CHARACT OF SPECIFICITY OF ANTIBODIES TO TRYPSINOGEN ACTIVATION PEPTIDE LE

胰酶原激活肽(TAP)抗体具有潜在的 作为研究细胞内加工的标志物具有重要价值 急性胰腺炎早期胰酶原(TG)对胰酶的影响 胰腺炎。然而，这需要这些抗体具有非常强的 与TAP中更丰富的TG相比，TAP的高选择性 腺泡细胞。TAP抗体是通过免疫兔而产生的 用含有大鼠甘油三酯N末端的合成肽 (LPLDDDDDK)与甲状腺球蛋白偶联。抗体具有亲和力 使用用于免疫的多肽或其 C末端5个氨基酸(DDK)。由此产生的抗体制剂 分别命名为AB9和AB5。关于免疫印迹，AB5 识别TG的水平仅为AB9观察到的约5%。 由于故障导致的酶联免疫吸附试验的潜在局限性 在TAP表位暴露的情况下，水溶液中的Tg 用质谱仪确认抗体的特异性。 准确分析免疫共沉淀的分子质量 含有AB9、AB5或商用兔抗甘油三酯的产品 抗体，在这些产物与蛋白A结合后 琼脂糖珠。与免疫沉淀法测定TG的结果比较 兔抗甘油三酯抗体，AB9与甘油三酯的结合较少； 与对照组相比，AB5与TG的结合量显著降低 使用AB9。在等摩尔量的竞争性结合实验中 Tg和含有TAP表位的多肽，AB5与 检测甘油三酯。利用免疫沉淀/质谱学 续..。建立抗体选择性的方法，我们有 确定了抗体的产生证明了相当大的 TAP对TG的选择性需要亲和纯化 短肽紧邻TG活化部位还原 Tg远N端的识别。首页--期刊主要分类--期刊细介绍--期刊细介绍 郭福生，L.马兹雷利，R.Wang，J.H.Grendell(1997)， 胰腺15(4)，431。

Antibodies to trypsinogen activation peptide (TAP) are potentially of great value as a marker for studying the intracellular processing of trypsinogen (TG) to trypsin in the early stages of acute pancreatitis. However this requires that these antibodies have a very high selectivity for TAP compared to the much more plentiful TG in the acinar cell. Antibodies to TAP were generated by immunizing rabbits with a synthetic peptide containing the N-terminal of rat TG (LPLDDDDDK) coupled to thyroglobulin. Antibodies were affinity purified employing either the peptide used for immunization or its C-terminal 5 amino acid (DDDDK). The resulting antibody preparations were designated AB9 and AB5 respectively. On immunoblot, AB5 recognized TG at a level only about 5% of that observed with AB9. Because of the potential limitations of ELISA resulting from breakdown of TG in aqueous solution with exposure of the TAP epitope, the specificities of the antibodies were confirmed using mass spectrometry to precisely analyze the molecular masses of the immunoprecipitated products involving AB9, AB5, or commercially available rabbit anti-TG antibodies, following binding of these products to the protein A agarose beads. Compared to results of immunoprecipitation of TG with rabbit anti-TG antibody, binding of TG by AB9 was substantially less; and AB5 binding to TG was markedly reduced further compared to that with AB9. In competitive binding experiments with equal molar amounts of TG and a peptide containing the TAP epitope, no binding of AB5 to TG was detected. Using the immunoprecipitation/mass spectrometry cont... approach to establish antibody selectivity, we have determined that generation of antibodies that demonstrate considerable selectivity for TAP over TG requires affinity purification with a short peptide immediately adjacent to the TG activation site to reduce recognition of the far N-terminal of TG. S.M. Chepilko, T. Otani, F.S. Gorelick, L. Mazzrrelli, R. Wang, and J.H. Grendell (1997), Pancreas 15(4), 431.