Macht M, Fiedler W, Kürzinger K, Przybylski M
University of Konstanz, Germany.
Biochemistry. 1996 Dec 10;35(49):15633-9. doi: 10.1021/bi961727w.
Monoclonal antibodies are widely used analytical tools in biochemical research. The knowledge of their corresponding epitopes is of major interest. One possible approach for epitope characterization is the application of protein antigen proteolysis in combination with mass spectrometric peptide mapping analysis. Two complementary analytical strategies were applied: (a) limited proteolysis of antibody-bound antigen followed by removal of nonbound peptides and detachment of the antigenic peptides (epitope excision) and (b) enzymatic digest of the antigen followed by extraction of the antigenic peptides with the antibody and detachment of antigenic peptides after removal of nonbinding fragments (epitope extraction). In the few examples published so far, immobilized antibodies were used for these studies. In this study we present a method for characterization of the epitope sequences without prior immobilization of the monoclonal antibody. The separation of nonepitope peptides from antibody-bound peptides was carried out by ultrafiltration. The epitope and nonepitope fractions were analyzed by MALDI-MS without further purification, and the epitope sequences were identified. The method was developed using a model system consisting of the synthetic C-terminal cyanogen bromide fragment CB3 of myoglobin and the commercial monoclonal anti-myoglobin MG1. In further investigations the epitope sequence of a synthetic 32 amino acid peptide derived from heart muscle protein troponin T toward a monoclonal antibody MAb-M7, which was raised against the intact protein, was characterized. With this approach the epitope binding site of this antibody was determined, and selective shielding of potential cleavage sites in the immune complex could be observed. Furthermore, statements about the three-dimensional structure of the bound antigen were made.
单克隆抗体是生物化学研究中广泛使用的分析工具。了解它们相应的表位备受关注。表位表征的一种可能方法是将蛋白质抗原蛋白酶解与质谱肽图谱分析相结合。应用了两种互补的分析策略：(a) 对抗体结合的抗原进行有限蛋白酶解，然后去除未结合的肽并分离抗原肽（表位切除）；(b) 对抗原进行酶解，然后用抗体提取抗原肽，并在去除非结合片段后分离抗原肽（表位提取）。在迄今为止发表的少数例子中，这些研究使用了固定化抗体。在本研究中，我们提出了一种无需预先固定单克隆抗体即可表征表位序列的方法。通过超滤将非表位肽与抗体结合的肽分离。表位和非表位部分无需进一步纯化即可通过基质辅助激光解吸电离质谱（MALDI-MS）进行分析，并鉴定表位序列。该方法是使用由肌红蛋白的合成C末端溴化氰片段CB3和市售单克隆抗肌红蛋白MG1组成的模型系统开发的。在进一步的研究中，对源自心肌蛋白肌钙蛋白T的合成32氨基酸肽针对针对完整蛋白产生的单克隆抗体MAb-M7的表位序列进行了表征。通过这种方法确定了该抗体的表位结合位点，并观察到免疫复合物中潜在裂解位点的选择性屏蔽。此外，还对结合抗原的三维结构进行了阐述。
