T. Lea

Quantification of the Terminal Complement Complex in Human Plasma by an Enzyme‐Linked Immunosorbent Assay Based on Monoclonal Antibodies against a Neoantigen of the Complex

The fluid‐phase terminal complement complex (TCC), consisting of the components C5b, C6, C7, C8, C9, and the S‐protein, has recently been detected in normal human plasma by using antibodies against native terminal complement components. Increased amounts of TCC were then found in several patients with in vivo activation of complement. We now describe a sensitive, specific, and reliable enzyme‐linked immunosorbent assay for quantification of the TCC, based on monoclonal antibodies against a neoantigen of the complex. The results indicate that the TCC is present in normal human plasma and in increased amounts in patients with complement activation in vivo, thus confirming previously obtained results. The assay is easy to perform and can be used for examination of large numbers of plasma samples.

Magnetic Monosized Polymer Particles for Fast and Specific Fractionation of Human Mononuclear Cells

Magnetic monodisperse polymer particles were developed and the necessary conditions established to use them for both quantification and fractionation of human peripheral blood mononuclear cell populations. The particles consist of a styrene divinylbenzene core into which magnetite has been deposited by an in situ oxidation process. Thereafter the core has been coated with a hydrophilic polymer containing epoxy and hydroxyl groups. The particles have strong nonspecific binding capacity for protein and can he coaled with the appropriate antibodies by physical adsorption only. However, the hydroxyl groups on the outer polymer also make covalent coupling possible. After appropriate blocking they can be used in a rosette assay for quantification of mononuclear leukocytes previously sensitized with monoclonal antibodies. Furthermore, a suitable magnet makes it possible to deplete the cell suspension efficiently of the rosette‐forming cells. We have thoroughly investigated the functional properties of human mononuclear cells depleted of T lymphocytes by this technique. Our results show that T cells are virtually completely eliminated, as demonstrated by flow cytometry and various functional assay systems.

Characterization of Human Mononuclear Cells after Positive Selection with Immunomagnetic Particles

We have investigated the possibility to employing magnetic monodisperse polymer particles for positive selection of human peripheral blood mononuclear cell populations. By carefully titrating the ratio between particles and cells we succeeded in isolating a number of cell populations that could be cultivated subsequently in vitro for functional studies. The success of the procedure is partly dependent on the properties of the monoclonal antibodies used to sensitize the cells. Provided these antibodies do not read with membrane structures involved in the transduction of activating signals, highly purified, quiescent cell populations can be recovered in a single fractionation step. In most instances panicles will detach from the isolated cells by overnight culture, and the particles can then be removed from the system by a suitable magnet. T lymphocytes, subpopulations of I lymphocytes, and B lymphocytes have been isolated in this way and studied in a variety of functional assay systems. Comparison with cells obtained after negative selection clearly demonstates the usefulness of this technique, especially if the membrane marker selected for it is not directly engaged in the activation processes.

cAMP-dependent protein kinase (PKA) inhibits T cell activation by phosphorylating Ser-43 of Raf-1 in the MAPK/ERK pathway

cAMP-dependent protein kinase (PKA) has been suggested to interfere with T-cell activation by inhibiting interleukin (IL-2) receptor alpha-chain (CD25) expression and IL-2 production. The Ras/MAP kinase pathway has been found to be necessary for induction of the IL-2 production. In this study, we have scrutinized the Ras/MAP kinase pathway in Jurkat T-cells to attempt to identify any sites for PKA-mediated regulatory phosphorylations. Here we unambiguously demonstrate that PKA directly inhibits anti-CD3-induced MAP kinase activation. In vitro phosphorylation experiments showed that Raf-1 was extensively phosphorylated by PKA, while ERK2 and MEK were not. Phosphopeptide mapping identified Ser-43 of Raf-1 as the only site phosphorylated by PKA in the Ras/MAPK pathway. Transient transfection experiments demonstrated that mutations of Ser-43 of the Raf-1 kinase were rendered insensitive to cAMP-mediated inhibition.

Purification and characterization of two protein antigens from the heterogeneous BCG85 complex in Mycobacterium bovis BCG

The heterogeneous BCG85 complex is a major component of BCG culture fluid. BCG85A and BCG85B were purified by combining ammonium sulphate precipitation with chromatography on hydroxyapatite, DEAE-Sephacel and phenyl-Sepharose columns. Twenty percent of BCG85B was recovered. The chromatographic separation procedures were monitored by fused rocket immunoelectrophoresis. The BCG85 complex was found to consist of three antigens, which were heterogenous with regard to electrophoretic mobility, molecular weight (MW), hydrophobic and immunological properties. They were designated A, B and C in increasing order, according to their electrophoretic mobilities. Thus BCG85A had the lowest electrophoretic mobility, BCG85C the highest. The MW of BCG85A was found to be 31,000, while BCG85B had a slightly lower MW, 29,000, as determined by SDS-PAGE. The antigenic relationship between the components was evaluated by crossed immunoelectrophoresis and double diffusion, and reactions of partial identity between the antigens were found. The BCG85 complex occurs in far lower concentration in sonicates of BCG than in culture fluid.

Detection and Quantification of the Terminal C5b‐9 Complex of Human Complement by a Sensitive Enzyme‐Linked Immunosorbent Assay

An enzyme‐linked immunosorbent assay for detection and quantification of the terminal complexes (SC5b‐9 and membrane attack complex) of human complement is described. We separate the complex from the native complement components, to use antibodies against the native components in a ‘double‐antibody sandwich’ technique. It is thereby possible to detect the terminal complement complex in solution without the requirement of specific antibodies against the neoantigens. The results show that the assay is both sensitive and specific. Evidence is presented that a terminal complement complex occurs in a normal plasma pool. The terminal complement complex may be valuable for evaluating both the physiology and palhophysiology of the complement system in vivo.

Carbamazepine: Effect on IgG Subclasses in Epileptic Patients

Summary: IgG subclass concentrations were determined in sera from 20 epileptic patients before carbamazepine therapy and after 6 weeks of treatment. Subclass‐specific monoclonal antibodies were used in an ELISA technique. Carbamazepine reduced the IgG2 concentration in 13 patients, and the mean value fell from 3.21 to 2.47 g/L during the carbamazepine treatment (p < 0.01). This IgG2 decrease was maintained after 4 and 12 months of carbamazepine therapy. IgGl, IgG3, and IgG4 concentrations did not change. IgG2 deficiency was not seen. The reduction in IgG2 was not related to the carbamazepine serum concentration within the therapeutic range, to the type of epilepsy, or to the age of the patient. The carbam‐azepine‐induced reduction of IgG2 in serum may indicate an altered immune response to certain antigens, preferentially to carbohydrates.

Heterogeneity of Human Amyloid Protein AA and Its Related Serum Protein, SAA

The heterogeneity of the human amyloid proteins SAA and AA was studied. Both proteins could be separated into several fractions by ion‐exchange chromatograpby. Amino acid analysis of the ion‐exchange‐chromatographed fractions of protein AA showed that the main difference was in the length of the polypeptide. Thus, it seems that the original AA preparation consists of a mixture of AA proteins with length ranging from 66 to 78 amino acid residues. By enzymatic degradation of three different forms of SAA with kallikrein, fragments were formed with a molecular weight very similar to thai of protein AA.

Enrichment of Human Epidermal Langerhans Dendritic Cells

Langerhans cells (I, C) were enriched from a human epidermal cell (EC) suspension by a rosette‐forming technique. First EC were sensitized with the monoclonal antibody OKT6, and then they were mixed with ox erythrocytes (ORBC) coated with affinity‐purified rabbit IgG anti‐mouse IgG antibodies. Rosette‐forming LC were then separated from non‐rosetting EC by means of Percoll flotation. After hypotonic lysis of the ORBC, the cells obtained had a viability greater than 90%, and 674–98% of the viable cells were OKT6‐positive. In functional studies the enriched cells were strongly stimulatory for allogeneic lymphocytes and were able to function as antigen‐presenting cells for the T‐cell response to the soluble antigen purified protein derivative.

Human complement-activating immunoglobulin (Ig)G3 antibodies are essential for porcine endothelial cell activation

Sæthre M, Lea T, Borgen MS, Fiane AE, Michaelsen TE, Thorsby E, Haraldsen G, Mollnes TE. Human complement‐activating immunoglobulin (Ig)G3 antibodies are essential for porcine endothelial cell activation.u2028Xenotransplantation 2006; 13: 215–223.