Skip to main content
Log in

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

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Antignac C, Knebelmann B, Drouot L, Gros F, Deschênes G, Hors-Cayla M-C, Zhou J, Tryggvason K, Grünfeld J-P, Broyer M, Gubler M-C (1994) Deletions in theCOL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression. J Clin Invest 93:1195–1207

    PubMed  Google Scholar 

  • Bächinger HP, Fessler LI, Fessler JH (1982) Mouse procollagen IV. Characterization and supramolecular association. J Biol Chem 257:9796–9803

    PubMed  Google Scholar 

  • Barker DF, Hostikka SL, Zhou J, Chow LT, Oliphant AR, Gerken SC, Gregory MC, Skolnick MH, Atkin CL, Tryggvason K (1990) Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science 248:1224–1226

    PubMed  Google Scholar 

  • Brazel D, Oberbaumer I, Dieringer H, Babel W, Glanville RW, Deutzman R, Kuhn K (1987) Completion of the amino acid sequence of the α1 chain of human basement membrane collagen (type IV) reveals 21 non-triplet interruptions located within the collagenous domain. Eur J Biochem 168:529–536

    Article  PubMed  Google Scholar 

  • Brazel D, Pollner R, Oberbaumer I, Kuhn K (1988) Human basement membrane collagen (type IV). The amino acid sequence of the α2(IV) chain and its comparison with the α1(IV) chain reveals deletions in the α1(IV) chain. Eur J Biochem 172:35–42

    Article  PubMed  Google Scholar 

  • Butkowski R, Wieslander J, Wilson BJ, Barr JF, Noelken ME, Hudson BG (1985) Properties of the globular domain of type IV collagen and its relationship to the Goodpasture antigen. J Biol Chem 260:3739–3745

    PubMed  Google Scholar 

  • Butkowski RJ, Langeveld JPM, Wieslander J, Hamilton J, Hudson BG (1987) Localization of the Goodpasture epitope to a novel chain of basement membrane collagen. J Biol Chem 262:7874–7877

    PubMed  Google Scholar 

  • Duncan KG, Fessler LI, Bächinger HP, Fessler JH (1983) Procollagen IV. Association to tetramers. J Biol Chem 258:5869–5877

    PubMed  Google Scholar 

  • Geysen HM, Rodda SJ, Mason TJ, Tribbick G, Schoofs PG (1987) Strategies for epitope analysis using peptide synthesis. J Immunol Methods 102:259–274

    Article  PubMed  Google Scholar 

  • Hostikka SL, Tryggvason K (1988) The complete primary structure of the α2 chain of human type IV collagen and comparison with the α1(IV) chain. J Biol Chem 263:19488–19493

    PubMed  Google Scholar 

  • Hostikka SL, Eddy RL, Byers MG, Höytya M, Shows TB, Tryggvason K (1990) Identification of a distinct type IV collagen α chain with restricted kidney distribution and assignment of its gene to the locus of X chromosome-linked Alport syndrome. Proc Natl Acad Sci USA 87:1606–1610

    PubMed  Google Scholar 

  • Hudson BG, Wieslander J, Wisdom BJ Jr, Noelken ME (1989) Goodpasture syndrome: molecular architecture and function of basement membrane antigen. Lab Invest 61:256–269

    PubMed  Google Scholar 

  • Hudson BG, Kalluri R, Gunwar S, Noelken ME, Mariyama M, Reeders ST (1993a) Molecular characteristics of the Goodpasture autoantigen. Kidney Int 43:135–139

    PubMed  Google Scholar 

  • Hudson BG, Reeders ST, Tryggvason K (1993b) Type IV collagen: Structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J Biol Chem 268:26033–26036

    PubMed  Google Scholar 

  • Johansson C, Butkowski R, Wieslander J (1991) Characterization of monoclonal antibodies to the globular domain of collagen IV. Connect Tissue Res 25:229–241

    PubMed  Google Scholar 

  • Kalluri R, Gunwar S, Reeders ST, Morrison KC, Mariyama M, Ebner KE, Noelken ME, Hudson BG (1991) Goodpasture syndrome: localization of the epitope for the autoantibodies to the carboxyl-terminal region of the α3(IV) chain of basement membrane collagen. J Biol Chem 266:24018–24024

    PubMed  Google Scholar 

  • Kamagata Y, Mattei M-G, Ninomiya Y (1992) Isolation and sequencing of cDNAs and genomic DNAs encoding the α4 chain of basement membrane collagen type IV and assignment of the gene to the distal long arm of human chromosome 2. J Biol Chem 267:23753–23758

    PubMed  Google Scholar 

  • Kashtan C, Fish AJ, Kleppel M, Yoshioka K, Michael AF (1986) Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport's familial nephritis. J Clin Invest 78:1035–1044

    PubMed  Google Scholar 

  • Kashtan CE, Atkin CL, Gregory MC, Michael AF (1989) Identification of variant Alport phenotypes using an Alport-specific antibody probe. Kidney Int 36:669–674

    PubMed  Google Scholar 

  • Kishiro Y, Kagawa M, Naito I, Sado Y (1995) A novel method of preparing rat-monoclonal antibody-producing hybridomas by using rat medial iliac lymph node cells. Cell Struct Funct 20:151–156

    PubMed  Google Scholar 

  • Kleppel MM, Santi PA, Cameron JD, Wieslander J, Michael AF (1989) Human tissue distribution of novel basement membrane collagen. Am J Pathol 134:813–825

    PubMed  Google Scholar 

  • Kühn K, Wiedemann H, Timpl R, Risteli J, Dieringer H, Voss T, Glanville RW (1981) Macromolecular structure of basement membrane collagens. Identification of 7s collagen as a cross-linking domain of type IV collagen. FEBS Lett 125:123–128

    Article  PubMed  Google Scholar 

  • Leinonen A, Mariyama M, Mochizuki T, Tryggvason K, Reeders ST (1994) Complete primary structure of the human type IV collagen α4(IV) chain. J Biol Chem 269:26172–26177

    PubMed  Google Scholar 

  • Maloy WL, Coligan JE, Paterson Y (1991) Production of antipeptide antisera. In: Coligan JE et al. (eds) Current protocols in immunology. Wiley, New York, chap 9.4.1

    Google Scholar 

  • Mariyama M, Kalluri R, Hudson BD, Reeders ST (1992) The α4(IV) chain of basement membrane collagen: isolation of cDNA encoding bovine α4(IV) and comparison with other type IV collagens. J Biol Chem 267:1253–1258

    PubMed  Google Scholar 

  • Mariyama M, Leinonen A, Mochizuki T, Tryggvason K, Reeders ST (1994) Complete primary structure of the human α3(IV) collagen chain: coexpression of the α3(IV) and α4(IV) collagen chains in human tissues. J Biol Chem 269:23013–23017

    PubMed  Google Scholar 

  • Miner JH, Sanes JR (1994) Collagen IV α3, α4, and α5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches. J Cell Biol 127:879–891

    Article  PubMed  Google Scholar 

  • Morrison KE, Germino GG, Reeders ST (1991) Use of the polymerase chain reaction to clone and sequence a cDNA encoding the bovine α3 chain of type IV collagen. J Biol Chem 266:34–39

    PubMed  Google Scholar 

  • Muthukumaran G, Blumberg B, Kurkinen M (1989) The complete primary structure for the α1-chain of mouse collagen IV. Differential evolution of collagen IV domains. J Biol Chem 264:6310–6317

    PubMed  Google Scholar 

  • Nakanishi K, Yoshikawa N, Iijima K, Kitagawa K, Nakamura H, Ito H, Yoshioka K, Kagawa M, Sado Y (1994) Immunohistochemical study of α1–5 chains of type IV collagen in hereditary nephritis. Kidney Int 46:1413–1421

    PubMed  Google Scholar 

  • Neilson EG, Kalluri R, Sun MJ, Gunwar S, Danoff T, Mariyama M, Myers JC, Reeders ST, Hudson BG (1993) Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen. J Biol Chem 268:8402–8405

    PubMed  Google Scholar 

  • Oohashi T, Sugimoto M, Matei M-G, Ninomiya Y (1994) Identification of a new collagen IV chain, α6(IV), by cDNA isolation and assignment of the gene to chromosome Xq22, which is the same locus forCOL4A5. J Biol Chem 269:7520–7526

    PubMed  Google Scholar 

  • Sado Y, Kagawa M, Naito I, Okigaki T (1991) Properties of bovine nephritogenic antigen that induces anti-GBM nephritis in rats and its similarity to the Goodpasture antigen. Virchows Arch [B] 60:345–351

    Google Scholar 

  • Sado Y, Kagawa M, Rauf S, Naito I, Moritoh C, Okigaki T (1992) Isologous monoclonal antibodies can induce anti-GBM glomerulonephritis in rats. J Pathol (Lond) 168:221–227

    Google Scholar 

  • Saus J, Wieslander J, Langeveld JPM, Quinones S, Hudson BG (1988) Identification of the Goodpasture antigen as the α3(IV) chain of collagen IV. J Biol Chem 263:13374–13380

    PubMed  Google Scholar 

  • Saus J, Quinones S, MacKrell A, Blumberg B, Muthukumaran G, Pihlajaniemi T, Kurkinen M (1989) The complete primary structure of mouse α2(IV) collagen. Alignment with mouse α1(IV) collagen. J Biol Chem 264:6318–6324

    PubMed  Google Scholar 

  • Soininen R, Haka-risku T, Prockop DJ, Tryggvason K (1987) Complete primary structure of the α1-chain of human basement membrane (type IV) collagen. FEBS Lett 255:188–194

    Google Scholar 

  • Sugimoto M, Oohashi T, Ninomiya Y (1994) The genesCOL4A5 andCOL4A6, coding for basement membrane collagen chains α5(IV) and α6(IV), are located head-to-head in close proximity on human chromosome Xq22 andCOL4A6 is transcribed from two alternative promoters. Proc Natl Acad Sci USA 91:11679–11683

    PubMed  Google Scholar 

  • Timpl R, Dziadek M (1986) Structure, development and molecular pathology of basement membranes. Int Rev Exp Pathol 29:1–112

    PubMed  Google Scholar 

  • Timpl R, Wiedemann H, Van Delden V, Furthmayr H, Khün K (1981) A network model for the organization of type IV collagen molecules in basement membranes. Eur J Biochem 120:203–211

    Article  PubMed  Google Scholar 

  • Wieslander J, Barr JF, Butkowski RJ, Edwards SJ, Bygren P, Heinegard D, Hudson BG (1984) Goodpasture antigen of the glomerular basement membrane: localization to noncollagenous regions of type IV collagen. Proc Natl Acad Sci USA 81:3838–8343

    PubMed  Google Scholar 

  • Yamaguchi N, Sato N, Ko JS, Ninomiya Y (1991) Cloning of α1(IV) and α2(IV) collagen cDNAs from rabbit corneal endothelial cell RNA. Invest Ophthalmol Vis Sci 32:2924–2930

    PubMed  Google Scholar 

  • Yoshioka K, Michael AF, Velosa J, Fish AJ (1985) Detection of hidden nephritogenic antigen determinants in human renal and non-renal basement membranes. Am J Pathol 121:156–165

    PubMed  Google Scholar 

  • Yoshioka K, Hino S, Takemura T, Maki S, Wieslander J, Takekoshi Y, Makino H, Kagawa M, Sado Y, Kashtan CE (1994) Type IV collagen α5 chain: Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody. Am J Pathol 144:986–996

    PubMed  Google Scholar 

  • Zheng K, Thorner PS, Martano P, Baumal R, McInnes RR (1994) Canine X chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the α5 chain of collagen type IV. Proc Natl Acad Sci USA 91:3989–3993

    PubMed  Google Scholar 

  • Zhou J, Hertz JM, Leinonen A, Tryggvason K (1992) Complete amino acid sequence of the human α5(IV) collagen chain and identification of a single-base mutation in exon 23 converting glycine 521 in the collagenous domain to cysteine in an Alport syndrome patient. J Biol Chem 267:12475–12481

    PubMed  Google Scholar 

  • Zhou J, Mochizuki T, Smeets H, Antignac C, Laurila P, Paepe A de, Tryggvason K, Reeders ST (1993) Deletion of the paired α5(IV) and α6(IV) collagen genes in inherited smooth muscle tumors. Science 261:1167–1169

    PubMed  Google Scholar 

  • Zhou J, Ding M, Zhao Z, Reeders ST (1994) Complete primary structure of the sixth chain of human basement membrane collagen, α6(IV): isolation of the cDNAs for α6(IV) and comparison with five other type IV collagen chains. J Biol Chem 269:13193–13199

    PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sado, Y., Kagawa, M., Kishiro, Y. et al. Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen. Histochem Cell Biol 104, 267–275 (1995). https://doi.org/10.1007/BF01464322

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01464322

Keywords

Navigation