Megumi Kagawa

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen

A group of rat monoclonal antibodies recognizing the six different α chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen α chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of α chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen

SummaryThe nephritogenic antigen that induces antiglomerular basement membrane antibody-induced glomerulonephritis (anti-GBM nephritis) in rats was isolated from collagenase-solubilized bovine renal basement membranes. Purification was achieved using antibody-coupled affinity columns which were originally used for the purification of trypsin-solubilized nephritogenic antigen (Sado et al. 1984a). The nephritogenic antigen was a heteropolymer composed of P2 (Mr 28 kDa) and P3 (Mr 30 kDa) polypeptides as monomers and their dimers in sodium-dodecyl-sulfate (SDS) polyacryl amide gel electrophoresis. The P3 polypeptide was considered to be the nephritogenic epitope, since a fraction composed of the P2 polypeptide alone was not nephritogenic.The properties of the nephritogenic epitope were the same as those of the Goodpasture epitope (M2*), which is a noncollagenous domain of the α3 chain of type IV collagen (Butkowski et al. 1985; Saus et al. 1988), indicating that the nephritogenic antigen is the same as the Goodpasture antigen.

Purification and characterization of human nephritogenic antigen that induces anti-GBM nephritis in rats.

Human nephritogenic antigen induces anti‐glomerular basement membrane antibody glomerulonephritis in rats. This antigen was purified from collagenase‐solubilized renal basement membrane by means of gel filtration and affinity chromatography using a rabbit antibody. Western blots of the purified nephritogenic antigen using epitope‐defined monoclonal antibodies showed that it contains the NC1 domains of the α1 to α6 chains of type IV collagen. Nephritogenicity was thought to be a feature of the NC1 domains of the α3 to α5 chains, because the α6 chain is not located in the glomerular basement membrane, and because an NC1 fraction consisting of the NC1 domains of the α1 and α2 chains was poorly nephritogenic. Autoantibodies in the sera of patients with Goodpastures syndrome were detected by ELISA using the purified nephritogenic antigen. These results indicate that the nephritogenic antigen contains the Goodpasture antigen, defined as the antigen reactive with sera from patients with Goodpastures syndrome.

Absence of α6(IV) collagen in kidney and skin of X-linked Alport syndrome patients

Abstract. To identify the abnormalities of the type IV collagen α6 chain, α6(IV), in Alport syndrome, we examined renal and skin tissue using rat monoclonal antibodies against non-consensus amino acid sequences of α6(IV). Immunofluorescence of normal human kidney and skin tissue revealed linear α6(IV) staining in the basement membrane (BM) of Bowman’s capsule, in some tubules, and also in the epidermal BM. Renal specimens from five male patients of four families with X-linked Alport syndrome showed no reactivity for α6(IV) in Bowman’s capsules and tubules. In these patients, α1(IV) and α2(IV) were normal, whereas α3(IV), α4(IV), and α5(IV) were absent from the BMs of the kidney. In skin tissue of male patients, neither α5(IV) nor α6(IV) were detected. The epidermal BM of female heterozygotes with X-linked Alport syndrome showed a mosaic staining for α5(IV) and α6(IV). These findings indicate that, in addition to a disturbed α3(IV)-α4(IV)-α5(IV) network, patients with X-linked Alport syndrome have abnormalities in α6(IV) of the renal and epidermal BMs at the protein level.

Nephritogenicity and α-chain composition of NC1 fractions of type IV collagen from bovine renal basement membrane

Nephritogenicity (anti-GBM-nephritis-inducing activity) and α-chain composition of globular-domain (NC1) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different α chains of type IV collagen. A purified nephritogenic fraction from renal BM contained α1–α6(IV)NC1, whereas a non-nephritogenic fraction contained only α1–α2(IV)NC1. Renal and pulmonary NC1 had strong nephritogenic activity; placental NC1 had weak activity. The renal and pulmonary fractions contained α1–α6(IV)NC1, and the placental fraction had a large amount of α1–α2(IV)NC1 and a very small amount of α3–α6(IV)NC1. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained α1–α5(IV) chains, but not the α6(IV) chain. The absence of α6(IV) chain in glomerular BM in bovine and in humans indicates that α6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of α3–α5(IV)NC1.