RESUMEN
In an attempt to improve the accuracy of sexing bovine embryos, new anti-H-Y monoclonal antibodies were produced and selected, using an extended screening procedure. In addition to the commonly used screening of soluble H-Y antigen sources, such as testis supernatant and Daudi supernatant, the binding specificity to cell surface H-Y antigen was tested also. A radioimmunoassay (RIA) employing male and, as a control, female bovine lymphocytes, and enzyme-linked immunosorbent assays (ELISAs) on solubilized membrane fractions resulted in the selection of a number of clones producing monoclonal antibody (mAb) with male-enhanced binding. Four of the anti-H-Y mAb were assessed for binding to Day 7 or 8 bovine embryos. The accuracy of sexing bovine embryos ranged from 58% to 71%. Two of the four antibodies did not react with presumed soluble H-Y antigen-containing sources in an ELISA. These results raise doubts about the suitability of the presumed soluble H-Y antigen sources, Daudi, TM4 and testis supernatant, to be used in screening tests for anti-H-Y antibodies.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Embrión de Mamíferos/fisiología , Antígeno H-Y/inmunología , Diferenciación Sexual/inmunología , Animales , Bovinos , Línea Celular , Membrana Celular/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Humanos , Inmunoglobulina G/inmunología , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Radioinmunoensayo/veterinaria , Testículo/inmunologíaRESUMEN
In vitro cultures of intact chick gonads (organ cultures) and reaggregation cultures of dispersed gonad cells (roller cultures) were made. Gonads or gonad cells from 7-day-old chick embryos, at the stage when sex-specific differentiation begins, were cultured in the presence of presumed H-Y antigen-containing supernatants, or co-cultured in the presence of H-Y antigen-producing cell lines. The H-Y antigen-producing cells tested were of human, mouse, bovine and chicken origin. During organ culture, addition of supernatant of the human lymphoma cell line Daudi, or co-culture with Daudi cells, stimulated a clear proliferation of the germinal epithelium in male gonads, indicating feminization. A similar effect was obtained by treatment with estradiol. In reaggregation culture, the increase in nuclear size of germ cells was chosen as a parameter for feminization. A significant increase of germ cell nuclear size was observed in gonads cultured in the presence of Daudi supernatant. In both organ cultures and reaggregation cultures, other tested H-Y antigen sources and semi-purified H-Y antigen fractions did not exert significant effects on differentiation of the gonads or on the average area of the germ cell nuclei. These findings suggest that it is not H-Y antigen, but another protein produced by Daudi cells, that might be responsible for the sex-reversing effects.
Asunto(s)
Trastornos del Desarrollo Sexual , Gónadas/citología , Antígeno H-Y/farmacología , Linfoma de Células B/inmunología , Animales , Bovinos , Agregación Celular/efectos de los fármacos , Agregación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Núcleo Celular/ultraestructura , Embrión de Pollo , Estradiol/farmacología , Femenino , Gónadas/embriología , Gónadas/fisiología , Antígeno H-Y/análisis , Humanos , Linfoma de Células B/patología , Linfoma de Células B/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Ovario/citología , Ovario/embriología , Ovario/fisiología , Testículo/citología , Testículo/embriología , Testículo/fisiología , Células Tumorales CultivadasRESUMEN
Post-weaning anoestrus was studied in eighteen primiparous sows, selected from a breed showing a high proportion of anoestrous sows. The sows were studied from late lactation, through weaning at day 29 post-partum (p.p.), until day 21 post-weaning (p.w.). Blood samples were taken once daily, and frequently (every ten minutes) on several days before and after weaning. Out of a total of ten anoestrous sows, three were exposed to a boar and seven were given gonadotropins (PG600) on day 21 p.w.. Serial blood samples were analysed for LH only and daily samples were additionally analysed for oestradiol-17 beta and progesterone, by validated radioimmunoassay procedures. Analysis of variance of the basal level, pulse frequency, pulse amplitude and mean level of LH showed, retrospectively, that during lactation the basal and mean levels of LH were significantly lower in anoestrous than in oestrous sows (P < or = 0.05). Furthermore, the post-weaning basal and mean levels of LH were also significantly lower in anoestrous than in oestrous sows (P < or = 0.05). However, because of the small number of oestrous animals (n = 3), these results should be interpreted with caution. Exposure of anoestrous sows to a boar did not result in oestrus and/or ovulation within seven days, but did increase LH pulse frequency. Injection of gonadotropins resulted in an LH surge, oestrus and ovulation in only three sows, but oestradiol levels were increased in six sows. From our experiments and from reports in the literature we conclude that a lowered secretion of LH may play a role in the aetiology of post-weaning anoestrus in the sow.
Asunto(s)
Anestro/fisiología , Gonadotropinas/farmacología , Hormona Luteinizante/sangre , Conducta Sexual Animal , Porcinos/fisiología , Anestro/sangre , Anestro/efectos de los fármacos , Animales , Estradiol/sangre , Estro/fisiología , Femenino , Gonadotropinas/administración & dosificación , Inyecciones Intramusculares/veterinaria , Lactancia , Masculino , Ovulación/fisiología , Progesterona/sangre , Radioinmunoensayo/veterinaria , Porcinos/sangre , DesteteRESUMEN
In the present experiments the efficacy of murine and bovine monoclonal antibodies for passive immunization in cattle was compared. The in vivo immunoneutralization of pregnant mare serum gonadotrophin (PMSG) by murine and bovine antibodies after repeated administration was chosen as a model for this study. Results indicate that repeated injections of murine monoclonal antibodies against PMSG (mMCA) alone did not, or only to a small extent, elicit an anti-mouse immune response. The simultaneous administration of mMCA and PMSG resulted in relatively high levels of anti-mouse antibodies after the second injection, leading to a decrease in neutralizing activity of mMCA. The results suggest that the neutralizing activity of mMCA is inhibited more by anti-idiotypic than by anti-isotypic antibodies against mMCA. In vivo, the bovine monoclonal antibody against PMSG (bMCA) only partially neutralizes PMSG. After repeated administration of bMCA, either alone or in combination with PMSG, no anti-bMCA antibodies could be detected in our assay system. In addition, no change in plasma levels of bMCA and PMSG compared with levels after the first injection was observed. Although it has to be confirmed by further experiments whether our findings can be generalized, the present results suggest that for repeated passive immunization in cattle homologous antibodies are to be preferred above heterologous antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Bovinos/inmunología , Gonadotropinas Equinas/inmunología , Inmunización Pasiva/veterinaria , Animales , Anticuerpos Monoclonales/administración & dosificación , Reacciones Cruzadas/inmunología , Femenino , Inmunización Pasiva/métodos , Ratones , Pruebas de Neutralización/veterinariaRESUMEN
Bovine-murine heteromyeloma cell lines were prepared by fusing lymphoid cells from a bovine leukemia virus (BLV)-infected cow with mouse myeloma cells. Selection of hybrid cell colonies was based on the ratio of bovine and murine chromosomes, the presence of cell-surface immunoglobulins and growth characteristics. First-generation fusion partners were compared for fusion efficiency and the number of antigen-specific antibody-producing clones generated. Hybrid cell colonies that initially secreted antibodies were selected from first-generation heteromyelomas to function as second-generation fusion partners. Although fusion efficiencies for both generations did not differ, the second-generation heteromyelomas yielded a higher number of specific antibody-producing clones. Fusion of hteromyelomas with either lymph node cells or splenocytes indicated that fusion with lymph node cells results in a higher number of specific antibody-producing clones, whereas fusion efficiency was found to be higher with splenocytes. The optimal time intervals between the final booster injection and fusion were found to be 4 days for splenocytes and 7 days for lymph node cells. Finally, the characterization of bovine monoclonal antibodies against bovine rotavirus and pregnant mare serum gonadotrophin and their neutralizing capacities in vitro are described.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Fusión Celular , Gonadotropinas Equinas/inmunología , Células Híbridas/inmunología , Rotavirus/inmunología , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Cariotipificación , Virus de la Leucemia Bovina , Leucemia Experimental , Linfocitos , Masculino , Ratones , Ratones Endogámicos , Mieloma Múltiple , Células Tumorales CultivadasRESUMEN
Monoclonal antibodies against the H-Y antigen were produced using spleen cells from female C57BL/6 mice hyperimmunized with cells from syngeneic males. Anti-H-Y positive clones were detected by enzyme immunoassays. Supernatant fluids from Daudi cell cultures and testicular cell preparations taken from mice, rabbits or calves served as presumptive sources of H-Y antigen. In addition, testis supernatant from genetically sterile mice was used. Male specificity was ascertained by the fact that the antibodies could be absorbed with spleen cells from male but not from female mice. Binding of the antibodies to H-Y antigen on the surface of male and female cells, obtained from a number of tissues and species, was confirmed by an indirect immunofluorescence assay. Several monoclonal antibodies appeared to be positive in all assays tested, suggesting that the molecule conferring the H-Y antigenicity lacks species-specificity and appears to be identical for soluble and membrane-bound H-Y antigen.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno H-Y/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Bovinos , Células Cultivadas , Femenino , Hibridomas/inmunología , Masculino , Ratones , Conejos , Caracteres Sexuales , Testículo/inmunologíaRESUMEN
The hybridoma technology for production of monoclonal antibodies circumvents many of the constraints associated with the use of conventional antisera, and consequently broadens the areas of application of antibodies in animal sciences. In the present review, the potential usefulness of monoclonal antibodies in animal production - with emphasis on reproduction - is discussed, including the inherent limitations of the current technology and the improvements that can be foreseen within the next few years. Because of their unique specificity and the fact that they can be produced in virtually unlimited quantities, monoclonal antibodies are an important tool in diagnostics. However, the use of these antibodies does not always guarantee absolute specificity, and the low affinity of many monoclonal antibodies will impose a number of limitations on their use. Monoclonal antibodies can also be used to optimize physiological processes such as growth and reproduction. For this, homologous antibodies will probably offer several advantages over their murine counterparts in terms of effectiveness for passive immunization. Some success has already been achieved in the development of monoclonal antibodies from livestock species. Finally, it is shown that monoclonal antibodies are becoming extremely powerful research tools.
La technologie des hybridomes pour produire des anticorps monoclonaux évite la plupart des contraintes associées à l'utilisation des antisérums conventionnels. Cela élargit en conséquence l'aire des applications des anticorps dans les sciences animales. Dans cette mise au point on discute l'utilité potentielle des anticorps monoclonaux dans la production animale en mettant l'accent sur la reproduction, y compris les limites inhérentes de la technologie actuelle et les améliorations qui peuvent être envisagées pour les prochaines années. A cause de leur spécificité unique, et du fait qu'ils peuvent être produits en quantité virtuellement illimitée, les anticorps monoclonaux sont un important outil diagnostique. Cependant, leur utilisation ne garantit pas toujours une spécificité absolue et la faible affinité de nombre d'entre eux en limtera l'emploi. Les anticorps monoclonaux peuvent être aussi utilisés pour optimiser les processus physiologiques tels que la croissance ou la reproduction. Pour cela, les anticorps homologues présenteront probablement plusieurs avantages sur leurs contreparties murines en ce qui concerne leur efficacité pour l'immunisation passive. On a déjà obtenu un certain succès dans le développement des anticorps monoclonaux pour le bétail. On montre enfin que les anticorps monoclonaux sont des outils de recherche extrêmement puissants.
RESUMEN
Diagnostics in animal production is an important field for application of monoclonal antibodies (MCAs). Representative examples are discussed. MCAs can also be used to influence physiological processes with regard to optimization of growth, reproduction, etc., although there are some limiting factors in this area. Finally MCAs may contribute to fundamental research directed toward application in agriculture.