Tipton D A, Walker W S, Schonbaum G R
Dental Research Center, College of Dentistry, University of Tennessee, Memphis.
Hybridoma. 1990 Aug;9(4):319-30. doi: 10.1089/hyb.1990.9.319.
Nine mouse monoclonal antibodies (MAbs) to horseradish peroxidase (HRP) were used to develop an epitope map of the enzyme. The results of a competitive binding assay indicated three distinct patterns of reactivity. Two groups of MAbs (I and III) recognized epitopes located in separate antigenic regions on the molecule; another (II) bound to sites that overlapped with epitopes in either region I or III. Further definition of these regions was obtained by analyzing the MAbs for their binding to isolated heme, other peroxidases and heme-containing proteins, and to denatured and apo-HRP. None of the group I MAbs bound heme, suggesting that this region was removed from the active site of the enzyme. All of the group II and III MAbs bound heme as well as the other peroxidases and heme-containing proteins, indicating that they recognized heme-associated epitopes at or near the active site. Only one MAb (2A2) in groups II and III bound to apo-HRP but not to denatured HRP; it was also the only MAb in the entire panel that inhibited the catalytic activity of HRP. This suggests that the epitope recognized by 2A2 involves both the heme moiety and a conformationally dependent protein determinant near the active site.
使用九种针对辣根过氧化物酶（HRP）的小鼠单克隆抗体（MAb）绘制该酶的表位图谱。竞争性结合试验结果显示出三种不同的反应模式。两组单克隆抗体（I和III）识别位于分子上不同抗原区域的表位；另一组（II）与区域I或III中与表位重叠的位点结合。通过分析这些单克隆抗体与分离的血红素、其他过氧化物酶和含血红素蛋白质以及变性和脱辅基HRP的结合情况，对这些区域进行了进一步定义。I组单克隆抗体均不与血红素结合，这表明该区域远离酶的活性位点。所有II组和III组单克隆抗体均与血红素以及其他过氧化物酶和含血红素蛋白质结合，表明它们识别活性位点处或附近与血红素相关的表位。II组和III组中只有一种单克隆抗体（2A2）与脱辅基HRP结合，但不与变性HRP结合；它也是整个单抗组中唯一抑制HRP催化活性的单克隆抗体。这表明2A2识别的表位涉及血红素部分以及活性位点附近的构象依赖性蛋白质决定簇。
