Daniel Portetelle

One-step purification of mouse monoclonal antibodies from ascitic fluid by DEAE Affi-gel blue chromatography

Monoclonal antibodies can be purified directly from ascitic fluids by chromatography on a DEAE Affi-gel blue column. Optimal conditions were determined for the recovery of immunoglobulins free of contaminating protease and nuclease activities.

Monoclonal antibodies define eight independent antigenic regions on the bovine leukemia virus (BLV) envelope glycoprotein gp51

Abstract Fifteen monoclonal anti-BLV gp51 antibodies are characterized. Competition antibody binding assays show that they are directed against eight independent antigenic regions on the BLV gp51 molecule. Conformation or accessibility of some of these gp51 epitopes change with the test system used, namely the liquid phase radioimmunoassay with radiolabeled antigen or the solid phase enzyme immunoassay with plastic bound gp51 or BLV particles. A two-site immunometric assay using monoclonal antibodies directed against two independent epitopes allows detection of isolated gp51 molecules at a minimal concentration of 0.4 ng/ml and is also suitable for the detection of BLV particles.

Topographical analysis by monoclonal antibodies of BLV-gp51 epitopes involved in viral functions.

Abstract Mouse monoclonal antibodies, directed against eight independent antigenic sites on the bovine leukemia virus (BLV) envelope gp51 (sites A, B, C, D, E, F, G, H), were tested for their capacity to interfere with the biological activities of BLV which are associated with the gp51 molecule, as follows: the pseudotype inhibition test (PI) measures the ability of antibodies to inhibit infectivity of BLV-VSV pseudotypes; the early polykaryocytosis inhibition test (EPI) measures inhibition of syncytia-inducing activity of BLV-producing cells in the presence of these antibodies; and the complement-dependent cell lysis measures the cytoxic activity of antibodies toward BLV-producing cells in the presence of complement. Three of the eight antigenic sites (sites F, G, H) were shown to be involved in the biological activities tested for in infectivity and syncytia neutralization tests. At least one of these three regions is exposed on the surface of BLV-producing cells (site G) and can be the target for complement-dependent cytotoxicity. Limited protease digestion of gp51 showed that a peptide fragment of MW 15,000 contained at least a part of sites FGH involved in the biological activities analyzed and of site E which is inactive. The major carbohydrate residues are localized on a fragment of apparent molecular weight 35,000, also containing the antigenic sites ABCD which are not involved in the biological activities tested for.

Bovine Leukemia Virus Involvement In Enzootic Bovine Leukosis

Publisher Summary Enzootic bovine leukosis is a contagious disease induced by bovine leukemia virus (BLV). It is a chronic disease that develops over a long period and can be schematically divided into three phases: (1) from birth to infection (some animals are already infected at birth), (2) from viral infection to tumorous transformation (a number of infected animals do not develop tumors before being slaughtered), and (3) from tumorous transformation to death. Bovine leukotic lymphocytes, kept in vitro in suitable medium, release detectable amounts of BLV. Short-term cultures of leukocytes from cows with persistent lymphocyotsis or long-term cultures of BLV-infected cells were used to study the morphogenesis of BLV, wherein one system was made of degenerating cells, the other made of healthy fast growing cells. Detection of anti-BLV antibodies includes immunodiffusion using p 24 , p 15 , immunofluorescence (IF), complement fixation (CF), and radioimmunoassay. All these methods are indirect detection methods. They detect BLV infection through the immune reaction of the host. The chapter presents the experiments performed to understand the transmission of BLV to the homologous host.

Biologically active epitopes of Bovine Leukemia Virus glycoprotein gp51 : their dependence on protein glycosylation and genetic variability.

A panel of monoclonal antibodies to the bovine leukemia virus envelope glycoprotein (BLV gp51) has previously demonstrated the association of the biological activities of the virus (infectivity, syncytia induction) with three out of eight epitopes of gp51. In BLV-infected cells, the unglycosylated homolog of the precursor to the BLV envelope glycoproteins (gPr72env) is a 47,000-MW polypeptide. Immunoprecipitation studies with monoclonal antibodies show that the neutralizing antibody-inducing sites, although present in gPr72env, are not conserved in the 47,000-MW unglycosylated homolog. Finally, it is demonstrated that the neutralizing antibody-inducing sites of gp51 are subject to antigenic variation among BLV isolates of the same or different geographical origins.

Epitopes of bovine leukemia virus glycoprotein gp51 recognized by sera of infected cattle and sheep

Sera of BLV-infected cattle and sheep are tested for their reactivity with different gp51 subregions by competition with radiolabeled monoclonal antibodies directed against 8 different gp51 epitopes. Sheep antisera are found to be very polyvalent, since they are able to displace the fixation of any of the mouse monoclonal antibodies to gp51. Bovine antisera do not display significant competition with monoclonal antibodies direct against 5 out of 8 gp51 epitopes. The bovine antibody response to gp51 is focused to a limited subregion of this molecule, bearing 3 epitopes (F, G and H) recognized by antibodies with virus-neutralizing activity. The differential reactivity of cattle and sheep antisera to BLV gp51 is discussed in relation to the pathology of BLV infection in these two species.

The diagnosis of enzootic bovine leukosis

This paper reviews the clinical and virological diagnostic procedures for enzootic bovine leukosis (EBL). The clinical diagnosis must be always confirmed by a specific laboratory test for Bovine Leukaemia Virus (BLV). Many virological tests were proposed. The sensitivity of all the diagnostic methods is sufficient to do an early detection of a BLV infection on an individual base. Advantages of the highly sensitive methods like RIA and ELISA appear when the samples to be tested have naturally very low antibody titers (individual milk, bulk milk, pooled sera).

Diagnostic tests of bovine leukemia: Comparison between an hematological test and the serological diagnosis☆

Abstract The classical hematological test (lymphocytes counts) was applied to 5 herds of cattle where the enzootic form of bovine leukemia was diagnosed. In the same five herds, a search for antibodies directed against bovine leukemia virus (BLV) p 24 was carried out using an agar gel immunodiffusion (AGID) technique. Among the 245 animals screened, 30 reacted positively to both tests, 11 were positive by hematological test only and 15 were positive by serological test only. From these results we would advise to eliminate from so heavily infected herds, all the animals showing abnormal blood pictures and all these reacting positively to the immunological test.

Solid-phase enzyme immunoassay of urokinase using monoclonal antibodies

Using two monoclonal antibodies directed against urokinase, we have developed a micro enzyme-linked immunosorbant assay (ELISA) to detect and measure urokinase in biological fluids. The system presents the following characteristics: simple and rapid procedure, reproducibility, sensitivity (urokinase levels down to 1 ng/ml) and evaluation of the enzyme in biological fluids such as urine, pleural elfusions, and ascitic fluids without preliminary purification.

DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line POTENTIAL INVOLVEMENT OF DIRECT INHIBITION OF cAMP-RESPONSIVE ELEMENT (CRE)-BINDING PROTEIN/CRE MODULATOR/ACTIVATION TRANSCRIPTION FACTOR BINDING

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2′-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5′-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator TaxBLV decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267LTaxSN 5′-LTR compared with the L267 5′-LTR. Interestingly, DNA methylation inhibitors and TaxBLV synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the −154 or −129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at −129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5′-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.