Funderburgh J L, Cintron C, Covington H I, Conrad G W
Division of Biology, Kansas State University, Manhattan 66506.
Invest Ophthalmol Vis Sci. 1988 Jul;29(7):1116-24.
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)
使用三种先前已鉴定的抗体，对兔实验性穿透性角膜伤口瘢痕组织中的角膜硫酸角质素蛋白聚糖（KSPG）在损伤后2至8周进行了分析。通过使用针对硫酸化KS表位的单克隆抗体进行间接免疫荧光法，在2周龄的瘢痕和正常角膜组织中鉴定出硫酸角质素（KS）。使用“夹心”酶联免疫吸附测定（ELISA）法，对瘢痕和正常角膜组织的未分级提取物中的KSPG进行测量。在2周龄瘢痕的提取物中，与两种不同抗KS单克隆抗体反应的KSPG分子含量分别为正常组织提取物中的55%和82%。对组织提取物进行离子交换高效液相色谱（HPLC）分析发现，瘢痕组织和正常组织中KSPG抗原的洗脱曲线在定性上相似。对HPLC纯化的KSPG进行直接ELISA检测显示，来自正常组织和瘢痕组织的针对核心蛋白和KS的抗体具有相同的定量结合。HPLC纯化提取物中的KS对内切β-半乳糖苷酶消化敏感，而核心蛋白抗原不受该酶影响，这与预期一致。使用固定化抗KS单克隆抗体的竞争性免疫测定法检测到瘢痕KSPG抗原特性的改变。2周、6周和8周龄瘢痕提取物中的KS竞争效力仅为正常角膜KS的5%-11%，尽管瘢痕提取物中的核心蛋白抗原竞争效力为正常角膜的61%-8%。（摘要截断于250字）
