Immunoanalysis of keratan sulfate proteoglycan from corneal scars.
Corneal keratan sulfate proteoglycan (KSPG) from scar tissue of experimental penetrating corneal wounds in rabbits was analyzed 2-8 weeks after injury using three previously characterized antibodies. Keratan sulfate (KS) was identified in 2 week scars and normal corneal tissue by indirect immunofluorescence using a monoclonal antibody against sulfated KS epitopes. KSPG was measured in unfractionated extracts of scar and of normal corneal tissue using a "sandwich" enzyme-linked immunosorbent assay (ELISA). In extracts of 2 week scars, KSPG molecules reacting with two different anti-KS monoclonal antibodies were 55% and 82% as abundant as in normal tissue extracts. Ion exchange high performance liquid chromatography (HPLC) of tissue extracts found qualitatively similar elution profiles of KSPG antigens from both scar and normal tissues. Direct ELISA of the HPLC-purified KSPG showed identical quantitative binding of antibodies against core protein and KS from normal and scar tissue. KS in the HPLC-purified extracts was sensitive to digestion with endo-beta-galactosidase, whereas core protein antigens were not affected by this enzyme, as expected. Alteration of the antigenic characteristics of the KSPG of scars was detected with a competitive immunoassay using immobilized monoclonal antibodies against KS. KS in extracts from 2, 6, and 8 week scars competed only 5-11% as effectively as KS from normal cornea, although core protein antigens in the scar extracts competed 61-80% as well as those of normal cornea.(ABSTRACT TRUNCATED AT 250 WORDS)
Funderburgh
J L
JL
Division of Biology, Kansas State University, Manhattan 66506.
Cintron
C
C
Covington
H I
HI
Conrad
G W
GW
D000818:Animals; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002921:Cicatrix / Q000378:metabolism*; D003315:Cornea / Q000032:analysis*; D003316:Corneal Diseases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006025:Glycosaminoglycans / Q000032:analysis*; D007632:Keratan Sulfate / Q000032:analysis*; D000071234:Lumican; D011509:Proteoglycans / Q000032:analysis*; D011817:Rabbits
D011508:Chondroitin Sulfate Proteoglycans; D006025:Glycosaminoglycans; D000071234:Lumican; D011509:Proteoglycans; D007632:Keratan Sulfate