Antimeasles immunoglobulin G in sera of patients with otosclerosis is lower than that in healthy people.

BACKGROUND: There is some evidence for an inflammatory process as a driving force in otosclerosis. Two popular hypotheses for the induction of this chronic inflammation have been proposed: an autoimmune phenomenon induced by an otic capsule specific antigen and measles virus infection. METHODS: Antibodies against measles virus hemagglutinin, polymerase, nucleocapsid, and matrix proteins were evaluated in sera from otosclerotic patients and in sera from healthy age-and sex-matched controls by use of the Western blot analyses. RESULTS: Significant differences were not detected between healthy men and women or between otosclerotic men and women. There were significantly stronger reactions against all viral proteins in the group of healthy women as compared with otosclerotic women despite a high standard deviation. The group of healthy male blood donors demonstrated significantly stronger reactions against polymerase and nucleocapsid proteins. Healthy blood donors again demonstrated stronger reaction compared with respective otosclerotic patients in a separate reaction for viral matrix protein. CONCLUSION: Our observation is consistent with viral participation in otosclerotic pathogenesis, but it is difficult to say if the diminished antimeasles humoral response is a consequence or the cause for a local measles infection. In light of the present data, we can discuss autoantibodies in otosclerosis as a sign of autoimmunity triggered by measles virus.

Increased adhesion of lymphoid cells to glycated proteins.

BACKGROUND AND AIMS: The advanced glycation end-products are involved in the pathogenesis of vascular damages and other clinical complications in diabetic patients. The aim of this study was to investigate the adhesion of lymphoid cells to nonenzymatically glycated proteins in comparison with the unmodified substances. METHODS: Two cell lines (monocyte-macrophage line U937 and the T-cell line Jurkat) were used throughout the experiments. The cells were left to adhere to nonenzymatically glycated and native proteins coated on a 96-well flat-bottom plates and the cellular adhesion was registered as absorption at 550 nm following the method described by Ivanov and Kyurkchiev [G. Ivanov, S. Kyurkchiev, Effect of advanced glycosylation end-products on the activity of integrins expressed on U937 cells, Hum. Immunol. 59 (1998) 325-330.]. RESULTS: It was found that the monocytes had increased adhesion to nonenzymatically glycated proteins such as collagen, fibronectin and bovine serum albumin, whereas the T-cells had increased adhesion to the glycated collagen and bovine serum albumin but reduced adhesion to advanced glycated fibronectin. Experiments with different stimulating agents showed that phorbol-myriastate, acetate (A550 = 0.672 +/- 0.068, S.E.M., n = 40), glucose (A550 = 0.593 +/- 0.051, S.E.M., n = 40) and TNF-alpha (A550 = 0.580 +/- 0.042, S.E.M., n = 40) increased the adhesion of U937 cells to advanced glycated bovine serum albumin in comparison with the adhesion of the untreated cells (A550 = 0.260 +/- 0.046, S.E.M., n = 40). This is probably due to an upregulation of the expression or the activity of the receptors for the advanced glycation end-products. CONCLUSION: Based on the results obtained it is concluded that the receptors for nonenzymatically glycated proteins expressed on the surface of lymphoid cells could act also as cell adhesion molecules.

Elevated autoantibodies in sera from otosclerotic patients are related to the disease duration.

In this study an indirect ELISA with patients' sera was performed using human collagen type II, double- (dsDNA) and single-stranded DNA (ssDNA), thyroid microsomal antigen, insulin and lysozyme as antigens. Since many preoperated otosclerotic patients demonstrated the signs of myringosclerosis (n=7). they were classified separately and compared with otosclerotic patients without myringosclerosis (n=28), with healthy controls (n=42) and with patients with tympanosclerosis (n=5) of other origin. The otosclerotic patients had serum antibodies to antigens tested similar to normal controls. However, elevated antibody levels to human collagen type II, dsDNA and ssDNA were observed only in patients with a disease duration between 3 and 5 years as compared to other otosclerotic patients. The same duration association was observed in the level of the total serum alkaline phosphatase activity. These observations would suggest that the enzymatic bone resorption is the driving force in human otosclerosis. Elevated serum autoantibodies during tissue reparation in the otosclerotic stage may be a transient response to sustained excess antigen turnover in the primary lesion.

Human sperm surface glycoprotein involved in sperm-zona pellucida interaction.

To identify peptide-specific antibodies which define sperm surface antigens, hybridomas were derived from the splenocytes of mice immunized with swollen human spermatozoa which had been subjected to limited proteolytic cleavage under reducing conditions prior to immunization. A total of 13.7% of the hybrid clones secreted antibodies which reacted with deglycosylated human seminal plasma glycoproteins when screened by an ELISA. A monoclonal antibody, designated mAb 4A8 sp., specifying a peptide epitope of human epididymal and a sperm surface glycoprotein was selected which inhibited human sperm-egg binding in a dose-dependent manner, and totally blocked sperm penetration in vitro. This inhibition did not result from an effect of the antibody on the motility of spermatozoa, nor was it due to premature induction of the acrosome reaction. Exclusion of oligosaccharide chains by chemical hydrolysis with trifluoromethane sulphonic acid (TFMS), enzymatic degradation and binding of lectins, did not abrogate the reactivity of mAb 4A8 to the cognate epitope whereas antibody binding was precluded upon digestion with proteolytic enzymes. In Western immunoblots of human seminal plasma glycoproteins, the antigen presented as a set of immunoreactive polypeptides, a major glycoprotein of M(r) 78 kDa and less prominent bands of M(r) 56 and 44 kDa. Immunocytochemical staining of a number of human reproductive and somatic tissues revealed strong immunostaining of the luminal epithelium of the epididymis as well as of spermatozoa in the lumen. Immunolocalization to the plasma membrane of ejaculated human spermatozoa was demonstrated by immunofluorescence, although on undigested spermatozoa the antigen epitope was less accessible. Upon capacitation the antigen persisted on the sperm surface and was present on the head of capacitated acrosome-intact spermatozoa. The pronounced peripheral immunostaining of the sperm head was accentuated after DTT/trypsin treatment, implicating the dynamic accessibility of the epitope on the plasma membrane of capacitated spermatozoa. It is suggested that the protein in question appears on the sperm membrane as a consequence of its modification in the epididymis (insertion and processing), and may be involved in the processes leading to sperm attachment and interaction with the human zona pellucida.

Cell specific nuclear antigens of boar spermatozoa.

Demembranated boar sperm heads were differentially extracted at conditions involving high salt-urea, proteolysis and DNase I cleavage that mimic the conditions promoting the in vivo decondensation of the fertilizing sperm nucleus in the egg ooplasm. The sperm-unique subset of proteins was studied which remained bound in the residual salt-resistant nuclear structure operationally defined as sperm nuclear matrix. By means of polyvalent antisera the immune specificity of the sperm nucleoprotein complex was estimated using ELISA and microcomplement fixation test as compared to somatic type dehistonized chromatin of boar liver. To define immunologically specific sperm DNA-associated proteins hybridomas were generated by fusing lymphocytes immunized with boar sperm protein/DNA complex. Monoclonal antibodies were selected (Mab 1A8, 1B3, 2B5, 2H5 and 3A4) which identified protein moieties in the sperm DNA-tight binding proteins complex resistant to cleavage with DNase I and sensitive upon digestion with high concentration of proteases. No appreciable reactivity was recorded of the antibodies to somatic chromatin and no significant binding to ssDNA. A polypeptide in the residual sperm nuclear structure of apparent Mr 27 kDa was recognized by Mab 3A4 as detected by Western blotting. The enhanced reactivity to the DNase I digested sperm nuclear fraction (except for Mab 2H5) suggests that DNA protected from nuclease digestion by a protein might be essential for immune reactivity and full antigenic integrity as well as the dependence of the cognate proteins on the binding to DNA for antigenicity and immune specificity. The functioning of the identified putative sperm specific proteins is anticipated in the structural rearrangement of chromatin in the zygote.

Characterization of a sperm nuclear protein.

PROBLEM: The molecular identity of sperm DNA-binding structural proteins contributing to the integrity of a sperm residual high salt/nuclease resistant nuclear structure is studied by cDNA cloning and monoclonal antibodies to the recombinant polypeptide. This structure, which is likely to be transferred unimpaired in the oocyte and is anticipated as a molecular correlate of the nuclear scaffold (nuclear matrix/envelope) in somatic cells, may be essential with respect to its DNA organization for the recovery and assembly of somatic-type chromatin in the zygote. Recently, a cDNA encoding one of these proteins has been cloned and the recombinant polypeptide expressed in E. coli as a beta-galactosidase fusion protein. The main objective of the present study is the identification of the native sperm antigen by monoclonal antibodies raised against the recombinant molecule. METHODS: We evaluated the possibility of immunizing by direct intrasplenic deposition in BALB/c mice of the recombinant fusion protein available as transblotted on nitrocellulose membrane carriers or as nitrocellulose protein-bearing particles. Isolated sperm DNA/tight binding protein complexes were used in ELISA and Western blotting for selection of monoclonal antibodies specific to self epitopes of the nuclear antigen, as well as immunofluorescence of swollen human spermatozoa subjected to in situ extraction with high salt/beta-mercaptoethanol/DNase I and proteolysis, and of a cultured fibroblast cell line L-929. RESULTS: A monoclonal antibody, Mab 2C4, was selected which recognized a 52 kDa protein in the fraction of sperm high salt/urea resistant proteins. The target polypeptide was detected on swollen spermatozoa primarily to the post-acrosomal and/or equatorial regions whereas in nonextracted sperm cells the epitope was exceedingly unavailable. The somatic cell location of the cognate epitope was confined to the nuclear envelope displaying a cap-like pattern of staining, and also in a juxtanuclear cytoplasmic randomly coiled filamentous network and in compact bodies. CONCLUSIONS: A nuclear protein salt-stably bound to the sperm residual structure has been identified. The antigen appears localized in sperm exclusively to perinuclear subacrosomal sites that may be anchored at the male nuclear envelope, given the occurrence of the target epitope in somatic cells as well in nuclear and cytoplasmic sites adjacent to the nuclear membrane.

Redistribution of nuclear envelope associated antigen during the mitotic cycle.

Murine hybridomas were generated to DNA/tight binding proteins complex isolated from the residual nuclear structure following a procedure analogous to that yielding "empty" shells of nuclear envelope. A monoclonal antibody designated 2A8 was selected because of its differential immunostaining of mitotic cells of a synchronized mouse fibroblast cell culture L-929. The target antigen was rendered insoluble by a sequence of extractions of isolated nuclei of diverse cell types with detergents, urea, DNase I and alkali thus reproducing some solubility properties of proteins constituting an operationally defined residual nuclear matrix. The cognate polypeptide was localized on a subset of proteins of M(r) 58-65 kDa, 70 kDa in isolated fibroblast nuclear matrices. The functional implication of the antigen in mitosis-related disassembly-assembly process of the nuclear matrix/envelope was detected. At prophase the antibody decorated the nuclear periphery and nuclear envelope fixed inward filaments. A fibrous network of cytoplasmic localization was stained in metaphase. At anaphase the antigen was dispositioned into peripheral fibrogranular clusters of polar orientation predominantly on one side of the nucleus. Proceeding to telophase a spreading fluorescence was manifested over the entire contour of the nuclear periphery to delineate the reforming nucleus. By immunogold electron microscopy of interphase cells the antigen was identified as evenly distributed in chromatin and interchromatin regions. At initiation of chromosome condensation in mitosis the label was detected predominantly in the chromosomal area.

Base specificity and binding of anti-ss/dsDNA monoclonal antibody. Application to the determination of ssDNA.

An anti-DNA murine hybridoma was generated and selected in a fusion of Sp2/0 myeloma cells with splenocytes derived from BALB/c mice immunized with DNA/protein complex. The monoclonal antibody designated as 4C7 was assessed by competitive immunoassay for binding to denatured, structurally single-stranded (ss)DNA, double-stranded (ds)DNA and oligodeoxynucleotides. The monoclonal antibody exhibited a marked preference for the ss conformation. Competitive inhibition assay performed with nucleosides and homopolynucleotides indicated that the deoxyadenosine residue was essential for antibody recognition and binding. The monoclonal antibody was purified by poly(A)-Sepharose chromatography and biotinylated. The ssDNA detection limit in the enzyme-linked immunosorbent assay with the biotinylated antibody ranged from 1 to 16 ng/ml. A dot immunobinding test was developed which was sensitive for the quantitation of picogram amounts of single-stranded DNA deposited on nitrocellulose filters and applied to the immunoenzymatic detection in human sera of target DNA.

An express immunological method for detection of human seminal plasma.

Monoclonal antibodies (Mabs) against human seminal plasma (HSP) were produced and during screening procedures dissociation constants of the antigen/antibody complexes were determined. Mab 1E5 was selected for further studies because of its high reactivity in an enzyme-linked immunoassay (ELISA) and high affinity for its corresponding antigen. The specificity of Mab 1E5 was checked in absorption ELISA with human organ extracts and some biological secretions. It was established that the 1E5-corresponding epitope was a thermostable peptide moiety which could be detected in HSP, only. This monoclonal antibody was used for the development of an express method for detection of human semen. The assay was applied for screening of 57 cases of suspected rape. A complete correlation was found between the results obtained by the proposed test and by routine microscopic methods. The newly designed immunoassay is reliable, it is easily performed and it is less time-consuming.

Identification of human ventricular myosin heavy chain fragments with monoclonal antibody 2F4 in human sera after myocardial necrosis.

An ELISA assay with monoclonal antibody (Mab 2F4) raised against human ventricular myosin heavy chains was developed and used to investigate human sera after myocardial infarction. The monoclonal antibody 2F4 was selected for its high affinity to soluble fragments of myosin heavy chains (subfragment-1) and for its appropriate tissue specificity. By including Mab 2F4 in a simple and rapid dot immunobinding assay sera from patients with acute chest pain and of persons without a history of heart disease were tested. Myosin was detected only in the sera of the patients with myocardial necrosis, confirmed by electrocardiographic data. Negative reactions in all control cases were found. The serum myosin fragments reactive with Mab 2F4 were characterized by immunoblot experiments and protein bands in the region about 43 kDa were found. It was concluded that the myocardial infarction can be demonstrated by detection of cardiac myosin heavy chain fragments in the patients' sera.

Trichinella spiralis: a 76-kDa excretory/secretory larval antigen identified by a monoclonal antibody.

Spleen cells from BALB/c mice were fused with myeloma cells following infection of the mice with Trichinella spiralis larvae and an ip booster injection with larval homogenate antigen. A monoclonal antibody (Mab), designated as TS 3G6 which did not react with sera or tissue extracts from noninfected mice, rats, and guinea pigs, was selected for further studies because of its high activity and specificity. When tested in ELISA TS 3G6 did not cross-react with Ascaris suum, A. lumbricoides, Toxocara canis, E. granulosus (larvae), Trichiuris suis, or T. ovis. Western blot analysis showed that Mab 3G6 recognized an antigen of 76 kDa located in the stichosome of the larvae as well as on the surface of the larval cuticle. Digestion of a larval extract with different enzymes suggests that the Mab TS 3G6 corresponding epitope is a polypeptide. The TS 3G6 antigen was detected in culture supernatants of Trichinella muscle larvae and in sera of experimentally infected animals using a sensitive ELISA assay. This secretory antigen also seemed to induce a specific immune response in the host since sera from infected animals could block the binding of Mab TS 3G6 to its target antigen when tested in a competitive ELISA.

Generation of monoclonal anti-digoxin antibodies.

Anti-digoxin monoclonal antibodies are a useful model for basic immunochemical studies, for investigation of endogenous digoxin-like substances and in immunoassay for cardiac glycosides. The complete phenotypic characterization is a requisite for the selection of antibodies with desired binding parameters for different purposes. Twenty-two high-affinity monoclonal antibodies specific for digoxin were obtained in two fusion experiments. Treatment of antigen-antibody complex with potassium thiocyanate (KSCN) and absorption ELISA were used for the selection of high-affinity antibodies at the earliest stages of hybridoma growth. The true affinity constants of selected antibodies were determined in ELISA. They had proven to vary between 10(-7) and 10(-10) M. The fine specificity of 21 anti-digoxin monoclonal antibodies was determined by cross-reactivity experiments with 25 structurally related compounds. Cardiac glycosides, digoxin metabolites, endogenous steroids and spironolactone were used in the elucidation of the antigenic recognition pattern of antibodies. The elucidation of the binding characteristics of anti-digoxin monoclonal antibodies makes possible the selection of antibodies possessing binding characteristics appropriate for a wide range of designations.

Selection of monoclonal anti-digoxin antibodies with appropriate binding characteristics for immunodiagnostic purposes.

Digoxin is the third most commonly prescribed drug in the United States and is routinely monitored in clinical chemistry laboratories. Polyclonal antisera used up until now in immunoassays for digoxin are not satisfactory and lead to poor precision and problems with standardization. Monoclonal antibodies would certainly be preferable because of high reproducibility and possibility of standardization which they ensure. Twenty-two anti-digoxin monoclonal antibodies (MoAbs) were selected by an ELISA absorption at the earliest stages of hybridoma growth. Dissociation constants of selected MoAbs were determined by ELISA. Detection limit in this system was also assessed. Specificity of twelve antibodies with sufficiently high-affinity constants to detect therapeutic and subtherapeutic levels of digoxin was determined by cross-reactivity experiments with 25 steroid compounds-cardiac glycosides, digoxin metabolites, steroid hormones, spironolactone. On the basis of these data, MoAbs with suitable binding parameters for immunodiagnostic purposes might be selected.

Application of an immunological method to detect trace amounts of dried human semen.

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.

Monoclonal antibodies to porcine zona pellucida that block the initial stages of fertilization.

Zona pellucida (ZP) is thought to be of utmost biological importance in the early stages of fertilization and implantation. Current hybridoma technology was used to produce monoclonal antibodies (MAbs) against specific antigens to porcine ZP. Two monoclonal antibodies (4F2 and 2D9) were raised that reacted against ZP antigens shared by human and porcine ZP. These antibodies were shown to block fertilization of human oocytes in in vitro fertilization (IVF). It is likely that MAb 4F2 recognized a protein epitope localized on the outer surface of ZP. These antibodies may be quite useful immunologic probes for studying the precise mechanisms of the early events of fertilization in mammals.

A human-mouse hybridoma producing monoclonal antibody against human sperm coating antigen.

Since anti-sperm antibodies were first discovered in the sera of women, the relationship of these antibodies to sterility has been studied by many investigators. In order to determine the antigens of spermatozoa responsible for raising antibodies to spermatozoa in humans, many studies have been carried out by purifying human spermatozoa cell membrane and seminal plasma components. Since it was found that the purification was difficult by physiochemical procedures, the immunoaffinity chromatography bound monoclonal antibody (Mab) to spermatozoa antigens was attempted for this purpose. The establishment of hybridomas producing Mabs to human seminal plasma and human spermatozoa was reported by Shigeta et al. (1980), Isojima, Koyoma & Fujiwara (1982), Lee et al. (1982) and Isahakia & Alexander (1984). The ordinary approaches to obtain the Mabs consisted of xenogenic immunization with human semen and cell fusion of immunized spleen cells with mouse myeloma cells. However, the antigenic epitopes of human spermatozoa, which induced antibody production, are xenogenic for the mouse, and therefore there is a possibility that there is a difference in recognized antigenic epitopes in humans as isotypic and in mice as xenogenic. In order to study these antigenic epitopes, which correspond to antibodies against spermatozoa in women, the establishment of human-mouse hybridomas, which produced anti-semen antibodies as produced in sterile women, became essential. In these studies, we used recently developed cell fusion techniques to fuse immunized human peripheral lymphocytes with mouse myeloma cells.

Identification and immunocytochemical localization of a human adult brain-specific antigen (HABSA).

A new protein with a molecular weight of 669,000, identified in brain extracts from 4 to 69 years old subjects has been isolated and immunochemically characterized. The antigen is found in human adult brain but not in the brains of human fetuses and newborn infants or in the brains of several other species tested. Immunocytochemically, using the PAP method, the antigen is localized at the surface of some nerve cells and on astrocytes and oligodendrocytes of the cerebral cortex, corpus striatum, pons and medulla. The Golgi epithelial cells with Bergmann's fibers, and the velate and ordinary astrocytes in the cerebellum show immunoreactivity as well.