Thibault G, Milne R, Cantin M
Clinical Research Institute of Montreal, Quebec, Canada.
Peptides. 1988 Sep-Oct;9(5):1059-65. doi: 10.1016/0196-9781(88)90089-7.
A "two-site" immunoradiometric assay (IRMA) was developed to specifically measure ANF (1-126), the precursor of ANF. This assay is based on the simultaneous use of antibodies against two different antigenic determinants: murine monoclonal antibody (2H2), which recognizes positions 101 through 103 of ANF, is linked to Immunobeads and employed to extract any ANF C-terminal; a second antibody, which is directed against positions 11 through 37, is radioiodinated and allows binding to any C-terminal-2H2-Immunobead material which bears the N-terminal antigenic site. A curvilinear relationship was obtained between radioactivity and the amount of proANF (1.5 to 400 fmol) added. Optimisation of IRMA was determined by the amount of 2H2-Immunobeads and labeled antibody used, incubation time as well as possible interference by both ANF (99-126) and ANF (1-98). Tissue extracts were used to validate the assay. proANF was detected in decreasing amounts in heart atria, heart ventricles, lungs, kidneys and adrenal glands. Its presence was further confirmed by reverse-phase HPLC followed by radioimmunoassay. IRMA is a simple and rapid method for the direct measurement of proANF in tissue extracts and chromatographic fractions. The presence of proANF in tissues strongly suggests local synthesis.
开发了一种“双位点”免疫放射分析（IRMA）来特异性测量心房钠尿肽（ANF）的前体ANF（1-126）。该分析基于同时使用针对两种不同抗原决定簇的抗体：识别ANF第101至103位的鼠单克隆抗体（2H2）与免疫磁珠相连，并用于提取任何ANF的C末端；第二种抗体针对第11至37位，经放射性碘化后可与任何带有N末端抗原位点的C末端-2H2-免疫磁珠材料结合。放射性与添加的前体ANF（1.5至400飞摩尔）量之间获得了曲线关系。IRMA的优化取决于所用2H2-免疫磁珠和标记抗体的量、孵育时间以及ANF（99-126）和ANF（1-98）可能的干扰。使用组织提取物验证该分析。在心房、心室、肺、肾和肾上腺中检测到前体ANF的量逐渐减少。通过反相高效液相色谱随后进行放射免疫测定进一步证实了其存在。IRMA是一种直接测量组织提取物和色谱馏分中前体ANF的简单快速方法。组织中前体ANF的存在强烈提示局部合成。
