CHARACT OF SPECIFICITY OF ANTIBODIES TO TRYPSINOGEN ACTIVATION PEPTIDE LE

胰蛋白酶原激活肽Le抗体的特异性特征

• 批准号: 6118316
• 负责人: JAMES H GRENDELL
• 金额: $ 0.09万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-10 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6118316
• 关键词: biomedical resource; human tissue; mental disorders; spectrometry

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.

胰蛋白酶原激活肽（TAP）的抗体可能是 作为研究细胞内加工的标记物具有重要价值 胰蛋白酶原（TG）的胰蛋白酶在急性 胰腺炎 然而，这需要这些抗体具有非常 高选择性TAP相比，更丰富的TG在 腺泡细胞 免疫家兔制备抗TAP抗体 用含有大鼠TG N-末端的合成肽 （LPLDDDDDK）偶联甲状腺球蛋白。 抗体亲和力 使用用于免疫的肽或其 C-末端5个氨基酸（DDDDK）。 所得抗体制剂 分别命名为AB 9和AB 5。 在免疫印迹上，AB 5 识别TG的水平仅为用AB 9观察到的水平的约5%。 由于酶联免疫吸附试验的潜在局限性， 在暴露TAP表位的情况下， 使用质谱法确认抗体的特异性 为了精确分析免疫沉淀物的分子量， 涉及AB 9、AB 5或市售兔抗TG的产品 抗体，在这些产物与蛋白A结合后 琼脂糖珠。 与TG免疫沉淀的结果相比， 兔抗TG抗体，TG与AB 9的结合显著较少; AB 5与TG的结合进一步显著降低， 在AB 9 在等摩尔量的竞争性结合实验中 TG和含有TAP表位的肽，AB 5与 检测TG。 使用免疫沉淀/质谱法 我... 建立抗体选择性的方法，我们有 确定产生的抗体显示出相当大的 TAP相对于TG的选择性需要亲和纯化， 与TG活化位点紧邻的短肽， TG的远N-末端的识别。 S.M. Chepilko，T.大谷 F.S.戈雷利克湖马兹雷利河Wang和J.H. Grendell（1997年）， 胰腺15（4），431。