Immunoanalysis of keratan sulfate proteoglycan from corneal scars.
Immunoanalysis of keratan sulfate proteoglycan from corneal scars.
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发表时间：
1988-07
影响因子：
4.4
通讯作者：
J. Funderburgh;C. Cintron;H. Covington;G. Conrad
中科院分区：
文献类型：
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作者：
J. Funderburgh;C. Cintron;H. Covington;G. Conrad
使用三种先前已鉴定的抗体，在损伤后2 - 8周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）进行了分析。通过使用一种针对硫酸化硫酸角质素表位的单克隆抗体进行间接免疫荧光检测，在2周的瘢痕组织和正常角膜组织中鉴定出了硫酸角质素（KS）。使用“夹心”酶联免疫吸附测定（ELISA）对瘢痕组织和正常角膜组织的未分级提取物中的KSPG进行了测量。在2周瘢痕组织提取物中，与两种不同的抗 - KS单克隆抗体发生反应的KSPG分子含量分别为正常组织提取物中的55%和82%。对组织提取物进行的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抗原的洗脱曲线在性质上相似。对HPLC纯化的KSPG进行直接ELISA检测表明，来自正常组织和瘢痕组织的针对核心蛋白和KS的抗体结合量相同。正如预期的那样，HPLC纯化提取物中的KS对内 - β - 半乳糖苷酶消化敏感，而核心蛋白抗原不受该酶影响。使用固定化的抗 - KS单克隆抗体通过竞争免疫测定法检测到瘢痕组织中KSPG的抗原特性发生了改变。来自2周、6周和8周瘢痕组织提取物中的KS与来自正常角膜的KS相比，竞争效率仅为5 - 11%，尽管瘢痕组织提取物中的核心蛋白抗原与正常角膜的核心蛋白抗原竞争效率为61 - 80%。（摘要截断至250字）
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)