sepharose and Lupus-Erythematosus--Systemic

Systemic lupus erythematosus (SLE) is an autoimmune diseases characterized by the presence of antiphospholipid and anti-dsDNA autoantibodies in the sera of patients. These autoantibodies and their subclasses have received increasing attention by medical community due to their association with recurrent venous thrombosis, fetal loss and thrombocytopenia. In particular, attention has been paid to IgG subclasses in SLE. The biological and functional properties together with the subclass distribution might therefore influence the course of SLE. The separation and elimination of these autoantibodies from sera of patients can be effective in clinical therapy. In the present study, histidine based pseudobioaffinity adsorbents have been used for the selective adsorption and separation of anti-double stranded DNA (anti-dsDNA), anticardiolipin (aCL) and anti-β2-glycoprotein-I (anti-β2-GPI) antibodies from sera of patients with SLE. For this purpose histidine acting as a pseudobiospecific ligand has been coupled to bisoxirane activated sepharose CL-6B for the adsorption and separation of these autoantibodies. The removal of autoantibodies was carried out under gentle adsorption and elution chromatographic conditions at pH values 7.0 and 8.0. Autoantibodies isotypes and subclasses distribution in the separated fractions were studied by enzyme-linked immune-sorbent assay. The obtained results showed that the separated anticardiolipin and anti-β2-glycoprotein-I autoantibodies belong to IgG1, IgG2 and IgG3subclasses, while those of anti-dsDNA belong to IgM isotype and were shown to have a DNA hydrolyzing activity that hydrolyzes plasmid DNA. The results also indicate a total IgM and IgG recovery superior to 90% of the fraction loaded at pH 7.4 and pH 8.0 respectively.

Topics: Adsorption; Adult; Aged; Autoantibodies; Chromatography, Liquid; DNA; Epoxy Compounds; Female; Histidine; Humans; Immunoglobulin G; Immunoglobulin M; Lupus Erythematosus, Systemic; Male; Middle Aged; Sepharose; Young Adult

The production of autoantibodies against a vast array of self antigens, most notably double stranded (ds) DNA, characterized systemic lupus erythematosus (SLE). The purpose of this work is to study specific Ig fractions isolated from normal human serum (NHS) and their effect on the binding of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies (Abs) to dsDNA. A fraction named immunoglobulin G (IgG)-reactive IgG was purified from total NHS IgG by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules (IgG-Sepharose column). IgG-reactive IgG was co-incubated with systemic lupus erythematosus (SLE) patient's serum and binding of the anti-dsDNA Abs to dsDNA was measured by enzyme-linked immunosorbent assay (ELISA). Co-incubation of SLE patient's serum with IgG-reactive IgG resulted in a dose-dependent reduction in binding of anti-dsDNA Abs to dsDNA. A reduction greater than 70% was observed at a concentration of 300 μg of IgG-reactive IgG per mL of a 400-fold diluted SLE patient's serum whereas total NHS IgG, at the same concentration, resulted in a 10% reduction in binding. The purification process used to isolate IgG-reactive IgG was based on interactions between intact Ig rather than on interactions between F(ab')(2) portions. IgG(2) is the predominant immunoglobulin (Ig) subclass in IgG-reactive IgG. Thus, IgG(2) might have an important role in the connectivity characteristics of NHS IgG. The capacity of IgG-reactive IgG to inhibit anti-DNA Ab binding to dsDNA may have potential application in the treatment of SLE. This targeted biological approach may provide an alternative strategy to immunosuppressants.

Topics: Adult; Antibodies, Anti-Idiotypic; Antibodies, Antinuclear; Antibody Specificity; Autoantigens; Binding Sites; Binding, Competitive; Chromatography, Affinity; DNA; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Immunotherapy; Lupus Erythematosus, Systemic; Male; Middle Aged; Protein Binding; Sepharose

Systemic blockade of TNF-α via monoclonal antibodies and soluble receptors has shown considerable effects against several typical autoimmune disorders, but remains unconvincing for the treatment of lupus. Based on our previous study, a CD169(+) macrophage-specific therapy using TNF-α antisense oligonucleotides (ASO) was tested for its efficacy in MRL/lpr lupus-prone mice. ASO-containing cationic agarose hydrogel were injected into mice subcutaneously. Tissue distribution and cellular localization of ASO were determined. The therapeutic effects and possible mechanism were further studied in MRL/lpr lupus-prone mice. The results showed that specifically accumulation of the anti-TNF-α ASO in CD169(+) macrophages could significantly reduce TNF-α expression in CD169(+) macrophages and inhibit lymphocytes over-proliferation, finally resulted in the relief of the lupus-like symptoms of the animals. The nucleic acid drug based on CD169(+) macrophage-specific TNF-α regulation represents a potential therapeutic approach that may be valuable for lupus therapy.

Topics: Animals; Cell Proliferation; Disease Progression; Drug Carriers; Drug Delivery Systems; Implants, Experimental; Lupus Erythematosus, Systemic; Lymph Nodes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Inbred MRL lpr; Oligonucleotides, Antisense; Sepharose; Sialic Acid Binding Ig-like Lectin 1; Spleen; Tissue Distribution; Tumor Necrosis Factor-alpha

The aim of this study was to prepare a DNA immunoadsorbent for the specific, extracorporeal removal of anti-DNA antibodies from the blood of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Two kinds of cellulose beads were applied as a carrier. Calf thymus DNA was covalently coupled to the carrier using the epichlorohydrin method. Efforts were focused on optimization of conditions for activation and coupling, trying to couple as much DNA as possible to a certain amount of carrier. It was found that the activation level increased with the increase of NaOH concentration and the amount of epichlorohydrin used. The mole of epichlorohydrin must be in excess of that of NaOH because excess NaOH could react further with the epoxy groups in the beads resulting in a decrease of activation level. High activation level could be obtained in a medium of 3.0 M NaOH. The DNA coupling was found to be mainly temperature and pH dependent. Using 0.1 M Tris-HCl buffer, pH 8 at a temperature of 50-90 degrees C, more than 3 mg of DNA could be coupled to 1 ml of wet beads. Prolonging the coupling reaction under 50 degrees C to 72 h resulted in the same coupling capacity as that obtained under 90 degrees C. To evaluate the adsorption ability for anti-DNA of this immunoadsorbent, batch and circulation tests were applied using SLE patient plasma. The immunoadsorbents showed excellent adsorption capacity, especially the cellulose with smaller size (200-300 microm). The incubation of 20 ml of patient's plasma with 1 ml of adsorbent resulted in an 80% decline in the anti-DNA antibody level. In the circulation tests, 30 ml of plasma was circulated through a column containing 3 ml of adsorbent. The maximum decline in anti-DNA level, 80%, was obtained after 60 min. Such high adsorption capacity and high adsorption rate suggest this immunoadsorbent may be used for treatment. For comparison, 1,4-butanediol diglycidyl ether activation method and other DNA sources were tested with the same protocol.

Topics: Adsorption; Arthritis, Rheumatoid; Cellulose; DNA; Humans; Immunosorbents; Lupus Erythematosus, Systemic; Particle Size; Sepharose

To remove anti-DNA antibodies from a patient's plasma with systemic lupus erythematosus (SLE), a DNA immunoadsorbent was developed by covalently coupling calf thymus DNA on activated Sepharose 4FF. Sepharose 4FF was activated with 5-norbornene-2,3-dicarboximido carbonochloridate (Cl-CO-ONB), which was proven to be a very effective method for preparation of affinity chromatographic adsorbents. The activation was carried out in dry acetone using 4-(dimethylamine)pyridine (DMAP) and triethylamine (TEA) as catalysts at 4 degrees C or at room temperature. The coupling of DNA to the activated support was investigated as a function of pH, temperature, time, concentration of DNA, and activation level. It was found that the pH for optimal coupling is 3.0, and the amount of coupled DNA increases with an increase either in the concentration of DNA or the activation level. The maximum amount of coupled DNA could reach 1.0 mg DNA/ml support. The incubation of 5 to 20 ml of SLE plasma with 1.0 ml of adsorbent resulted in an 80 to 90% decline in the anti-DNA antibody level. Nonspecific adsorption for normal IgG and total protein is less than 15%.

Topics: Acetone; Adsorption; Animals; Antibodies, Antinuclear; Biocompatible Materials; Blood Proteins; Cattle; Chromatography, Affinity; DNA; Ethylamines; Gels; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunoglobulin G; Immunosorbents; Lupus Erythematosus, Systemic; Norbornanes; Pyridines; Sepharose; Temperature; Time Factors

Immunoadsorption onto protein A sepharose can be applied to eliminate immunoglobulins, autoantibodies and circulating immune complexes from the circulation. In vivo kinetic studies showed that all IgG subclasses are removed from the patient's plasma although IgG3 elimination is variable and dependent on the presence of other immunoglobulins. IgG elimination half time was 4.8 days during intermittent and 2.9 days for daily therapy in the absence of immunosuppression. Autoantibodies and immune complexes can be cleared effectively but administration of intravenous immunoglobulins should be avoided because of competition for protein A binding sites. Redistribution/ denovo synthesis (half time 2.1-8.8 d for IgG 1-4) occurred in the absence of immunosuppression.

Topics: Antigen-Antibody Complex; Case-Control Studies; Humans; Immunoglobulins; Immunosorbent Techniques; Kinetics; Lupus Erythematosus, Systemic; Sepharose; Staphylococcal Protein A

Autoantibodies from the MRL/lpr mice react with numerous proteins on neuronal cell surfaces. The purpose of this study was to isolate and characterize a population of autoantibodies reactive preferentially or exclusively with nervous system tissue. Using a purified plasma membrane preparation from brain cortex of balb/c mice coupled to diaminopropylamine agarose gel, we affinity-isolated antineuronal antibodies from pooled MRL/lpr immunoglobulins. The isolated immunoglobulins reacted with brain cortex plasma membranes and neuroblastoma cells (but not liver, kidney, or fibroblasts) by Western blot and indirect immunofluorescence with confocal microscopy. By Western blot, the epitopes in the brain cortex were proteins of apparent molecular weights 101, 63, 53, 43, 39, and 33, kd; the epitopes in the neuroblastoma cells were 63, 57, and 53 kd. Lectin column isolation revealed that the 101 and 63 kd epitopes were glycosylated. Indirect immunofluorescence revealed that the antibodies bound to the cell soma more intensely than to the cell processes of viable cultured neuroblastoma cells. The cell surface localization of this binding was confirmed by confocal microscopy. Within the central nervous system the antibodies bound more intensely to primary cultures of isolated neurons from fetal cortex than to hippocampal or neostriatal cells. With these antibodies we can begin studies of their potential pathogenic effects.

Topics: Animals; Antibody Specificity; Antigen-Antibody Reactions; Autoantibodies; Autoimmune Diseases; Blotting, Western; Brain Neoplasms; Cell Membrane; Cerebral Cortex; Chromatography, Affinity; Disease Models, Animal; Female; Fibroblasts; Fluorescent Antibody Technique; Gels; Immunosorbent Techniques; Kidney; Liver; Liver Neoplasms, Experimental; Lupus Erythematosus, Systemic; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Molecular Weight; Nerve Tissue Proteins; Neuroblastoma; Neurons; Organ Specificity; Sepharose

Naturally occurring autoantibodies against native DNA (nDNA) in SLE sera showed diverse antigen binding characteristics. The antibodies isolated by affinity chromatography using nDNA linked to Sepharose 4B exhibited specificity towards nDNA and showed strong reactivity with DNA-psoralen photoadduct by direct binding assay and competitive ELISA. The anti-DNA antibody belong to both IgG and IgM immunoglobulin classes and their ratio was 5:1. The possible significance of altered conformation of nDNA in the etiology of SLE has been discussed.

Topics: Antibodies, Antinuclear; Antibody Diversity; Antibody Specificity; Autoimmune Diseases; Binding, Competitive; Chromatography, Affinity; Cross Reactions; DNA; DNA, Single-Stranded; Enzyme-Linked Immunosorbent Assay; Ficusin; Humans; Immunoglobulin G; Immunoglobulin M; Lupus Erythematosus, Systemic; Nucleic Acid Conformation; Sepharose

We developed a sensitive and quantitative method for assaying lupus anticoagulants. The method was based on the inhibition of fibrin formation in a plasma-agarose gel plate, which was described as a method for assaying hemophilic factor VIII-inhibitor (Bird, 1975). The final concentration of plasma in agarose gel was set up 30% instead of 50%. Fibrin formation was stopped, when the clear zone of normal plasma as negative control disappeared and that of 0.039 units heparin as positive appeared. Then this improved the precision of measurement. The method was not only more sensitive than the dilute KPTT of a 1:1 mixture with normal plasma, but had no false positive. The standard curve was linear at heparin units from 0.039 to 100. The assay value could be estimated with heparin titer. This method seem to be useful for quantitative assaying and for determining low titer in lupus anticoagulants.

Topics: Blood Coagulation Factors; Humans; Immunodiffusion; Lupus Coagulation Inhibitor; Lupus Erythematosus, Systemic; Plasma; Sepharose

Current population studies indicate that the HAKATA antigen is one of the normal plasma proteins not yet completely characterized. The frequency of Japanese donor, patients and Swedish patients was 100%, 99.99% and 99.98%, respectively. Anti-HAKATA antibody production was found in three patients, all with systemic lupus erythematosus (SLE). Transient HAKATA antigen deficiency was found in 13 patients and appeared to be strongly associated with SLE (11 out of 13). None of the 14 SLE patients had a history of transfusion. It is therefore concluded that anti-HAKATA antibody is produced as one of the autoantibodies in SLE.

Topics: Antibody Formation; Antigen-Antibody Complex; Autoantibodies; Gels; Humans; Lupus Erythematosus, Systemic; Precipitin Tests; Sepharose

Agarose-poly-L-lysine (Ag-(lys)n-DNA) has been used to bind DNA for assay of anti-DNA antibodies (ab). In this work, an algorithmic approach has been used to classify antinuclear ab (ANA) as being directed against native DNA (dsDNA), denatured DNA (ssDNA), DNA-protein complexes (deoxyribonucleoprotein; DNP), and against antigens which are independent of DNA (iDNA). These ab were subjected to Ag-(lys)n-DNA, and the selectivity of this adsorbent for the various specificities of ab was determined. The DNA on the columns was left untreated or treated with S1 nuclease, this being effected either by treating the DNA prior to introducing it onto the columns or by adding S1 nuclease to the columns after the DNA was bound. Ag-(lys)n-DNA adsorbs ab directed against ssDNA and DNP as well as ab to dsDNA; iDNA ab are not adsorbed. S1 nuclease treatment does not effectively remove ssDNA regions from the Ag-(lys)n-DNA, but it does result in the abolition of the adsorption of a population of ab which are in the anti-DNP sera and contribute to the total ANA load. While anti-iDNA ab are not adsorbed onto the columns, they do contribute to the ANA titer, unlike anti-ssDNA ab which are adsorbed onto the Ag-(lys)n-DNA but do not contribute to the ANA titer. We conclude that Ag-(lys)n-DNA bears antigenic sites for dsDNA, ssDNA, and DNP ab and suggest that our understanding of the characteristic ab-binding profile of this versatile immunoadsorbent may have applications in the study of autoimmune diseases.

Topics: Antibodies, Antinuclear; Arthritis, Rheumatoid; DNA, Single-Stranded; Epitopes; Humans; Immunosorbent Techniques; Lupus Erythematosus, Systemic; Mixed Connective Tissue Disease; Polylysine; Sepharose

Human monoclonal antibodies against DNA were specifically purified from the culture medium of an EBV transformant of SLE patients' lymphocytes using a DNA-coupled Sepharose 4B affinity column. The monoclonal antibodies were eluted from the column with 5% dimethylsulfoxide (pH 10.7) containing 0.5 M NaCl without loss of immunological activity and without contamination by other proteins.

Topics: Antibodies, Antinuclear; Antibodies, Monoclonal; Cell Transformation, Viral; Cells, Cultured; Chromatography, Affinity; Culture Media; Dimethyl Sulfoxide; DNA; Herpesvirus 4, Human; Humans; Lupus Erythematosus, Systemic; Lymphocytes; Sepharose

Specificity of antibodies to Victoria strain of Newcastle disease virus (NDV) found in infectious mononucleosis (IM) and other pathologic sera was investigated by agglutination of NDV-modified human O red blood cells, as well as by immunodiffusion and enzyme immunoassay with various preparations of the virus. These studies clearly demonstrated that the NDV antibodies are distinct from P-B or H-D antibodies. The unexpected observation that guinea pig kidney (GPK) tissues absorbed NDV antibodies allowed their classification into a group of 'GPK-positive' heterophile antibodies. The simultaneous occurrence of the NDV antibodies and H-D antibodies in IM and other diseases suggests the possibility that multiple new antigenic determinants, especially those of carbohydrate nature, may appear due to the alteration of self-antigens as a result of various pathologic processes.

Topics: Antibodies, Viral; Antibody Specificity; Gels; Humans; Immunodiffusion; Infectious Mononucleosis; Leprosy; Lupus Erythematosus, Systemic; Multiple Sclerosis; Neoplasms; Newcastle disease virus; Sepharose; Syphilis

High resolution two-dimensional (2D) gel electrophoresis is useful for analysis of constituents of immune complexes (IC) in serum, provided that the samples for the analysis are prepared by a standardized and effective purification protocol. The details of the protocol, which involve gel permeation chromatography and adsorption with protein A-Sepharose, were worked out with a model system of radiolabeled antigen bound to antibody. With this protocol one can attain an over 50% recovery of the antigen, in a protein preparation purified over 1000-fold with respect to starting amounts in an initial 0.5 ml serum. With silver staining of the 2D gel, the model antigen was detectable at levels of 100 ng initial input. The analysis of eight normal sera and eight sera of patients with systemic lupus erythematosus (SLE) showed no clearly demonstrable differences, suggesting that the latter sera did not contain homogeneous antigens exceeding 100 ng within IC. In addition to IgG, albumin was seen in all preparations, probably due to complexing with immunoglobulin. Trace amounts of other constituents of the samples appeared in some gels, and the presence of C3 related material was detectable only by Western blotting.

Topics: Antigen-Antibody Complex; Gels; Humans; Immunoelectrophoresis, Two-Dimensional; Immunoglobulin G; Isoelectric Focusing; Lupus Erythematosus, Systemic; Microchemistry; Sepharose; Serum Albumin; Staphylococcal Protein A

We describe a technique for estimating the mass of anti-DNA antibodies by immunonephelometry of serum immunoglobulins (IgG, IgA, IgM) before and after adsorption onto DNA bound to agarose-polylysine columns. Sixteen patients with systemic lupus erythematosus and 16 age- and sex-matched controls were studied. Precision was determined for high-value (in 10 patients) and low-value (in nine controls) ranges for each of the immunoglobulins. Within-run CVs ranged from 3.0% (IgG, controls) to 11.8% (IgA, patients); between-run CVs ranged from 15.5% (IgG, patients) to 25.2% (IgM, patients). We found anti-DNA antibody concentrations (mean +/- SD) in systemic lupus erythematosus of 1.981 +/- 1.015 g/L for IgG (controls: 0.243 +/- 0.231, p less than 0.001), 0.257 +/- 0.215 g/L for IgA (controls: 0.038 +/- 0.035, p less than 0.001), and 0.282 +/- 0.234 g/L for IgM (controls: 0.191 +/- 0.165, p greater than 0.05). Sensitivity and linearity are such that fivefold dilutions of patients' serum with either a buffered albumin solution or control serum yielded values close to the expected values for IgG. Similarly diluted sera gave inordinately high values in the radiometric binding assay. Neither parametric (linear regression) nor nonparametric correlation methods (Spearman's rank and Kendall's tau) show a significant correlation between patients' data obtained by the present technique and that by a radiometric binding assay (p greater than 0.05), although combined data from patients and controls demonstrate a significant nonparametric correlation (p less than 0.005 for Spearman's and p less than 0.02 for Kendall's).

Topics: DNA-Binding Proteins; Humans; Hypergammaglobulinemia; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Immunosorbent Techniques; Lupus Erythematosus, Systemic; Nephelometry and Turbidimetry; Polylysine; Radiochemistry; Sepharose

Purification of anti-DNA antibodies from systemic lupus erythematosus (SLE) serum is usually achieved by DNA-affinity chromatography. However, using DNA-cellulose, the present study has found that this technique results in very low yields of DNA binding activity, much of which is contaminated with DNA simultaneously released during chromatography. In comparison it has been found that the anionic dyes Cibracon blue F3GA and Procion red HE3B, bound to crosslinked agarose, give more than 80% recovery and purification of DNA binding activity from whole SLE serum of 11- and 7-fold respectively. The majority of serum immunoglobulin did not bind to the dyes, but that which did, including anti-DNA antibody, bound by ionic interaction. Dye-ligand chromatography is therefore suggested as a useful technique for high yield recovery of partially purified DNA binding activity which can be subjected to further purification procedures, such as preparative isoelectric focusing.

Topics: Antibodies, Antinuclear; Binding Sites, Antibody; Chromatography, Affinity; DNA; Humans; Immunoelectrophoresis, Two-Dimensional; Lupus Erythematosus, Systemic; Sepharose; Triazines

The solid-phase C1q binding assay for circulating immune complexes has been evaluated. The assay provides a rapid, sensitive (detecting as little as 1 microgram of aggregated IgG) and reproducible procedure for the detection of immune complexes in biological fluids. Using artificially prepared immune complexes, the assay detects complexes at four-times antigen-excess. Gel filtration over Sepharose 6B showed that these complexes were distributed over a range of molecular weights from greater than 4 x 10(6) to 300,000 daltons. Using radiolabelled anti-BSA, antigen (BSA) could be detected in these complexes. Screening of gel-filtered SLE showed that the assay detects complexes of both high and low molecular weight, but does not detect all complexes in the SLE sera. Clinical studies showed that immune complexes are frequently found in the sera of patients with SLE and measurement of the concentrations of complexes provides a more sensitive index of disease activity than either serum C3 or C4 concentrations or DNA binding capacity. In patients with RA concentrations of immune complexes were generally higher in synovial fluid than serum, although a patient with systemic rheumatoid disease with hypocomplementaemia had an extremely high level of circulating immune complexes. The assay only infrequently detects circulating immune complexes in glomerulonephritis and in renal transplant recipients. It is concluded that the assay provides a useful clinical tool, but detects only a limited species of immune complexes. It can be used in the detection of antigens in complexes.

Topics: Antigen-Antibody Complex; Arthritis, Rheumatoid; Chromatography, Gel; Complement C1q; Complement System Proteins; DNA; Glomerulonephritis; Humans; Immunoassay; Kidney Transplantation; Lupus Erythematosus, Systemic; Molecular Weight; Reproducibility of Results; Sensitivity and Specificity; Sepharose; Serum Albumin, Bovine