Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway.

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.

Standardization of measurement of beta-amyloid(1-42) in cerebrospinal fluid and plasma.

The standardization and clinical validation of the measurement of beta-amyloid(1-42) (Abeta42) in cerebrospinal fluid (CSF), plasma and urine is described using a commercially available sandwich-type ELISA with 21F12 and 3D6 as monoclonal antibodies. The INNOTEST beta-amyloid(1-42) allows the specific and reliable measurement of(1-42) amyloid peptides in CSF and plasma. The Abeta42 concentrations in serum and urine were below the detection limit. In plasma, no differences were found in Abeta42 levels between controls and patients with different neurodegenerative disorders (Alzheimer's disease (AD), Lewy body disease (LBD), others). In contrast, CSF-Abeta42 concentrations were lower in AD and LBD patients as compared to controls. No correlation was found in AD patients between CSF and plasma concentrations of Abeta42 or between CSF Abeta42 levels and blood-brain-barrier function. The quantitative outcome of the test is in part dependent on confounding factors such as tube type, freeze/thaw cycles, temperature of incubation, standard preparation protocol, and antibody selection. Notwithstanding these aspects, it emerged that Abeta42 is a useful biochemical marker for the diagnosis of AD patients, but there is a need for an international Abeta standard, a universally accepted protocol for CSF preparation, and a thorough evaluation of assay performance in function of the boundary conditions.

Study of the muscarinic receptor subtypes in N1E 115 mouse neuroblastoma cells.

Muscarinic receptors in N1E 115 mouse neuroblastoma cells were characterized by competition binding experiments using three agonists and five antagonists, including 4-DAMP and AF-DX 116, and by studying the effect of agonist stimulation on the cellular cAMP and cGMP content. The results of the binding studies with the antagonists suggest that only one single homogeneous binding site of the M1 muscarinic receptor subtype is present. For the binding with the agonists, two binding sites were detected, one with high affinity for the ligand (between 53 and 77% of the total binding sites depending on the agonist) and one with low affinity. In contrast to the results obtained with the binding experiments using antagonists, the study of the cellular cyclic nucleotide response upon carbachol stimulation suggested the presence of both the M1 and M2 subtypes as there was an increase in cyclic GMP concentration while at the same time, the prostaglandin-stimulated synthesis of cyclic AMP was inhibited. Considering both binding and functional data we suggest that in N1E 115 cells a majority of M1 and a minority of M2 muscarinic receptors are present; there is no evidence for the presence of M3 muscarinic receptors.

Gradient distribution pattern of muscarinic receptors in N1E 115 mouse neuroblastoma cells.

The distribution pattern of muscarinic receptors in N1E 115 mouse neuroblastoma cells after linear and non-linear gradient centrifugation was investigated. In untreated cells, at least two forms of the receptors, with different densities, were found.

Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.