Biomedical subjects

I Kehayov

Publications and source records attributed to I Kehayov.

12 recordsLinked to original sources

PubMed1998

Cross-reacting idiotypes on anti-insulin autoantibodies in autoimmune diseases, identified by monoclonal antibodies.

Investigations on the specific idiotypes of autoantibodies are supposed to help with the understanding of the control mechanisms participating in the pathogenesis of autoimmune diseases. This study describes three monoclonal antibodies (Mabs) that recognize distinct idiotypic determinants on anti-insulin autoantibodies. The preabsorption by IAA-positive sera of insulin inhibits their subsequent binding to the anti-Id, thus suggesting that the Mabs recognize epitopes located at or near the binding site of insulin autoantibodies (IAA). These idiotypes are detected in sera from patients with insulin-dependent diabetes mellitus (IDDM), which are IAA-negative, also. It is possible that the expression of the idiotypes recognized might generally be associated with induction of autoantibodies, since they were found in sera from patients with rheumatoid arthritis (RA), autoimmune thyroid disease (AITD), and cataract (K). It can be assumed that the corresponding idiotypes of these Mabs, or similar structures (sequential or conformational), are expressed on autoantibodies with various antigen-binding specificities. These data suggest that some autoimmune diseases are preceded by the secretion of autoantibodies which express a common or similar pathological idiotype.

Adolescent↗

PubMed1995

Selective effect of methoxyindoles on the lymphocyte proliferation and melatonin binding to activated human lymphoid cells.

Three pineal methoxyindoles (melatonin (Mel), 5-methoxytryptamine (5-MTA) and 5-methoxytryptophol (5-MTO)) were studied for their ability to influence the proliferative response of human peripheral blood lymphocytes (PBL) and tonsillar lymphocytes (TL) following activation with concanavalin A (ConA) in vitro. The ConA-stimulated DNA synthesis was affected in a different dose-dependent mode by the methoxyindoles tested. Melatonin and 5-MTO inhibited and 5-MTA increased the ConA-induced [3H]thymidine incorporation in PBL and TL. The initial screening for 2-[125I]iodomelatonin binding using a single point assay revealed significantly increased specific binding to PBL and TL after 72-h stimulation with ConA as compared to the non-activated cell cultures. Coincubation of separate lymphocyte cultures with ConA and Mel or 5-MTO resulted in inhibition of the specific 2-[125I]iodomelatonin binding (85% and 74%, respectively). The specific binding determined in the presence of 5-MTA did not differ from control values. Series of saturation and competition experiments were performed to examine the binding characteristics of ConA-stimulated lymphocytes for 2-[125I]iodomelatonin. The radioligand labelled binding sites of high affinity (Kd = 0.14 +/- 0.03 nM) and low capacity (Bmax = 6.8 +/- 1.5 fM/mg protein). Competitive studies with a variety of indoles determined the following order of relative potency for inhibition of 2-[125I]iodomelatonin binding in TL: 2-iodomelatonin > melatonin > > 5-methoxytryptophol. 5-Methoxytryptamine did not show displacement potency for the labelled ligand. Collectively, our data suggest that pineal hormones might be directly involved in the regulation of the T-lymphoproliferative response of human lymphoid cells. We show the availability of melatonin receptors, which seem to be an intrinsic characteristic of activated human lymphocyte populations. While the effects of Mel and 5-MTO can be linked to the binding sites described, it is unlikely that serotonin agonists like 5-MTA may act through the same sites to influence the mitogen-stimulated lymphocyte proliferation.

5-Methoxytryptamine↗

PubMed1995

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody.

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other murine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.

Animals↗

PubMed1995

Human hybridomas secreting monoclonal antitreponemal antibodies raised after in vitro stimulation of human lymphocytes.

Peripheral blood lymphocytes from patients with syphilis were stimulated in vitro against Treponema pallidum extract and consequently were fused with mouse myeloma cells to raise heterohybridomas secreting specific human antibodies. In these experiments, 5 heterohybridomas were selected which were shown to secrete monoclonal antibodies which recognized treponemal antigens. Some of the monoclonal (Mab 1C12, Mab 1D11) react with antigens specific to T. pallidum while others (Mab 2A2, Mab 2C8, Mab 2C11) bind to treponemal components which demonstrated group specificity.

Animals↗

PubMed1993

Conformation-sensitive immunoassay: optimisation, validation and evaluation.

A conformation-sensitive immunoassay (CSI) has recently been developed (Pfund and Bourdage, 1990). This paper describes the optimal laboratory protocol which makes this method useful as an early screening procedure for conformation-sensitive monoclonal antibodies. The method was validated with a panel of 12 monoclonal antibodies. Results from our experiments confirm that this variant of CSI can be applied in the analysis of purified proteins and glycoproteins, as well as complex mixtures of antigens.

Antibodies, Monoclonal↗

PubMed1993

An anti-digoxin monoclonal antibody seems to express more than one functional paratope.

An anti-digoxin monoclonal antibody (mAb 4G3) has been produced and characterized with respect to its fine specificity and affinity. In an independent series of experiments anti-idiotypic monoclonal antibody (mAb 7G9) was selected which reacted with the antigen-binding center of an anti-human chorionic gonadotropin monoclonal antibody (anti-hCG mAb 1B10). In detailed studies on its binding characteristics it has been shown that mAb 4G3 binds to an anti-idiotypic monoclonal antibody mAb 7G9 in solution. Western blotting experiments showed that mAb 4G3 reacted against antiidiotypic antibody under non-reducing conditions, only. Moreover, mAb 4G3 has been shown to express self-binding properties. Absorption with saturating amounts of its specific hapten, i.e. digoxin, did not change the binding of mAb 4G3 to anti-idiotypic antibody and its self-binding ability. It is speculated on the basis of these data that mAb 4G3 possesses more than one functional paratope.

Animals↗

PubMed1991

Antigenic differences between Trichinella spiralis and T. pseudospiralis detected by monoclonal antibodies.

Antigenic differences between Trichinella spiralis and T. pseudospiralis were established using two monoclonal antibodies (mAbs) that show different specificities to muscle larvae of the two variants. Enzyme-linked immunosorbent assay (ELISA) revealed that mAb 3G6 reacts positively against T. spiralis, T. nelsoni, T. nativa and T. pseudospiralis, whereas mAb 3E10 does not react with T. pseudospiralis under the same experimental conditions. These antigenic differences were confirmed after preabsorption of the antibodies with serial dilutions of extracts of T. spiralis or T. pseudospiralis muscle larvae. The indirect immunofluorescence technique showed that the antigen corresponding to mAb 3G6 is located in the stichosomes and the cuticle surface of both T. spiralis and T. pseudospiralis. In contrast, mAb 3E10 positively stained cryostat sections of T. spiralis, forming a dense reaction product on the surface of the whole larvae and the surrounding capsule. This antibody can be quite useful as a specific probe for distinguishing T. spiralis from T. pseudospiralis in taxonomic studies. Using an avidin-biotin system, we could prove that mAb 3G6 recognizes an excretory/secretory-type antigen.

Animals↗

PubMed1991

Identification and characterization of human acrosomal antigen defined by a monoclonal antibody with blocking effect on in vitro fertilization.

A monoclonal anti-human sperm antibody (Mab 1A1) has been produced by fusion of myeloma cells with splenocytes from a BALB/c mouse immunized with in vitro capacitated human spermatozoa. Immunofluorescence studies with Mab 1A1 show that it recognizes an antigen(s) (Ag 1A1) which is located in the acrosome of human spermatozoa. As shown by Western blotting experiments, 1A1 antigen represents a family of proteins with Mr ranging from 20 kDa to 34 kDa. Immunofluorescence observations on epitope exposure and location suggest that during in vitro capacitation of human spermatozoa, Mab 1A1 epitope-bearing molecules are concentrated in regularly arranged granules in the acrosome. After long-term incubation the epitope is exposed on the apical acrosome surface exhibiting a spot-like arrangement. The 1A1 epitope is widely distributed among mammalian species: boar, ram, mouse and rat acrosome is intensively stained by Mab 1A1. The antibody inhibits in vitro fertilization mainly by blocking sperm attachment to and penetration through the zona pellucida when included in the medium for the in vitro fertilization of mouse, porcine and human oocytes.

Acrosome↗

PubMed1990

Characterization of NAD(P)H diaphorase from boar spermatozoa using specific monoclonal antibodies.

1. After immunization of BALB/c mouse, four monoclonal antibodies against soluble NADH diaphorase from ejaculated boar spermatozoa were produced and characterized. The monoclonal antibodies were designated as follows Mab 1F2, Mab 4E2, Mab 5B8, Mab 5D8. 2. These monoclonal antibodies react with other enzyme forms-sedimentary NADH and NADPH and soluble NADPH and inhibit (although not completely) their activity. It is supposed that different forms of the enzyme share some common epitopes. 3. Treatment of ejaculated boar semen with 2O-methylcholanthrene causes an increase of the activity of the soluble diaphorase form only. 4. These results lead to the assumption that the sperm diaphorase is a dynamic enzyme system consisting of four immunologically similar isoenzymes although their functions are different.

Animals↗

PubMed1990

Production and application of monoclonal antibody specific for human chorionic gonadotropin.

Spleen cells from mice immunized with human chorionic gonadotropin (hCG) were fused with mouse myeloma cells and four hybridomas, secreting monoclonal antibodies, were selected for further studies. In cross-check tests against tissue extracts and peptide hormones it was established that mAb IB10 (IgG1) reacted positively against hCG only but not with other hormones. Ascites from this hybridoma was fractionated by ammonium sulphate precipitation and by FPLC to isolate the IgG fraction. Subsequently, the purified mAb was conjugated with horseradish peroxidase and used for development of a simple test for pregnancy diagnosis. Some preliminary experiments have shown that the test is very specific and reproducible.

Animals↗

PubMed1976

Demonstration of a phase (stage)-specific embryonic brain antigen in human meningioma.

The water-soluble antigens of human embryonic brain were studied using a rabbit antiserum to human 8- to 10-week embryonic brain (non-absorbed and absorbed with extracts of human embryonic and adult organs). A phase (stage)-specific brain antigen(s) characteristic for the human 8- to 10-week-old embryo was detected by double immunodiffusion and immunoelectrophoresis in agarose gel. The testing of this anti-embryonic brain serum against saline extracts of human brain tumours (meningiomas, glioblastomas, astrocytomas, neurinomas and brain metastasis of carcinoma) revealed the presence of an "embryonic" antigen in the human meningioma only. Immunochemically this meningioma-associated antigen is identical to a phase (stage)-specific antigen of the 8- to 10-week embryo.

Antigens, Neoplasm↗

PubMedDate not supplied

Purification and characterization of sperm-coating antigen, identified by a monoclonal antibody.

A procedure was designed for purification of a human seminal plasma-specific antigen (HSP-antigen) identified by a monoclonal antibody produced in this laboratory (mAb 4E6). Pooled human seminal plasma was fractionated by consecutively applied methods: affinity chromatography on Lentil lectin sepharose, gel chromatography on Ultrogel AcA 34 and immunoaffinity on mAb 4E6 coupled CNBr-Sepharose 4B. The antigen-containing fraction obtained after this procedure was proved to be homogeneous when analysed by electrophoresis in polyacrylamide gel. After the consecutive purification procedures the degree of purification was 128 times as compared to the starting material. Electrophoretic analysis of the purified 4E6 antigen under reducing conditions showed that it consisted of 3 polypeptide subunits with molecular weight 70 kDa, 64 kDa and 60 kDa respectively. On the basis of the data obtained from competitive ELISA testing of sera from infertile patients it has been suggested that the identified antigen may be involved in pathogenesis of immunologic infertility.

Adult↗