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D Llanes

Publications and source records attributed to D Llanes.

At least 19 recordsLinked to original sources
PubMed1997

Purification and characterization of the pig analogue of human membrane cofactor protein (CD46/MCP).

A panel of mAbs were raised against pig lymphocytes. Seven mAbs immunoprecipitated a 50- to 60-kDa membrane-bound protein. This protein, termed JM4C8-Ag, was expressed on a wide variety of cells, including all circulating cells and cells of fibroblast, epithelial, and endothelial origin. The JM4C8-Ag was transmembrane-anchored and glycosylated. One of the Abs was used in immunoaffinity chromatography to isolate JM4C8-Ag from erythrocyte membranes. N-terminal amino acid analysis through the first 28 residues showed a 43% homology with the human complement regulatory molecule membrane cofactor protein (MCP; CD46). The purified protein had cofactor activity for factor I-mediated cleavage of human and pig C3b, confirming its identity as the pig analogue of human MCP. The purified protein also strongly inhibited lysis of rabbit erythrocytes by human and pig complement after activation of the classical or alternative pathway. This is the first report of a nonprimate analogue of MCP. The presence of a resident MCP on pig cells capable of acting as a cofactor in the control of human complement activation has consequences for the use of pig organs in xenotransplantation.

Amino Acid Sequence↗
PubMed1997

Characterization of the porcine homologue to human platelet glycoprotein IIb-IIIa (CD41/CD61) by a monoclonal antibody.

Human gpIIb-IIIa or CD41/CD61 is a Ca(2+)-complex dependent heterodimer, abundant on platelets, that plays a key role in hemostasis. This report describes a murine monoclonal antibody, JM2E5, able to recognize and immunoprecipitate the gpIIb/IIIa surface glycoprotein from porcine platelets. Immunoprecipitation analysis showed an antigen molecular weight of 115 and 85 kDa under nonreducing conditions, and of 110, 100 and 25 kDa under reducing conditions. Immunohistochemistry analyses of frozen sections from several porcine lymphoid organs gave specific staining on platelets. EDTA treated platelets were studied by flow cytometry indicating that the epitope recognized was Ca(2+)-complex independent. Western-blotting experiments with porcine platelet extracts gave an antigen molecular weight of 85 kDa under nonreducing conditions, thus localizing the epitope recognized by JM2E5 on the complex light chain gpIIIa or CD61. JM2E5 was also cross-reacting with human, bovine and horse platelets, as shown by flow cytometry. This mAb would allow further studies on this important adhesion molecule on horses, ruminants and pigs, and it should be especially useful as a general anti-porcine platelet reagent.

Animals↗
PubMed1996

Ruminant cluster WC13.

Five monoclonal antibodies (mAbs) belonged to preliminary cluster 27; however, only two mAbs, Buf13 (3W-506) and Co-3D1D4 (3W-202), were shown to detect the same surface antigen and belong to WC13. The other three mAbs, IVA120 (3W-323), IVA197 (3W-533) and IVA198 (3W-290), detected a 45 kDa molecule that could be fibrinogen (Mateo A., Perez de la Lastra, J., Moreno, A., Dusinsky, R., Bilka, F., Simon, M., Horovska, L., Naessens, J. and Llanes, D., 1996. Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies. Vet. Immunol. Immunopathol., 52: 363-370; Perez de la Lastra, J.M., Mateo, Dusinsky, R., Bilka, Simon, M., Horovska, L. and Llanes, D., 1996. Two monoclonal antibodies from the platelet panel recognize sheep plasma fibronigen. Vet. Immunol. Immunopathol., 52:).

Animals↗
PubMed1996

Platelet activation studies with anti-CD41/61 monoclonal antibodies.

The purpose of this study was to monitor platelet activation by following alpha IIb beta 3 integrin (GpIIb/IIIa complex or CD41/CD61) on the platelet surface by flow cytometry (FCM) analysis using workshop mAbs. The results obtained with the mAbs showed increased expression of the GpIIb/IIIa complex (about 40%) on the platelet membrane surface under thrombin stimulation.

Animals↗
PubMed1996

Biochemical characterization of antigens detected with anti-platelet monoclonal antibodies.

A panel of 18 monoclonal antibodies (mAbs) defined by the third workshop as specific for platelets, clustered in three preliminary groups: PC7, PC13 and PC27. These mAbs were further analysed by immunoprecipitation using extracts of iodinated and biotinylated peripheral blood mononucleated cells (PBMC) and platelets. We could confirm the existence of mAbs with specificities to WC9 (in PC7) and CD41/61 (in PC13). Two mAbs formed a new cluster, WC13, which may be homologous to human CD31 (in PC27). The influence of EDTA and thrombin on the expression of the different antigens on the platelet membrane was assessed by flow cytometry (FCM) analysis, as well as cross-reactivity with platelets from different species.

Animals↗
PubMed1996

Two monoclonal antibodies from the platelet panel recognize sheep plasma fibrinogen.

Among the monoclonal antibodies (mAbs) submitted to the third Workshop, two mAbs, IVA120 (3W-323) and IVA198 (3W-290), could be identified to recognize the sheep fibrinogen molecule. The apparent molecular weight of the immunoprecipitated 48-60 kDa cell surface protein under reducing conditions suggested this antigen could be the fibrinogen molecule. ELISA and immunoblotting assays, performed with commercially available sheep plasma fibrinogen, confirmed that these two mAbs recognize two different epitopes present on the sheep fibrinogen molecule.

Animals↗
PubMed1996

A monoclonal antibody to ruminant fibrinogen produced through immunization with ovine peripheral blood leukocytes.

By immunizing BALB/c mice with peripheral blood leukocytes (PBL) we have obtained a monoclonal antibody (MAb) named Co.1C9D10, specific for the gamma chain of ruminant fibrinogen. This MAb immunoprecipitates a molecule of apparent molecular weight of 55/65 kDa under reducing conditions and of around 300 kDa under nonreducing conditions. Co.1C9D10 recognized the gamma chain of fibrinogen by immunoblotting techniques and blood vessel plasma from paraffin embedded tissues by immunocytological techniques. Flow cytometry analysis showed that fibrinogen appears adhered to monocytes and granulocytes being eliminated through EDTA treatment of the cells. This phenomenon explains why PBL used as immunogen was able to induce an MAb against fibrinogen.

Animals↗
PubMed1995

Characterisation of a membrane receptor on ruminants and equine platelets and peripheral blood leukocytes similar to the human integrin receptor glycoprotein IIb/IIIa (CD41/61).

This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band did not appear owing to the absence of labelling with sulpho-NHS-biotin. Co.35E4 and Co.20A1 recognised two different Ca2+ complex independent epitopes. The glycoprotein IIb/IIIa was present on ruminant and equine granulocytes, monocytes and platelets. However, binding on granulocytes and monocytes was due to the adsorption of membrane platelet fragments.

Animals↗
PubMed1995

Homotypic aggregation of ovine leucocyte induced by anti-leucosialin (CD43) monoclonal antibodies.

Four monoclonal antibodies directed against ovine/bovine leucocyte antigens (Co.44B8, Co.11F10, Co.33B3 and Co.26F4) were produced and all recognized an antigen with an apparent molecular weight of 105 kDa under reducing conditions. This molecule was considered to be the ovine/bovine analogue of human, rat and mouse CD43 or leucosialin. This conclusion was based on (a) the cellular distribution of the antigen which was similar to that described in other species, (b) a decrease of apparent M(r) (150 kDa) after treatment with neuraminidase, and (c) production of cell aggregation by the four monoclonal antibodies and the influence of temperature and metabolic inhibitors on this phenomenon. Co.44B8, Co.11F10, Co.33B3 and Co.26F4 all recognized and immunoprecipitated the molecule after neuraminidase treatment of the cells. The data indicate an important function of CD43 as a mediator of cell aggregation for ruminant cells.

Animals↗
PubMed1995

A monoclonal antibody to an ovine gp130 molecule inhibits homotypic aggregation induced by anti-CD43 monoclonal antibodies of ruminant leukocytes.

A monoclonal antibody (mAb) Co.3E1D4 which recognized a molecule present on the surface of ovine peripheral blood granulocytes, monocytes, platelets and a small percentage of lymphocytes, was able to block leukocyte aggregation induced by mAbs specific for the leukosialin or CD43 molecule. Co.3E1D4 precipitated an antigen with apparent molecular weight of 130 kDa (under reducing and non-reducing conditions). Endoglycosidase F digestion of the molecule resulted in reduction in the apparent molecular weight. These data suggest that the antigen recognized by Co.3E1D4 may be homologous to the human CD31 or platelet endothelial cell adhesion molecule-1 (PECAM-1). Aggregation produced by other inducers such as PMA was not blocked showing that only some cell-cell interactions were mediated by the gp130 molecule.

Animals↗
PubMed1995

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin.

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the Ba allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the Ba allele and does not react with BbBb homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.

ABO Blood-Group System↗
PubMed1995

The beta chain of the GPIIb molecule on ruminant leukocytes and platelets is not labelled by the sulfo-NHS-biotin method.

Glycoprotein (Gp)IIb/IIIa molecules have been immunoprecipitated from platelets and leukocytes of cattle, goat, horse, human, sheep and swine using specific monoclonal antibodies. The sulfo-NHS-biotin (sulfosuccinimidobiotin) method used to label the proteins has been found unsuitable for labelling the beta chain of the ruminant GPIIb/IIIa molecule. The beta chain was present on ruminant leukocytes and platelets when immunoprecipitates were silver stained.

Animals↗
PubMed1995

Serologically defined lymphocyte alloantigens in Spanish sheep.

Using segregation and population analysis of serological data prepared in our laboratory, we have tentatively defined five OLA antigenic specificities: CO1.1, CO1.2, CO2.1 and CO2.2, whose genetic control would fit a model of two class I OLA loci (CO1.1 and CO1.2 first locus, CO2.1 and CO2.2 second locus, and CO3.1, that could represent a subtype of CO2.2. Alloantisera were obtained from Spanish Merino sheep by intrafamilial cross-immunization and from serum of primiparous and multiparous ewes. Screening of alloantisera for antilymphocyte reactivity was carried out by a two-step dye exclusion microcytotoxicity test against 216 animals belonging to different local breeds: Spanish Merino, Merino Landschaft, Manchega and Churra. Lymphocyte antigen specificities were characterized by cluster and correlation analysis, as well as family studies. Comparison of CO alloantisera with reference reagents from Scotland (CA reagents) showed an excellent correlation between CO1.1 and CA2, and CO2.1 and CA5. The reagents described can be used in various breeds, a characteristic important for their future applications.

Animals↗
PubMed1992

An erythroid species-specific antigen of swine detected by a monoclonal antibody.

A monoclonal antibody (1AC11) has been produced which recognized the glycophorin of swine red blood cells. 1AC11 was specific for swine membrane erythrocytes. No other swine cells (leukocytes, macrophages, kidney and testis cells) nor red blood cells from all the tested mammalian species (goat, human, sheep, cattle, horse, rabbit, cat and guinea pig) were recognized. There was no blood group activity detected. Immunocytochemical analysis of blood vessel in the swine pituitary tissue showed that besides membrane erythrocytes, cytoplasmic molecules were recognized in some cells. Immunoblot analysis of both membrane and aqueous phase of chloroform/methanol fractions from swine erythrocytes showed that the monoclonal antibody 1AC11 reacts with the major sialoglycoprotein of apparent molecular weight 45,000 daltons.

Animals↗
PubMed1992

Sheep blood polymorphism and genetic divergence between French Rambouillet and Spanish Merino: role of genetic drift.

Rambouillet sheep originating from Spanish Merino have been maintained in France as a small and closed flock since their importation. After 190 years of independent evolution, the flock has markedly differentiated from its Spanish parental population. The observed differences between them were characterized by the fixation in Rambouillet of the Mb and F30 alleles, which occurred in Spanish Merino with frequencies of 0.90 and 0.80 respectively (at two distinct blood group loci M and F30) and by the absence in Rambouillet of other alleles or phenogroups (at the Tf and the A, B, C blood group loci) which were observed in Spanish Merino with frequencies ranging from 0.10 to 0.28. On the basis of their phenotypic distributions at 11 blood polymorphic loci, the two populations differed significantly from each other (total chi 2 values = 352.62, 23 df, P less than 0.001). By comparing the observed magnitude of gene frequency differences between Rambouillet and Spanish Merino with the estimate of inbreeding coefficient for Rambouillet obtained from pedigrees, it appeared that the observed genetic differences could be attributed to the evolutionary change due to random drift in the small and closed flock of Rambouillet.

Animals↗
PubMed1992

Mouse monoclonal antibodies to swine blood group antigens.

Eight mouse hybridomas with haemagglutination capacity to swine blood group antigens were obtained, three of them producing antibodies capable of being used as blood group reagents. Two detected the Ba factor and another the Fa factor. The others gave non-specific and weak reactions or cross-reaction with antigens present in more than one system. We conclude that mouse monoclonal antibodies are also suitable for use in swine as a complement of polyclonal reagents.

Animals↗
PubMed1991

Enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in goats.

Using a heat and sonicated Mycobacterium paratuberculosis Cordoba antigen (COA1) and the commercial protoplasmic-antigen (PPA-3) as antigens, an ELISA for detecting goat antibodies was standardized. When 2 reference populations, 1 positive (17 goats) and the other negative (63 goats) to disease, were used, this test showed 87.5% sensitivity and 93.6% specificity for COA1, and 88.2 and 95.2%, respectively for PPA-3. Absorption with M phlei was performed; no significant differences were found for COA1, but a lower sensitivity was found with PPA-3. This test was not especially affected by cross-reactivity with other mycobacterial disease because when 9 goats with M bovis infection were included in the M paratuberculosis control group, the specificity was only slightly different for absorbed (94.4%) and nonabsorbed sera (91.7%) for COA1, and (93.1 and 94.4%, respectively) for PPA-3. This test was used to study the percentage of seropositive goats for M paratuberculosis in 3 herds with different prevalences. Among 251 goats in southern Spain (Huelva), 40% were found positive for COA1 and 41% for PPA-3. Among 242 goats studied in southern Spain (Córdoba), 10.0% were positive for COA1 and 13.0% for PPA-3. In the Canary Island population of 176 goats, 3% were positive for COA1 and 0.5% for PPA-3. According to the accuracies of both positive and negative predictions, our test could be applied to populations with high prevalence to prevent additions to the herd and to cull infected animals (with 40% prevalence, the positive and negative predictive values are 90%), and to prevent adding infected animals to populations with moderate or low prevalence.

Animals↗