Ruurd Torensma

Comparison of immunomagnetic beads coated with protein A, protein G, or goat anti-mouse immunoglobulins Applications in enzyme immunoassays and immunomagnetic separations

Immunomagnetic beads were prepared using either protein A (PA) or protein G (PG) coupled to magnetic beads for binding antibodies at their Fc region. The performance of these beads was compared with commercially available beads coated with goat anti-mouse (G alpha M) immunoglobulins. Both the PA- and PG-beads possessed a higher binding capacity than the G alpha M-beads for the monoclonal antibodies tested, although, PA bound weakly with some IgG1 antibodies. PA-beads were compared with G alpha M-beads in a magnetic enzyme immunoassay for the detection of mouse immunoglobulins as an alternative to a conventional capture ELISA. The magnetic enzyme immunoassay was characterized by a detection time of less than 60 min and a linear assay range from 5-10 to 500 ng/ml for G alpha M-beads and 5-10 to 1000 ng/ml for PA-beads. The capture ELISA was linear from 10 to 250 ng/ml. For immunomagnetic separation of Salmonella with immunomagnetic beads, PA-beads were superior to both PG- and G alpha M-beads. For specific isolation of bacteria from heterogeneous suspensions by immunomagnetic separation, PA- and PG-beads are preferable since G alpha M-beads crossreact with bacteria possessing proteins with Fc-binding activity.

Monoclonal antibodies that identify gram-negative bacteria using the magnetic immunoluminescence assay

Abstract Monoclonal antibodies against live gram-negative bacteria were prepared. The antibodies were tested in a newly developed Magnetic Immuno Luminescence Assay, ELISA and agglutination reaction. Bacteria were captured by specific monoclonal antibodies bound to magnetic beads coated with covalently bound goat anti-mouse immunoglobulin and detected in an ATP dependent luciferase-luciferin enzyme system. The system was evaluated with monoclonal antibodies specific for Klebsiella, Enterobacter and Proteus . Clinical isolates belonging to these species could be identified.

Multivalent binding of toxin A from Clostridium difficile to carbohydrate receptors

A specific monoclonal antibody against toxin A from Clostridium difficile was generated that did not show thermolabile binding. Nonspecific murine monoclonal antibodies bound toxin A at 4 degrees C, but less effectively at 37 degrees C. Nonspecific human monoclonal antibodies did not bind to toxin A at 4 degrees C. Cytotoxic properties of purified toxin A were not inhibited by Bandeiraea simplicifolia lectin. This points to a carbohydrate moiety on the cell surface and a multivalent nonspecific carbohydrate binding ligand on toxin A.

In vitro stimulation of immune spleen cells enhances the number of anti-lipid A-producing hybridomas

An in vitro stimulation method for the generation of hybridomas producing antibodies with specificity for the weakly immunogenic lipid A is described. Conditions influencing in vitro stimulation of immune spleen cells were investigated. Depending on the experimental conditions the percentage of specific antibody-producing hybridomas varied between 0 and 39%. Most successful was stimulation with both antigen and the synthetic adjuvant muramyl dipeptide (MDP) for 3 days. In vitro stimulation of spleen cells from animals classically immunized with Salmonella Re mutant enhanced the number of lipid A-specific IgG-producing hybridomas from six after direct fusion to 17 after stimulation. These experiments indicate that the synergistic action of antigen and MDP is caused by preferential action on antigen selected B cells.

Mechanism for monoclonal antibody mediated treatment of gram-negative shock

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