Roos G, Landberg G, Huff J P, Houghten R, Takasaki Y, Tan E M
Department of Pathology, University of Umeå, Sweden.
Lab Invest. 1993 Feb;68(2):204-10.
Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) are increasingly used for evaluation of cell proliferation especially in diagnostic pathology. To understand the different staining characteristics of such antibodies, we found it important to map the reactive epitopes in detail.
Overlapping, synthetic 15-mer peptides encoding the entire PCNA amino acid sequence were analyzed for reactivity with 6 anti-PCNA monoclonal antibodies using enzyme-linked immunosorbent assay and flow cytometric competition analysis. One immunodominant region was studied using a set of peptides with single amino acid substitutions. Further epitope localization was obtained by immunoblotting of fusion protein constructs.
The epitopes recognized by the antibodies could be assigned to specific peptides. Five monoclonal antibodies (19A2, 19F4, TO17, TO30, PC10) reacted with the same protein region (aa 111-125) whereas one antibody (TOB7) recognized a separate region of the protein (aa 181-195). The reactivity pattern with 8 recombinant PCNA-fusion proteins agreed with the peptide data. The immunodominant aa 111-125 peptide reactive antibodies differed concerning immunofluorescence patterns. These differences could be attributed to epitope microheterogeneity within this peptide as determined by reactivity with Ala-substituted peptides. The 19A2, 19F4 and PC10 antibodies showed nuclear fluorescence and similar but not identical peptide recognition patterns. Compared with 19A2 and 19F4, the PC10 antibody was directed against a simpler epitope. TO17 and TO30 gave cytoplasmic filamentous staining and their epitopes were different from those defined by the other monoclonal antibodies.
The results indicate that induced anti-PCNA antibodies recognize well defined linear epitopes, in contrast to PCNA autoepitopes which are strongly dependent on the protein conformation.
增殖细胞核抗原（PCNA）单克隆抗体越来越多地用于评估细胞增殖，尤其是在诊断病理学中。为了解此类抗体的不同染色特性，我们发现详细绘制反应性表位很重要。
使用酶联免疫吸附测定和流式细胞术竞争分析，分析编码整个PCNA氨基酸序列的重叠合成15聚体肽与6种抗PCNA单克隆抗体的反应性。使用一组具有单个氨基酸取代的肽研究一个免疫显性区域。通过融合蛋白构建体的免疫印迹获得进一步的表位定位。
抗体识别的表位可归为特定肽段。五种单克隆抗体（19A2、19F4、TO17、TO30、PC10）与同一蛋白区域（氨基酸111 - 125）反应，而一种抗体（TOB7）识别蛋白的另一个区域（氨基酸181 - 195）。与8种重组PCNA融合蛋白的反应模式与肽段数据一致。免疫显性氨基酸111 - 125肽反应性抗体在免疫荧光模式上有所不同。这些差异可归因于该肽段内的表位微异质性，这是通过与丙氨酸取代肽的反应性确定的。19A2、19F4和PC10抗体显示核荧光以及相似但不完全相同的肽段识别模式。与19A2和19F4相比，PC10抗体针对的是一个更简单的表位。TO17和TO30产生细胞质丝状染色，其表位与其他单克隆抗体定义的表位不同。
结果表明，诱导产生的抗PCNA抗体识别明确的线性表位，这与强烈依赖蛋白质构象的PCNA自身表位形成对比。
