM, directed to the cathode. The rate of the counterflow in the zone, occupied by the leading electrolyte, exceeds the migration rate of any protein possessing anode mobility and present in the zone. Under these circumstances EEF serves as a "conveyer belt" transferring immunological reagents (antibodies, immunoconjugates, peroxidase) through the protein bands, "printed" on NCM. The immunoblots were developed in a standard way with 4-ethyl-1-naphthol as a substrate for antibody-bound peroxidase. The counterflow immunoblotting makes it possible to reveal and characterize light chains of immunoglobulins when they are present in the urine in the range of 20 ng/ml.

Counterimmunoelectrophoresis↗
PubMed1987

[Mixed gel precipitation in the analysis of monoclonal antibodies against determinants of the carcinoembryonic antigen].

Mixed gel precipitation technique was used in the study of 5 monoclonal antibodies (MA) to carcinoembryonic antigen (CEA). The method is based on specific inclusion of MA into the precipitate formed by the antigen and polyclonal antibodies to it. The test-system for the determination of nonspecific cross-reacting antigen (NCA) has demonstrated that MA 2D10 are directed to the determinant, common for CEA and NCA, whereas MA 3C12 and 2G10 give no reaction with NCA. The specificity of MA 192 and 35 correlated with the previously established one. Mixed gel precipitation technique is recommended for primary screening and study of MA specificity to any precipitating antigen.

Animals↗
PubMed1984

Counterflow immunoisotachophoresis on cellulose acetate membranes.

Discontinuous electrophoresis on cellulose acetate membranes with the use of 0.06 M Tris-HCl (pH 6.7) as the leading electrolyte and 0.012 M Tris-beta-alanine (pH 8.6) as the terminating one results in concentration of the proteins present in the system on the Cl-/beta-alanine- boundary. If the antigen solution is placed in a "pocket" ahead of the moving boundary, a counterflow to the cathode arises due to electroendosmosis. At constant voltage the migration rate of the boundary drops and that of electroendosmosis does not change until they become equal. In such a stationary position, the antigen-containing solution is passing through the Cl-/beta-alanine- boundary to the cathode, while all the proteins are completely "absorbed" on the boundary as highly concentrated bands. Addition of ampholytes to the antigen solution contributes to the isotachophoretic separation of a protein mixture on the strip. The concentrated and separated antigens can be revealed by immunofixation, immunodiffusion, or crossed immunoelectrophoresis in gel. The technique is approximately 100 times more sensitive compared to the usual immunodiffusion and immunoelectrophoresis methods on cellulose acetate membranes, and is applicable to the detection of trace amounts of antigens in the urine, liquor, amniotic fluid, tears, and other biological fluids with low protein contents.

Animals↗
PubMed1979

[Counter-flow isotachophoresis on cellulose acetate membranes. Role of electroendosmosis].

A variant of counter-flow isotachophoresis of proteins on cellulose acetate membranes is proposed. The liquid counter-flow is created by electroendosmosis in the membrane. Proteins are concentrated at the Kolrausch boundary during isotachophoresis in the presence of ampholytes. The method permits one to make microanalysis of proteinic mixtures in diluted solutions, and it can be used in combination with immunodiffusion and immunoelectrophoretic methods of antigenic protein detection.

Antigens↗
PubMedDate not supplied

New approaches for the detection and characterization of alpha-fetoprotein epitope variants.

Alpha-fetoprotein (AFP) is an embryo-specific protein of mammalian sera that consists of a single glycosylated polypeptide chain (approximately 70 kD) with 5-6 epitope clusters located on the native molecule. We have elaborated three approaches for the separation of AFP into fractions (variants) that are different in the distinct epitope expression on the native molecule. For this, we have used three technologies largely based on immunoaffinity electrochromatography and electrophoresis/immunoblotting. The separation of these variants was facilitated using monoclonal antibodies previously characterized to the 5 epitope clusters and two individual epitopes on AFP in the ISOBM international workshop. We have shown that these approaches facilitate the identification of cryptic epitopes in AFP, and the relevance of these findings is discussed.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Determination of DNA-binding proteins by countercurrent isotachopheresis on nitrocellulose membranes. I. Antibodies to DNA and its adducts].

A very strong electroosmotic counterflow was produced on nitrocellulose membranes during isotachophoresis in a system of 0.06 M Tris-HCl (pH 6.7) as the leading electrolyte and 0.012 M Tris-beta-alanine (pII 8.6) as the terminating one. This counterflow was equal in rate and opposite in direction to the migration of the Cl-/beta-alanine boundary. The rate of counterflow was much higher than the rate of migration of any organic anions, including different proteins. Double-stranded and single-stranded DNA or its adducts were fixed on the nitrocellulose membrane, and the membrane was blocked with unrelated proteins. DNA-binding proteins, namely antibodies to DNA, followed by peroxidase-conjugated anti-IgG, were introduced into the counterflow, which transferred them one after another to the DNA spots. Thus, sequential binding and washing was performed automatically. In this way, antibodies were detected to ds- and ss-DNA, to BrdU-DNA, to Z-DNA, to biotinylated DNA and DNA modified with trans-Pt, as well as development of biotinylated DNA dots by steptavidin-peroxidase.

Antibodies, Antinuclear↗