Antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a disease characterized by venous and arterial thromboses or spontaneous abortions and the repeated detection of antiphospholipid antibodies (aPL). APS may be associated with another autoimmune disease (secondary APS), particularly systemic lupus erythematosus (SLE), or unrelated to an underlying disease (primary APS). APS affects almost all organs. In addition to the clinical criteria, lupus anticoagulant testing and immunological aPL determinations are required to establish the diagnosis of APS.

Development of a creatinine ELISA and an amperometric antibody-based creatine sensor with a detection limit in the nanomolar range.

Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology. Antibodies from various clones have been characterized with BIAcore 2000 with respect to the dissociation constant and specificity. Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74 microM) and the highest specificity for creatinine and were chosen for the development of a competitive ELISA and an amperometric creatinine sensor. The creatinine sensor was constructed by fixing a creatinine-modified membrane on the top of a platinum working electrode which was then incorporated into a stirred electrochemical measuring cell. For creatinine determination the creatinine-containing sample was incubated with B90-AH5 and anti-IgG(mouse)-glucose oxidase conjugate and applied to the measuring cell. After a washing step glucose was added and the produced hydrogen peroxide was registered at Eappl = +600 mV vs Ag/AgCl. The measuring range was 0.01-10 microg/mL. The highest sensitivity for creatinine was achieved at 330 ng/mL (3 microM) and the lower detection limit at 4.5 ng/mL (40 nM). This is far below the relevant clinical range, which is 5-17 microg/mL (44-150 microM) and allows a reliable determination of very low creatinine concentrations in serum, where standard methods cannot be applied. After each measurement the sensor was regenerated with 10 mM HCl without any loss in binding activity.

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody.

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.

C1q: a multifunctional ligand for a new immunoadsorption treatment.

C1q is a highly conserved protein with multiple functions involved in innate and adaptive immunity. It plays an important role in the activation of the classical pathway of the complement system to mediate the scavenging of infectious agents, apoptotic products, and immune complexes by the mononuclear phagocyte system (MPS). Exhibiting this function, C1q is able to bind various molecules (complexed IgG, IgM, fibrinogen, fibronectin, lipopolysaccharides, DNA, C-reactive protein [CRP], and viral proteins). Moreover, the collagen-like region of C1q is a target of autoantibodies. Immune complexes and anti-C1q autoantibodies are known to be involved in the pathogenesis of autoimmune diseases. Therefore, C1q is a promising candidate to extract waste material from the circulation. Following the development of the C1q immunoadsorbent, 8 patients with systemic lupus erythematosus (SLE) were treated in a first clinical trial. These preliminary results indicate that C1q immunoadsorption is a safe, compatible, and effective treatment for these patients.

Quantitative polymerase chain reaction using a DNA hybridization assay based on surface-activated microplates.

A method for DNA quantification on microplates based on the hybridization between single-stranded target and solid-phase bound capture DNA is presented. Binding of the capture DNA to the microplates was attained by surface activation using organosilanes. Detection of hybridized DNA was performed by an enzyme-linked assay taking advantage of the labeled target DNA. Basic test characteristics are described and application examples are given demonstrating its feasibility for a quantitative PCR (QPCR). The QPCR was carried out by coamplifying an immunoglobulin complementarity determining region 3 (CDR3)-coding plasmid DNA with a synthetic internal standard (IS). IS and plasmid CDR3 both shared primer binding sites and produced length-identical amplicons. The amplified DNA was quantified following differential hybridization with IS- and plasmid-specific capture probes. Based on the product ratios of plasmid to IS, the initial amounts of plasmid DNA were calculated. This way, QPCR was shown to be capable of detecting initial concentration differences of plasmid DNA of about 30%.

Development of a C1q-adsorbent for the selective removal of circulating immune complexes.

In body fluids circulating immune complexes possess a substantial pathogenic effect on a variety of diseases. Thus it seems to be reasonable to influence the pathogenic mechanism of such diseases by removing the circulating immune complexes out of the body fluids. The selective adsorbent for binding immune complexes were developed as possible alternative of plasmapheresis for specific removal of immune complexes out of the blood plasma. The principle is based on the biospecific binding of immune complexes to C1q that is immobilized by a covalent binding to different solid phases. Thus: so bound immune complexes can be separated by detaching the solid phases. The adsorbent may be regenerated by non-denaturated media and can be used manyfold. This adsorbent has a high biocompatibility and offers the possibility for clinical use.

Binding of C1q and DNA to support materials by means of a new coupling procedure.

Since several years matrix-bound biologically active substances are widely used in biosciences, biotechnology and medicine. We are presenting a simple and inexpensive activation procedure for support materials which allows stable binding of C1q and DNA. We could not found a remarkable leakage of the bound ligands. In connection with the good biocompatibility of our procedure, both are important for the application in extracorporeal immunoadsorption. The method is technologically simple and represents an alternative to the activation methods applied so far.

Development of a DNA-adsorbent for the specific removal of anti-DNA autoantibodies in systemic lupus erythematosus (SLE).

Autoantibodies against DNA are of primary importance for the diagnosis and pathogenesis of systemic lupus erythematousus (SLE). The level of anti-DNA antibodies correlates well with the disease activity and renal involvement. In such patients the removal of anti-DNA antibodies from plasma may lead to a clinical improvement. For this reason an adsorbent was made by covalent coupling of calf thymus DNA to a solid support based on ethylene dimethacrylate cross-linked hydroxethyl methacrylate. Up to 2.5 mg of DNA were immobolized to 1 ml of the support activated chemically by aminosilane and glutaraldehyde. The incubation of 40 ml of SLE plasma with 1 ml of the adsorbent resulted in a 50% decline in anti-DNA activity. There was no release of immobilized P-32-DNA into the plasma. Biocompatibility, sterilisation, and reapplication (without loss of binding capacity) of the adsorbent could be demonstrated. We concluded that the adsorbent may be suitable for treatment.

[Antibodies to anticoagulants in rheumatic autoimmune diseases]. / Hemmkörper der Blutgerinnung bei Autoimmunerkrankungen des rheumatischen Formenkreises.

The systemic lupus erythematosus (SLE) and the rheumatoid arthritis (RA) as the classic autoimmune diseases exhibit a great number of autoantibodies. Some of them are anticoagulants. Besides inactivating inhibitors against single coagulation factors interfering anticoagulants are known, belonging to the group of anti-phospholipid antibodies and detected as the lupus anticoagulants or anticardiolipin antibodies. Anti-phospholipid antibodies 184 patients with SLE or RA had been checked for. An enzyme immuno assay was used for detection of the anti-cardiolipin antibodies. The relations between occurrence of the anti-cardiolipin antibodies and vascular processes as well as other immunologic parameters had been tested for clinical relevancy.

[Rapid determination of C-reactive protein using latex agglutination]. / Die schnelle Bestimmung des C-reaktiven Proteins mittels Latex-Agglutination.

A simple and fast Latex agglutination assay for the determination of C-reactive protein (CRP) is described. The decision range of the assay presented is 7 mg/l; the measuring range extend from 7 mg/l up to 8,000 mg/l. Presented assay allows the cyto-determination of CRP in clinical routine work and therefore is suitable for the early diagnosis of infections in gynecology and obstetrics.

[Establishment and characterization of the pig aortic endothelial cell line BSEz-3 in a serum-reduced medium]. / Etablierung und Charakterisierung der Schweineaorten-Endothelzellinie BSEz-3 in einem serumreduzierten Nähr-medium.

The cell line BSEz-3 was established in a serum-reduced medium MEMPAS. The cells were isolated and cultivated under the same conditions as the calf aortic endothelial cell line BKEz-7. With regard to cell isolation and cell density in the stationary phase, BSEz-3-cells show characteristic differences from BKEz-7-cells. For the isolation of BSEz-3-cells an enzymatic-mechanical method was more successful than a mechanical technique only. The low saturation density in the stationary phase with an average number of 50,000 cells/cm2 of glass surface area is considered as a strong contact inhibition of cell proliferation. As a cell type specific marker the BSEz-3-cells contain factor-VIII-antigen and possess angiotensin-converting enzyme activity. For a practical use of the BSEz-3-cells in drug research we give recommandations regarding cell inoculum density for subcultivation and cryo-conservation in ampoules.

The use of glass as solid phase in enzyme immunoassay as exemplified by the detection of circulating immune complexes.

This article describes a solid-phase enzyme immunoassay for the quantitative determination of circulating immune complexes based on the covalent binding of proteins to glass. A quantitative comparison between adsorption to polystyrene and covalent binding to glass gave a protein higher concentration on the glass support. This leads, in connection with the strong attachment of the protein, to a higher sensitivity and precision in immunoassay. The binding of C1q to glass as solid phase offers the possibility to reuse the protein as well as the glass support after accomplishment of the immunoassay which can reduce the cost of the assay in routine work dramatically. Furthermore, the immobilization of C1q to glass gives an extreme stability of the protein which means that the c1q-coated glass supports can be used up to one year under routine conditions.

Immunoglobulin G binding to human erythrocytes.

The binding of 125iodine labelled human IgG to allogenic erythrocytes was studied at various ratios of free IgG per cell. At low concentrations of free IgG a high affinity binding was measured, which is with respect to its apparent association constant (2.8 x 10(13) mol-1) most likely a specific receptor binding and might indicate aged cells of those destined for elimination. At higher concentrations of free IgG the erythrocytes were coated by IgG in a non-saturable manner as reported by Grob et al. (Immunology 13, 189 (1967)). The erythrocyte-bound labelled IgG, which remained in the range from 15 to 400 molecules IgG per cell after intensive washings could not be chased by unlabelled IgG. This cell-bound IgG (high affinity binding) was increased 7-fold after complete ATP-depletion of erythrocytes and was 4 times higher after erythrocyte storage for 42 days. It appears that it is not the number of bound IgG molecules alone which is important for erythrocyte recognition by macrophages but also the arrangement of IgG molecules bound to membrane polypeptides.