Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents.

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.

A novel approach to the generation of antibodies against phylogenetically preserved sperm antigens.

Conventional methods for immunization of laboratory animals against human spermatozoa proved not to be efficient enough to identify phylogenetically conserved sperm-specific antigens. A combination of vasectomy and subcutaneous administration of autologous testis homogenates was tested in 5 New-Zealand rabbits, and in 7 Long-Evans and 8 Spraque-Dawley rats in an attempt to induce an autoimmune response against such antigens. This experimental procedure resulted in a generation of sperm autoantibodies cross-reactive with human, rabbit and rat spermatozoa, as demonstrated by sperm-agglutination, ELISA and flow cytometry (FCM). No specific binding to human seminal plasma was detected by ELISA, indicating that intrinsic sperm membrane antigens rather than sperm-coating antigens were involved in establishing cross-reactivity with human spermatozoa. This suggestion was confirmed by the finding that rabbit autoantisera reacted more strongly against epididymal than against ejaculated human spermatozoa as shown by FCM. Humoral antispermatozoal response correlated well with impaired spermatogenesis in rabbits. The autoimmunized rats revealed severe alterations in reproductive tissues, including testicular and epididymal sperm granulomas; however, they showed a lower incidence of circulating antibodies. The results indicate that the established experimental model in rabbits can be further used to identify and characterize evolutionary preserved intrinsic sperm membrane autoantigens, which are desirable candidates for contraceptive vaccine development.

[The sperm-agglutinating activity of the fractions of saline root extracts from Arum maculatum]. / Spermoaglutinirashta aktivnost na fraktsii ot korenishten vodno-solevi ekstrakt na Arum maculatum.

Our previous investigations have demonstrated that saline extract of Arum maculatum contains lectin (sperm agglutinin) which agglutinates human ejaculated spermatozoa of the type tail to tail and head to tail. By means of speed liquid chromatography (Mono S XP 5/5) eight fractions (I-VIII) were obtained. The sperm-agglutinating activity of the fractions was investigated against frog testicular spermatozoa and testicular and epididymal spermatozoa of man, rabbit, rat and mouse. The fractions did not agglutinate frog spermatozoa. Fraction I agglutinated rabbit, rat and murine spermatozoa; fraction II-rabbit and rat spermatozoa; fraction VII-only human spermatozoa, and fraction VIII-human, as well as animal spermatozoa. The fractions III, IV, V, VI had no sperm-agglutinating activity. These data could be explained by the assumption that the differences in the sperm-agglutinating activity of the fractions are due to isoforms (called also isolectins) of the phytospermagglutinin, contained in the saline extract of Arum maculatum.

[The composition and structure of isolectins from Arum maculatum]. / Prouchvane na sustava i strukturata na izolektini ot Arum maculatum.

The delipidized saline extract of Arum maculatum (AM) contains two selectively agglutinating human spermatozoa isolectinesphytospermoagglutinins (FSA1 and FSA2), which bind to various receptors of spermatozoon plasmalemma. It is established by means of disc polyacrilamide electrophoresis amino acid analysis and isoelectric focusing that FSA1 is tetrameter, but FSA2-octameter, which have similar amino-acid content and do not contain carbohydrates. The established structural differences probably cause also the capability of FSA1 and FSA2 to bind various receptors of spermatozoon plasmalemma. It is proven that FSA2 possesses antigenic properties.

[The effect of lectins on human spermatozoa in the capillary sperm penetration test]. / Prouchvane na vliianieto na lektini vurkhu choveshki spermatozoidi chrez kapiliarniia spermopenetratsionen test.

The effect of the commercial lectin (Con A, PHA, SRA, WGA and UEA) as well as of the prepared by us extract from roots of Arum maculatum (Am and AM 80 degrees C) was studied on migration of human spermatozoa in bovine estrous cervical mucus, using the capillary sperm penetration test of Kremmer. It was established that Con A, PHA and AM inhibited the migration of spermatozoa in a concentration, in which they agglutinated human spermatozoa in vitro. Nonagglutinating human spermatozoa SBA and AM 80 degrees C inhibited also the migration of spermatozoa in the mucus.

[Localization of receptors for phytospermoagglutinin from Arum maculatum on human spermatogenic cells and spermatozoa]. / Lokalizatsiia na retseptori za fitospermoaglutinin ot Arum maculatum vurkhu choveshki spermatogenni kletki i spermatozoidi.

Our precious studies showed that delipidized water-saline extract from roots of Arum maculatum (AM) contained lectin-phytospermoagglutinins (PSA), which caused agglutination, manifested by the type of a tail with a tail and a head with a tail. This extract we conjugated with fluorescein-isothiocyanate and by means of the conjugate we studied the localization of receptor molecules for PSA on the plasmalemma of human spermatogenic cells and human spermatozoa, obtained from the testis, epididymis and ejaculate. It was established that receptor molecules for PSA were distributed irregularly on the plasmalemma of spermatozoa, that the basic part of these molecules were synthesized on spermatogenic cells and that ejaculate spermatozoa adsorbed such molecules from the sperm plasma.

[Study of the biological properties of isolectin from Arum maculatum]. / Prouchvaniia vurkhu biologichni svoistva na izolektini ot Arum maculatum.

The delipidized water-saline extract of roots from Arum maculatum contains lectin-phytospermoagglutinins (PSA), which consist of two forms, isolectins--PSA1 and PSA2. The capability of these isolectins to agglutinate spermatozoa and erythrocytes and to precipitate serum of a frog, a rabbit and man was studied. It was established that PSA1 interacted with a receptor specific for human spermatozoa, but PSA2--with a receptor probably common for human and rabbit spermatozoa. But this mean that these isolectins could be used for isolation of spermatozoa antigens, which have no blood group specificity and are not found in human biological fluids.

Polymeric pyelotomy (experimental studies).

A non-toxic, soluble polymeric preparation MAG with controlled period of gelation was synthetized for the extraction of stones from the pyelocalyceal system during surgery for urolithiasis. With respect to toughness the preparation proved superior to the fibrinogen-thrombin mixture, used in the so-called "fibrin pyelotomy" according to Dees. Its biologic testing in animals (rabbits, dogs and pigs) yielded excellent results.