Production of monoclonal antibodies to human glomerular basement membrane.

Using the technique of somatic cell fusion, we produced monoclonal antibodies to collagenase-digested human glomerular basement membrane (GBM). Fourteen monoclonal antibodies which reacted with normal human kidney in indirect immunofluorescence (IIF) studies were produced. An analysis of the binding patterns indicated that the antigens recognized could be divided into six broad groups. Monoclonal antibody B3-H10 (Group 1) reacted with only GBM in a fine granular pattern. A5-B12 and B5-C2 (Group 2) reacted with GBM and peritubular capillary in a linear pattern. B2-A12 (Group 3) reacted with only epithelial cells. Al-C9 and A4-E2 (Group 4) showed a mesangial pattern in glomerulus and a lineal pattern in tubular basement membrane (TBM), Bowman's capsule and peritubular capillary. A1-E1, A1-E11, A2-E6, A3-B6, A4-F8 and B5-H2 (Group 5) recognized determinants common to GBM, TBM, Bowman's capsule and/or peritubular capillary. A3-F1 and B5-E10 (Group 6) reacted with TBM and Bowman's capsule. The staining pattern of B3-H10 (Group 1) was characteristic because it was not linear, but finely granular along the GBM. The staining pattern of B2-A12 (Group 3) was also characteristic because only epithelial cells were stained, and processes of epithelial cells were observed as fine fibrils. To the best of our knowledge, these two types of monoclonal antibodies have not been reported previously.</p

A study on cross-reactivity of anti-DNA antibody with glycosaminoglycans.

To study the pathogenesis of lupus nephritis, the cross reactivity between anti-DNA antibody and glycosaminoglycans (GAGs) was investigated. Monoclonal anti-DNA antibodies were obtained from hybridomas by the fusion of MRL/lpr/lpr splenocytes with murine myeloma cells. Some of these monoclonal anti-DNA antibodies showed cross reactivity with GAGs, such as hyaluronic acid, chondroitin sulfate and heparan sulfate. To elucidate the mechanism of cross reactivity, inhibition assays with propanol and polyethylenimine (PEI), a cationic agent, were carried out. Increase of the concentration of PEI (0.6-2.0% vol/vol) resulted in a dose dependent decrease in the binding ability of anti-DNA antibody to GAGs. Propanol, an organic reagent which disrupts the van der Waals bonds between epitopes and paratopes, showed little inhibitory effect on the binding activity of monoclonal anti-DNA antibody to GAGs. These results indicate that the binding of anti-DNA antibody to GAGs is due to a charge interaction rather than van der Waals forces. Anti-DNA antibody which can react with GAGs in the glomerular basement membrane seems to play an important role in the pathogenesis of lupus nephritis.</p

Molecular sieve in bovine descemets membrane as revealed by negative staining.

Descemet's membrane was isolated from the corneas of cows and observed by electron microscopy after negative staining with 1% phosphotungstic acid solution, pH 7.2. Ultrastructurally, bovine Descement's membrane had a very regular hexagonal pattern. Nodes were connected to the six others around each of them by thin filaments to form a hexagon. The distance between the nodes was approximately 120 nm, the diameter of the nodes approximately 30 nm, and the width of the connecting filaments approximately 10 nm. Bovine Descement's membrane was a molecular sieve composed of nodes and filaments substantiating our molecular sieve theory of basement membranes.</p

Study on the change of glomerular antigenicity in various renal diseases with anti-human renal monoclonal antibodies

We produced 22 different kinds of monoclonal antibody (Mab) by immunizing mice with human GBM antigens. In these Mabs, Mab-G1 to G5 recognized only GBM in the glomerulas, Mab-E1 and E2 recognized only glomerular epithelial cells, and Mab-M1 to M4 recognized mainly mesangium. The reactions of these Mabs with known GBM antigens such as type IV collagen, fibronectin and laminin were negative by immunoblotting. Using Mab-G1, Mab-E1 and Mab-M1, changes in the antigenicity of antigens recognized by Mabs were examined on kidney sections from the patients with various renal diseases by the indirect immunofluorescence test. When Mab-G1 recognizing GBM was used, there was no particular change of anti-genicity in minimal change nephrotic syndrome (MCNS) and IgA nephropathy (IgA), whereas in membranous nephropathy (MN) thickened GBM was found to maintain anti-genicity and the region of deposits was observed as negative punched-out region. In type I and III of membranoproliferative glomerulonephritis (MPGN), GBM was observed only outside of subendothlial deposits without showing double contour. In type II MPGN, GBM showed a double linear pattern and antigenicity of GBM in regions of dense deposits was not detected. When Mab-E1 recognizing glomerular epithelial cells was used, there was no change of antigenicity in the renal diseases. Further, in crescentic glomerulone-phritis, the region of the cellular crescents was not stained, When Mab-M1 recognizing mesangium was used, extensive staining was observed in the increased mesangium in IgA, MPGN, and diabetic nephropathy. We feel that it is of significance in elucidating the pathogenesis of renal diseases to study the changes of glomerular antigenicity in diseased kidneys by using anti-human renal monoclonal antibodies

Assay of GBM antigen in urine and serum with an anti-human renal monoclonal antibody

Using a monoclonal antibody (Mab-G3) recognizing glomerular basement membrane (GBM), we assayed GBM antigen (G3-Ag) in the urine and serum of renal disease patients by sandwich ELISA. The subjects included normal control (NOR), minimal change nephrotic syndrome (MCNS), IgA nephropathy (IgA), membranous nephropathy (MN), membranoproliferative glomerulonephritis (MPGN) and chronic renal failure (CRF). The urine and serum was used as the material. With urinary G3-Ag, there were no statistically significant differences among the NOR, MCNS, IgA, MN, MPGN and CRF groups. Although no correlation was observed with proteinuria, hematuria, serum creatinine, serum β2 microglobulin and urinary NAG, urinary G3-Ag showed a significant (p<0.05) increase in excretion in the group of progressive CRF patients with s-Cr more than 1.0 mg/dl/month as compared to the stationary CRF group with s-Cr<1.0 mg/dl/month. Serum G3-Ag showed lower values in almost all cases, and there were no significant differences among the renal disease groups. The above findings led us to believe that the assay of urinary G3-Ag was useful in determining the degree of GBM disorder. It was also presumed that assay of renal antigens in urine and serum with the respective anti-human renal monoclonal antibodies could be a new tool in diagnosing renal diseases