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M De Boer

Publications and source records attributed to M De Boer.

27 records · Page 2Linked to original sources
PubMed1990

Generation and characterization of monoclonal antibodies directed against noniodinated and iodinated thyroglobulin, among which are antibodies against hormonogenic sites.

In this paper we describe the preparation and characterization of three different groups of monoclonal antibodies (Mabs) raised against iodinated and noniodinated human thyroglobulin (hTg). One group (A) of three Mabs is directed against hTg without discrimination between different iodine contents of Tg. The second group (B) of three Mabs has a higher affinity for iodinated Tg than for noniodinated Tg, and the Mabs are not species specific. The last group (C) of two Mabs generated against noniodinated hTg shows a higher affinity for noniodinated hTg than for iodinated hTg. From competition experiments with T4 we conclude that the second group of Mabs is directed against hormonogenic sites in the protein. From these Mabs, probably two are directed against the N-terminal hormonogenic site, and one against one of the C-terminal hormonogenic sites in Tg. Reactivity of the Mabs of group B with Tgs of various degrees of iodination indicates that the N-terminal T4 is formed first.

Animals↗
PubMed1989

Optimal conditions for the generation of monoclonal antibodies using primary immunisation of mouse splenocytes in vitro under serum-free conditions.

We have further optimised the serum-free in vitro immunisation system described by Ossendorp et al. (J. Immunol. Methods 91, 257, 1986) for the generation of hybridomas secreting specific antibodies. Thyroglobulin and the hapten 2-phenyl-5-oxazolone coupled to chicken serum albumin were used as antigens. For an optimal outgrowth of antigen-specific B cells the presence of T cells, thymocyte-conditioned medium and antigen are required. The addition of supernatant from EL-4 cells (stimulated by phorbolmyristate acetate) inhibits the outgrowth of antigen-specific B cells. Using six-well plates with a surface area of 10 cm2 per well, an optimal IgM response was obtained when 10(7) splenocytes in a total volume of 2 ml/well were cultured for 3 days in the presence of antigen and thymocyte-conditioned medium. Increasing the concentration of cells whilst maintaining a constant surface area resulted in a decreased response.

Animals↗
PubMed1989

Efficient selection of high-affinity B cell hybridomas using antigen-coated magnetic beads.

A method for the selection of antigen-specific B cell hybridomas using antigen-coated magnetic beads is described. Stable B cell hybridoma cell lines directed against human thyroglobulin were incubated with thyroglobulin-coated beads. 2 h of incubation at 4 degrees C using bead-to-cell ratios of at least 3:1 were found to be the optimal conditions for rosette formation. Rosettes were efficiently isolated with a strong magnet. Rosette formation was antigen-specific since irrelevant hybridoma cell lines could not form rosettes, nor could BSA-coated or uncoated beads form rosettes. Free antibodies produced by the hybridoma cells were able to block rosette formation. Blocking of rosette formation permitted the identification of different and overlapping epitopes recognized by four different hybridomas. Using six stable hybridoma cell lines with different affinities for thyroglobulin, rosette formation appeared to be dependent on the affinity of the immunoglobulin membrane receptor for antigen. A correlation was observed between the affinity of the secreted antibodies and the capacity of the hybridomas to form rosettes, suggesting that this method is suitable for the selection of hybridomas producing antibodies with a high affinity for the antigen. Antigen-coated magnetic beads were found to be suitable for the efficient selection of thyroglobulin-specific hybridoma cells from bulk cultures shortly after fusion. A 300-fold enrichment of thyroglobulin-specific cells was obtained using this method.

Animals↗
PubMed1988

Requirements for the generation of memory B cells in vivo and their subsequent activation in vitro for the production of antigen-specific hybridomas.

We have studied the conditions required for the activation in vitro of memory B cells generated in vivo. BALB/c mice were immunised by a single injection of antigen emulsified in Freund's complete adjuvant. Splenocytes were isolated after different time intervals and cultured in a serum-free medium in the presence of antigen and thymocyte-conditioned medium. After 3 days the splenocytes were fused with myeloma cells. A minimum time interval of more than 2 weeks between priming in vivo and stimulation in vitro was required in order to obtain antigen-specific IgG-secreting hybridomas. After a time interval of 4 weeks or longer most of the antigen-specific hybridomas secreted IgGs. During stimulation in vitro the presence of antigen and of T cells was found to be essential for obtaining an antigen-specific IgG response. The addition of thymocyte-conditioned medium enhanced the IgG response.

Animals↗
PubMed1987

Purification of eosinophils from normal human blood, preparation of eosinoplasts and characterization of their functional response to various stimuli.

Eosinophils from the blood of normal individuals were purified by centrifugation over discontinuous Percoll gradients. Eosinophil suspensions were obtained with a mean purity of 96% and a mean recovery of 64% (n = 19). When incubated with phorbol-myristate acetate, eosinophils consumed twice as much oxygen as did neutrophils from the same donors. With serum-treated zymosan, 70% and 100% of the maximal oxidative response (i.e. the response to phorbol-myristate acetate) was obtained with eosinophils and neutrophils, respectively. The calcium ionophore A23187 is a weak stimulus that triggered only 2.5% of the eosinophil and 10% of the neutrophil oxidative capacity. The response of both cell types to formyl-methionyl-leucyl-phenylalanine (fMLP) was rapid, with a maximum after 3 min. The magnitude of this eosinophil reaction was half that of neutrophils. Although the activities of the granule enzymes beta-glucuronidase and arylsulphatase were 2.5 and 6 times higher in eosinophils than in neutrophils, respectively, the exocytosis of these enzymes in response to various stimuli was lower in eosinophils. The high yield of eosinophils from our separation method enabled us to prepare eosinoplasts by centrifugation of eosinophils over discontinuous Ficoll gradients that contained cytochalasin B. Eosinoplasts are plasma membrane vesicles derived from eosinophils, filled with cytoplasm but devoid of granules and nucleus. The eosinoplasts contained 30% of the cytoplasm and plasma membrane present in intact eosinophils. Eosinoplasts still possessed a functionally intact oxidase enzyme that could be stimulated with various stimuli. Therefore, eosinoplasts may provide a valuable tool to study separately the role of the oxidase products and that of the granule contents in eosinophil functions.

Arylsulfatases↗
PubMed1986

Production of murine monoclonal antibodies against human thyroglobulin using an in vitro immunization procedure in serum-free medium.

A serum-free in vitro immunization method for the generation of hybridomas producing specific antibodies to an antigen is described. The method was tested with human thyroglobulin as antigen. The serum-free medium used (Yssel et al., 1984) consisted of Iscove's modification of Dulbecco's modified Eagle's medium, supplemented with albumin, transferrin, insulin, ethanolamine and linoleic, oleic and palmitic acids. An optimal response was obtained when splenocytes from BALB/c mice were cultured for 3 days in the presence of 1.5 nM thyroglobulin and thymocyte-conditioned medium prior to fusion with SP2/0 myeloma cells and seeding of the fused cells in microtitre plates. The frequency of positive wells, defined as the number of wells secreting anti-(thyroglobulin) antibodies/number of viable cells used for the fusion, was 1.6 X 10(-6) +/- 0.25 X 10(-6) (mean +/- SD; n = 4). Eight stable clones producing anti-(thyroglobulin) antibodies were isolated. One clone (3D12) produced antibodies reacting only with human thyroglobulin. The antibodies produced by the other clones reacted with human, murine and porcine thyroglobulins. Seven of the clones produced antibodies of the IgM class and one clone produced IgG. The specificity of 3D12 (IgM) for human thyroglobulin and the absence of any reactivity with murine thyroglobulin provides evidence for a primary response of splenocytes in culture to the presence of an antigen.

Antibodies, Monoclonal↗
PubMed1981

Large-scale purification and cryopreservation of human monocytes.

Human blood monocytes were purified by a new method capable of handling 3 X 10(9) mononuclear leukocytes, which does not involve adherence of the cells and takes about 3 h to perform. The yield of monocytes is 70%, the purity about 90% and the viability 99%. Monocytes purified by this method were cryopreserved at -196 degrees C. The function of the cells was tested before freezing and after thawing. We found that the capacity of cryo-preserved monocytes to move to the source of a chemotactic gradient, to ingest particles, to mount a respiratory burst during phagocytosis, to kill intracellular bacteria, to lyse anti-D sensitized erythrocytes and to help autologous lymphocytes in a proliferative response to mitogens or antigens, was preserved by 70% or more as compared with non-frozen cells. Thus, cryopreservation of human blood monocytes is possible with maintenance of functional capacities.

Blood Bactericidal Activity↗