Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies

Hori Hisae, Douglas R. Keene, Lynn Sakai, Mary Wirtz, Hans Peter Bächinger, Maurice Godfrey, David W. Hollister

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the tN-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller tC-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4°C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.

Original languageEnglish (US)
Pages (from-to)759-770
Number of pages12
JournalMolecular Immunology
Volume29
Issue number6
DOIs
StatePublished - 1992

Fingerprint

Collagen Type III
Epitopes
Amino Acid Sequence
Monoclonal Antibodies
Antibodies
Cyanogen Bromide
Peptides
Immunoglobulin G
Enzyme-Linked Immunosorbent Assay
Immunoglobulin Fragments
Pepsin A
Collagenases
Dermis
Antigen-Antibody Complex
Population Groups
Gold
Fluorescent Antibody Technique
Larva
Electron Microscopy
Collagen

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies. / Hisae, Hori; Keene, Douglas R.; Sakai, Lynn; Wirtz, Mary; Bächinger, Hans Peter; Godfrey, Maurice; Hollister, David W.

In: Molecular Immunology, Vol. 29, No. 6, 1992, p. 759-770.

Research output: Contribution to journalArticle

Hisae, Hori ; Keene, Douglas R. ; Sakai, Lynn ; Wirtz, Mary ; Bächinger, Hans Peter ; Godfrey, Maurice ; Hollister, David W. / Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies. In: Molecular Immunology. 1992 ; Vol. 29, No. 6. pp. 759-770.
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abstract = "Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the tN-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller tC-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30{\%} inhibition of antibody binding to native type III collagen at 4°C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.",
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