References of "Journal of Immunological Methods"
     in
Bookmark and Share    
Full Text
Peer Reviewed
See detailAntibody production by injection of living cells expressing non self antigens as cell surface type II transmembrane fusion protein.
Nizet, Yannick; Gillet, Laurent ULg; SCHROEDER, Hélène ULg et al

in Journal of immunological methods (2011)

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps ... [more ▼]

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens. [less ▲]

Detailed reference viewed: 37 (12 ULg)
Full Text
Peer Reviewed
See detailGeneration of T lymphocytes from the epithelium and stroma of squamous pre-neoplastic lesions of the uterine cervix.
Jacobs, Nathalie ULg; Giannini, Sandra ULg; Al-Saleh, Walid et al

in Journal of Immunological Methods (1999), 223(1), 123-9

In this study, we have developed a simple and efficient technique for the isolation of viable lymphocytes from the epithelium and stroma of small pre-neoplastic squamous intraepithelial lesions (SIL) of ... [more ▼]

In this study, we have developed a simple and efficient technique for the isolation of viable lymphocytes from the epithelium and stroma of small pre-neoplastic squamous intraepithelial lesions (SIL) of the uterine cervix. Following the separation of the epithelium from the stroma using dispase II, both biopsy fragments were used to generate T lymphocytes. The stroma-derived lymphocytes were obtained by collecting and culturing the cells migrating out of the biopsy in the presence of IL2 (50 U/ml). An average of 0.7 x 10(6) and 1.4 x 10(6) lymphocytes could be obtained after 20 and 30 days of culture, respectively. For the expansion of lymphocytes derived from the pre-neoplastic epithelium (SIL) it was necessary to use a combination of irradiated peripheral blood mononuclear cells (PBMC) as a feeder layer with PHA (0.1%), in addition to IL2 (50 U/ml). Interestingly, these lymphocytes could be obtained using either allogeneic or syngeneic PBMCs. With this protocol, we were able to generate up to 100 x 10(6) lymphocytes from the epithelium, the majority of which were T lymphocytes. [less ▲]

Detailed reference viewed: 22 (3 ULg)
Peer Reviewed
See detailLymphoid cell apoptosis induced by trophoblastic cells: a model of active foeto-placental tolerance
Coumans, Bernard ULg; Thellin, Olivier ULg; Zorzi, Willy ULg et al

in Journal of Immunological Methods (1999), 224(1-2), 185-196

To test the hypothesis that CD95-L (Fas-L) present on trophoblastic cells plays a part in establishing foeto-placental tolerance by inducing apoptosis of immune defence cells, we cocultured trophoblasts ... [more ▼]

To test the hypothesis that CD95-L (Fas-L) present on trophoblastic cells plays a part in establishing foeto-placental tolerance by inducing apoptosis of immune defence cells, we cocultured trophoblasts with lymphoid cells and scored the frequency of cell death in these cultures. We prepared human trophoblastic cells from term placentas removed by C-section and placed them in culture for 48 h before introducing the lymphoid cells. We added Jurkat cells, a CD3 + lymphoid cell line, or purified T cells from human blood to the cultured trophoblasts and monitored apoptosis by electron microscopy and flow cytometry after TUNEL or annexin V labelling. The frequency of cell death in the CD3 + cell population was higher when the lymphoid cells were cocultured with trophoblastic cells than when they were cultured alone. This frequency increased with time but was reduced when anti-CD95-L antibodies were added to the culture medium. Cell death was less frequent in the lymphoid cell population when trophoblasts were replaced with human fibroblasts not expressing CD95-L. (C) 1999 Elsevier Science B.V. All rights reserved. [less ▲]

Detailed reference viewed: 35 (4 ULg)
Full Text
Peer Reviewed
See detailImproved IL-2 detection for determination of helper T lymphocyte precursor frequency in limiting dilution assay
Winandy, Marie ULg; Lewalle, Philippe; Deneys, Véronique et al

in Journal of Immunological Methods (1998), 215

In the context of allogeneic bone marrow transplantation, an accurate estimate of the risk of developing graft-versus-host disease GVHD. is of major interest. The pre-transplant frequency of donor’s ... [more ▼]

In the context of allogeneic bone marrow transplantation, an accurate estimate of the risk of developing graft-versus-host disease GVHD. is of major interest. The pre-transplant frequency of donor’s helper T-lymphocyte precursors HTLp. directed against host’s antigens may be helpful in predicting this risk. This technique relies on an indirect measurement of interleukin-2 IL-2. secreted by the HTLp, as assessed by the proliferation of an IL-2 dependent cell line. Many authors use the murine CTLL-2 cell line in this assay, but these cells do not respond to the presence of minute amounts of IL-2 in the culture medium, and thus do not discriminate between the absence or the presence of very low levels of IL-2. We therefore decided to compare CTLL-2 with another IL-2 dependent cell line, the murine A9.12 cell line. A comparison was made using serial dilutions of recombinant human IL-2, limiting dilutions of baby hamster kidney BHK. cells transfected with human IL-2 gene and in the context of clinical tests performed for the detection of pre-transplant HTLp. Both the sensitivity and reliability of the tests were better using A9.12. We conclude that the A9.12 cell line might be a more suitable tool for pre-transplant HTLp determinations before allogeneic bone marrow transplantation or whenever low IL-2 levels are to be measured. [less ▲]

Detailed reference viewed: 11 (2 ULg)
Full Text
Peer Reviewed
See detailOptimization of murine CD8+ cytotoxic T-lymphocyte responses to pseudorabies virus
Depierreux, C.; Graff, I.; Lancelot, V. et al

in Journal of Immunological Methods (1997), 203

Detailed reference viewed: 7 (1 ULg)
Full Text
Peer Reviewed
See detailImmunogold Labelling of Fatty Acyl Chains
Compère, Philippe ULg; Maneta-Peyret, Lilly; Goffinet, Gerhard ULg et al

in Journal of Immunological Methods (1995), 181(2), 201-9

For the first time, antibodies against a hydrophobic hapten have been used for immunogold labelling of a lipid antigen (BSA-C18:1 conjugate) coated on polystyrene. The labelling was visualised either ... [more ▼]

For the first time, antibodies against a hydrophobic hapten have been used for immunogold labelling of a lipid antigen (BSA-C18:1 conjugate) coated on polystyrene. The labelling was visualised either directly in transmission electron microscopy or in light microscopy after silver enhancement. Good recognition of the fatty acyl chain was obtained even after treatment of the antigen coat with various cross-linking fixatives used for electron microscopy, i.e. formaldehyde, glutaraldehyde and osmium tetroxide. [less ▲]

Detailed reference viewed: 7 (3 ULg)
Peer Reviewed
See detailFast double antibody radioimmunoassay of human granulocyte myeloperoxidase and its application to plasma.
Pincemail, Joël ULg; Deby-Dupont, G.; Deby, Christiane ULg et al

in Journal of Immunological Methods (1991), 137(2), 181-191

The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a ... [more ▼]

The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a radioimmunoassay (RIA) for this enzyme, a specific antiserum against human neutrophil MPO was raised in rabbits and used at an initial dilution of 1/10,000. MPO labelled with 125iodine by a technique of self-labelling in the presence of H2O2, had a specific activity of 24 mCi/mg. After incubation at room temperature (2 h) and separation by double antibody precipitation in the presence of polyethylene glycol, the sensitivity of the RIA was 21 ng/ml. The RIA showed good precision and accuracy with intra- and interassay coefficients of variation of less than 7% for MPO concentrations ranging from 100 to 800 ng/ml, and satisfactory recoveries of known amounts of exogenous MPO in plasma. For the measurement of MPO in blood, the best sampling technique was to collect blood into EDTA. Rapid centrifugation (within 20 min) was necessary for blood collected into heparin. Mean MPO values in normal individuals were 340 +/- 98 ng/ml in EDTA plasma (n = 152) and 332 +/- 82 ng/ml in heparinized plasma (n = 34). When MPO was measured 12-6 h after injury in critically ill patients high values (above 1000 ng/ml) were found in 6/15 patients with multiple injuries. In patients with sepsis (n = 22), MPO values were always above 1000 ng/ml. [less ▲]

Detailed reference viewed: 12 (0 ULg)
Full Text
Peer Reviewed
See detailThe ability of normal human monocytes to phagocytose IgG-coated red blood cells is related to the number of accessible galactosyl and mannosyl residues in the Fc domain of the anti-red blood cell IgG antibody molecules
Malaise, Michel ULg; Franchimont, P.; Mahieu, P. R.

in Journal of Immunological Methods (1989), 119(2), 231-239

The percentage of normal human monocytes (MCs) that are able to form rosettes with, and subsequently phagocytose, IgG-coated red blood cells (RBCs) has been determined in vitro using five batches of anti ... [more ▼]

The percentage of normal human monocytes (MCs) that are able to form rosettes with, and subsequently phagocytose, IgG-coated red blood cells (RBCs) has been determined in vitro using five batches of anti-RBC IgG antibodies. These antibodies differed from each other by their capacity to bind to lectins recognizing two of the oligosaccharide structures of the Fc domain, namely, peanut agglutinin (PNA) and concanavalin A (ConA) which specifically bind to beta-galactosyl and alpha-mannosyl residues, respectively. The threshold between high (H) and low (L) binding capacities (BC) was arbitrarily fixed at 15% of mean specific binding. For each level of RBC sensitization tested (1500-6000 Ab molecules/one RBC), the percentage of MCs binding at least three IgG-RBCs was similar whatever the IgG Ab preparations used. In contrast, the percentage of MCs capable of phagocytosing at least three IgG-RBCs coated with 3000, 4500 and 6000 IgG/cell, as well as the phagocytosis index (number of IgG-RBCs ingested/100 MCs) of IgG-RBCs coated with 1500, 3000, 4500 and 6000 IgG/cell, were significantly lower (P less than 0.01 at least) using IgG Ab molecules with either [(PNA-H)(ConA-H)] BC, [(PNA-L) (ConA-H)] BC or with [(PNA-L)(ConA-L)] BC than the corresponding values measured using RBCs coated with IgG Ab molecules exhibiting [(PNA-H)(ConA-L)] BC. The binding to MCs of 125I-labelled anti-RBC IgG Ab molecules exhibiting different binding profiles to PNA and to ConA was studied by Scatchard plot analysis. A single class of binding sites was observed in each case. MCs bound a mean of 23,000 IgG molecules with a mean association constant (Ka) for IgG binding of about 1.4 X 10(8) M-1. These data indicate that terminal (and/or accessible) galactosyl and mannosyl residues of IgG Ab molecules play a role in the ingestion of IgG-RBCs by human MCs, despite the fact IgG Ab binding to IgG(Fc) receptors is not significantly affected. Thus, when studying the phagocytosis of IgG-coated RBC by human MC monolayers, the assay should be performed not only using similar RBC/MC ratios and IgG coating values, but also with IgG antibodies having comparable mean PNA and ConA binding capacities. [less ▲]

Detailed reference viewed: 9 (0 ULg)
Full Text
Peer Reviewed
See detailComparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates
Jeanson, Antoinette; Cloes, Jean-Michel; Bouchet, Mireille et al

in Journal of Immunological Methods (1988), 111(2), 261-270

Four monoclonal antibodies belonging to different subclasses and with differing isoelectric points were coupled to horseradish peroxidase (HRP) and alkaline phosphatase (AP) using various conjugation ... [more ▼]

Four monoclonal antibodies belonging to different subclasses and with differing isoelectric points were coupled to horseradish peroxidase (HRP) and alkaline phosphatase (AP) using various conjugation procedures. The conjugates were tested by enzyme immunoassay and their efficiency was characterized by the antibody and enzyme concentrations needed to obtain an arbitrary OD value. The suitability of antibody for conjugation through NH2 groups was tested by fluorodinitrobenzene (FDNB). HRP conjugates were produced by two variants of the sodium periodate procedure and two variants of the glutaraldehyde method, as well as by the heterobifunctional linker N-succinimidyl 3-(2-pyridyldithio)pro-pionate (SPDP). Two of the four antibodies were coupled by a third variant of the periodate method, through their carbohydrate moieties. The periodate-mediated conjugations, using sugar moieties on the enzyme, provided the most efficient HRP conjugates, regardless of the antibody subclass or isoelectric point. The glutaraldehyde procedures consistently gave the worst results. AP conjugates were prepared using the same methods. The most efficient and reproducible AP conjugates with all four monoclonal antibodies were obtained using the SPDP procedure. The efficiency of the other methods differed from one antibody to another. [less ▲]

Detailed reference viewed: 36 (0 ULg)
Full Text
Peer Reviewed
See detailElucidation of non-parallel EIA curves
François-Gérard, C.; Gérard, Paul ULg; Rentier, Bernard ULg

in Journal of Immunological Methods (1988), 111(1), 59-65

Quantitative determinations by EIA can be only obtained by reverse regression when linear portions of sample and standard curves are parallel. However, analysis of complex biological fluids often yields ... [more ▼]

Quantitative determinations by EIA can be only obtained by reverse regression when linear portions of sample and standard curves are parallel. However, analysis of complex biological fluids often yields sigmoid curves displaying lower slopes, thus invalidating any quantitative interpretation. We hypothesized that this phenomenon was due to a competition effect between the target (for example an antigen) and related molecules for the binding sites (for example a capture antibody) immobilized onto the solid phase. This has been confirmed experimentally using various target-to-competitor ratios and formulated as a mathematical model. The slope decrease in target detection was related to the proportion of competitor, not in a linear, but in an exponential manner. This mathematical model has been computerized and can be used to correct aberrant sample curves provided the relevant parameters have been previously determined in the same systems. A competition effect should be suspected whenever non-parallel EIA sigmoid slopes are obtained. [less ▲]

Detailed reference viewed: 25 (4 ULg)
Full Text
Peer Reviewed
See detailAn enzyme-linked immunoassay for direct measurement of the gelatin-binding capacity of human plasma fibronectin
Damas, Pierre ULg; Adam, A.; Closset, Jean ULg et al

in Journal of Immunological Methods (1986), 91

A new solid-phase enzyme immunoassay measuring the gelatin-binding capacity of plasma fibronectin has been developed. This assay is based on the direct and high-affinity interaction between fibronectin ... [more ▼]

A new solid-phase enzyme immunoassay measuring the gelatin-binding capacity of plasma fibronectin has been developed. This assay is based on the direct and high-affinity interaction between fibronectin and gelatin coated to polyvinyl chloride plates. The amount of fibronectin bound to gelatin is then measured by sequential incubation with a specific rabbit anti-human fibronectin antiserum, with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies and with substrate. The final degradation of the substrate is read at 492-650 nm in an ELISA processor. The assay allows the accurate detection of fibronectin concentrations ranging from 1 to 20 micrograms/ml, is inhibited by the addition of gelatin to plasma, is highly reproducible (interplate CV less than 10%), requires 100 microliter of plasma only and has been fully automated. Significant linear correlations were noted between total antigenic fibronectin (measured by laser nephelometry) and fibronectin gelatin-binding capacity in plasma from 310 blood donors. Both parameters were higher in men than in women and significantly increased according to age. Dissociation between immunoreactive fibronectin and fibronectin gelatin-binding capacity was observed in two polytraumatized patients. This enzyme immunoassay therefore provides a new method to investigate functional alterations of the gelatin-binding domain of fibronectin in various pathological conditions. [less ▲]

Detailed reference viewed: 38 (25 ULg)
Peer Reviewed
See detailIsolation of follicular dendritic cells from human tonsils and adenoids. I. Procedure and morphological characterization.
Lilet, C.; Radoux, D.; Heinen, Ernst ULg et al

in Journal of Immunological Methods (1984), 66(2), 235-44

Follicular dendritic cells have been isolated from human tonsils and adenoids and characterized at the ultrastructural level. Follicles were dissected and digested with different hydrolytic enzymes. The ... [more ▼]

Follicular dendritic cells have been isolated from human tonsils and adenoids and characterized at the ultrastructural level. Follicles were dissected and digested with different hydrolytic enzymes. The cells were separated by sedimentation at unit gravity. By this procedure we obtained follicular dendritic cells enveloping lymphocytes with their cytoplasmic extensions in a way analogous to that described for isolated thymic nurse cells. The ultrastructural features of isolated follicular dendritic cells are similar to those observed in situ. Prolonged enzymatic action caused loss of the enveloped lymphocytes. [less ▲]

Detailed reference viewed: 39 (2 ULg)
Peer Reviewed
See detailColloidal gold, a useful marker for antigen localization on follicular dendritic cells.
Heinen, Ernst ULg; Radoux, D.; Kinet-Denoel, C. et al

in Journal of Immunological Methods (1983), 59(3), 361-8

Bovine serum albumin (BSA) bound to colloid gold particles (BSA-gold; 20 nm diameter) and injected into preimmunized mice was found at ultrastructural level in different locations of the lymph nodes. It ... [more ▼]

Bovine serum albumin (BSA) bound to colloid gold particles (BSA-gold; 20 nm diameter) and injected into preimmunized mice was found at ultrastructural level in different locations of the lymph nodes. It was detected particularly in the secondary lysosomes of macrophages and between the cytoplasmic processes of the follicular dendritic cells. Between these processes the gold particles were isolated or grouped in clusters; they were in close contact with cell membranes or embedded in dense material. Colloidal gold injected alone was not retained on these cells. The presence of anti-BSA antibodies in the serum was necessary for trapping of BSA-gold particles on follicular dendritic cells. Injections of BSA alone after BSA-gold had been administered to preimmunized mice eliminated most of the BSA-gold from the dendritic processes. BSA-gold is thus trapped in the form of immune complexes which behave characteristically. BSA-gold is thus a suitable marker for antigen localization. Being small and electron dense it permits more precise location than radioactive markers. [less ▲]

Detailed reference viewed: 19 (1 ULg)
Peer Reviewed
See detailMagnetic solid-phase enzyme immunoassay for the detection of anti-glomerular basement membrane antibodies.
Druet, E.; Mahieu, P.; Foidart, Jean-Michel ULg et al

in Journal of Immunological Methods (1982), 48(2), 149-57

A new enzyme immunoassay has been developed for the demonstration of antiglomerular basement membrane antibodies. Magnetically responsive polyacrylamide-agarose beads (Magnogel) activated with ... [more ▼]

A new enzyme immunoassay has been developed for the demonstration of antiglomerular basement membrane antibodies. Magnetically responsive polyacrylamide-agarose beads (Magnogel) activated with glutaraldehyde were used to bind sonicated insoluble rat glomerular basement membranes. Both the collagenous and the non-collagenous moieties were demonstrated to be fixed on the beads. Sera from brown Norway rats with anti-glomerular basement membrane antibodies induced by HgCl2 injections were incubated with the beads. After washing, the fixed rat IgG were revealed using alkaline phosphatase labelled Fab fragments from anti-rat IgG sheep, IgGs. Comparison with a radioimmunoassay showed that results were reliable. This enzyme immunoassay has several advantages which may render this assay of considerable clinical usefulness. [less ▲]

Detailed reference viewed: 11 (0 ULg)
Peer Reviewed
See detailOne step purification of mouse monoclonal antibodies from ascitic fluid by DEAE affigel blue chromatography.
Bruck, Claudine; Portetelle, Daniel ULg; Glineur, C. et al

in Journal of Immunological Methods (1982), 53

Detailed reference viewed: 12 (1 ULg)