Production and Characterization of Hybridoma Antibody to Native Human Type II Collagen

David W. Hollister, Lynn Sakai, Nicholas P. Morris, Linda H. Shimono, Robert E. Burgeson

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Using a sensitive RIA for collagenous proteins, we have applied a series of consecutive selection steps to construct a hybridoma that produces antibody against native human Type II collagen. The selection steps consisted of: 1) hybrid cell line selection; 2) co-selection of IgG class immunoglobulin reactive to the immunizing antigen; 3) selection for anticollagen antibody by complete inhibition of antibody binding to Type II collagen-coated plates by prior specific collagenase digestion; 4) selection for anti-helical antibody by complete inhibition of antibody binding to heat-denatured Type II collagen-coated plates; 5) clonal selection; 6) selection for absence of crossreactivity to 5–7 additional native collagen types; and finally 7) selection for antibody yielding indirect immunofluorescent staining of human cartilage matrix, but not adjacent bone or perichondrium. Crossreactivity assays against other native human collagen types revealed little crossreactivity. In addition, we have demonstrated that native Type II collagen inhibits antibody binding to Type II collagen-coated plates; that Type II collagen can be recovered from antigen-antibody complexes; that a sensitive radio immunoassay for Type II collagen can be developed; and that the antibody is useful for indirect immunofluorescent studies of human cartilage matrix.

Original languageEnglish (US)
Pages (from-to)197-210
Number of pages14
JournalTopics in Catalysis
Volume2
Issue number3
DOIs
StatePublished - Jan 1 1982
Externally publishedYes

Fingerprint

Collagen Type II
Hybridomas
Population Groups
Collagen
Antibodies
Cartilage
Antigens
Hybrid Cells
Immunoglobulin Isotypes
Collagenases
Antigen-Antibody Complex
Radio
Immunoassay
Digestion
Anti-Idiotypic Antibodies
Immunoglobulin G
Hot Temperature
Assays
Bone
Staining and Labeling

Keywords

  • collagen is used to denote AB collagen
  • DEAE
  • diethylaminoethyl cellulose
  • DMEM
  • Dulbecco's-modified Eagle media
  • HAT
  • HT
  • Hybridoma
  • hypoxanthine, aminopterin, thymidine
  • hypoxanthine, thymidine
  • immunofluorescence
  • monoclonal antibody
  • NaDodS0/PAGE
  • PBS
  • phosphate-buffered saline
  • polyoxyethylene (20) sorbitan monolaurate
  • radioimmunoassay
  • radioimmunoassay
  • RIA
  • sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • trademark of Atlas Chern. Ind., Inc.
  • TRIS
  • trishydroxymethylamine
  • Tween®
  • Tween® 20
  • Type V

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Rheumatology

Cite this

Production and Characterization of Hybridoma Antibody to Native Human Type II Collagen. / Hollister, David W.; Sakai, Lynn; Morris, Nicholas P.; Shimono, Linda H.; Burgeson, Robert E.

In: Topics in Catalysis, Vol. 2, No. 3, 01.01.1982, p. 197-210.

Research output: Contribution to journalArticle

Hollister, David W. ; Sakai, Lynn ; Morris, Nicholas P. ; Shimono, Linda H. ; Burgeson, Robert E. / Production and Characterization of Hybridoma Antibody to Native Human Type II Collagen. In: Topics in Catalysis. 1982 ; Vol. 2, No. 3. pp. 197-210.
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AB - Using a sensitive RIA for collagenous proteins, we have applied a series of consecutive selection steps to construct a hybridoma that produces antibody against native human Type II collagen. The selection steps consisted of: 1) hybrid cell line selection; 2) co-selection of IgG class immunoglobulin reactive to the immunizing antigen; 3) selection for anticollagen antibody by complete inhibition of antibody binding to Type II collagen-coated plates by prior specific collagenase digestion; 4) selection for anti-helical antibody by complete inhibition of antibody binding to heat-denatured Type II collagen-coated plates; 5) clonal selection; 6) selection for absence of crossreactivity to 5–7 additional native collagen types; and finally 7) selection for antibody yielding indirect immunofluorescent staining of human cartilage matrix, but not adjacent bone or perichondrium. Crossreactivity assays against other native human collagen types revealed little crossreactivity. In addition, we have demonstrated that native Type II collagen inhibits antibody binding to Type II collagen-coated plates; that Type II collagen can be recovered from antigen-antibody complexes; that a sensitive radio immunoassay for Type II collagen can be developed; and that the antibody is useful for indirect immunofluorescent studies of human cartilage matrix.

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