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首页> 外文期刊>Journal of Immunological Methods >Rapid and sensitive immunomagnetic-electrochemiluminescent detection of p53 antibodies in human serum.
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Rapid and sensitive immunomagnetic-electrochemiluminescent detection of p53 antibodies in human serum.

机译：快速，灵敏的免疫荧光电化学发光检测人血清中的p53抗体。

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The mutation of tumor suppressor p53 gene is common in malignant tumor. p53 antibodies are products of immunoresponse against abnormal p53 protein. It has been found that p53 antibodies are of importance in tumor's diagnosis, prognosis and relapse monitoring. However, current method for detecting p53 antibodies, i.e. enzyme-linked immunosorbent assay (ELISA), requires a long time with multiple steps, and the assay is only semi-quantitative. In this work, a protocol for quantitative detection of p53 antibodies in human serum using immunomagnetic electrochemiluminescence (IM-ECL) was devoloped. The immunoassay format consisted of a three antibody sandwich in which a biotinylated capture antibody, was banded with the commercial p53 protein. A detector antibody was added to bind the p53 protein at another site. Then, secondary antibody, labeled with ruthenium(II) tris-bipyridal, was added and, when bound to the bead immunocompiex, generated light in the presence of an excess of tripropylamine. The light was detected and measured by the analyzer made by us. Our experimental results indicate that the sensitivity of this assay was 10 pg of p53 antibodies per ml of reference serum (normal human serum). A stable calibration curve with a wide dynamic range was established. The calibration curve was linear from 0.01 to 1000 ng/ml, thus, making quantitation possible. An immunologic prozone effect was observed above 1000 ng p53 antibodies per milliliter of serum. Serum samples from lung and nasopharyngeal carcinoma patients were tested using the IM-ECL assay. The positive rate of p53 antibodies were 28.6% in lung carcinoma and 8.33% in nasopharyngeal carcinoma, respectively. p53 antibody concentration in the carcerous human sera were quantified from the calibration curve. In the case of lung carcinoma, a trend was found that a higher p53 antibody concentration in the serum was likely linked to a higher stage of the cancer. This trend was not found in nasopharyngeal carcinoma. The assay uses only 50 microl of sample per test and requires a 30-min incubation period in addition to a 50 s acquisition time. This assay has several advantages over the commonly used ELISA method in terms of sensitivity, linear range, and assay time. Results of the study suggest that IM-ECL is a feasible method for rapid and sensitive detection of p53 antibodies in human serum.
机译：抑癌基因p53的突变在恶性肿瘤中很常见。 p53抗体是针对异常p53蛋白的免疫反应产物。已经发现p53抗体在肿瘤的诊断，预后和复发监测中是重要的。然而，目前用于检测p53抗体的方法，即酶联免疫吸附测定法（ELISA），需要很长的时间且需要多个步骤，并且该测定法只是半定量的。在这项工作中，制定了使用免疫磁电化学发光（IM-ECL）定量检测人血清中p53抗体的方案。免疫测定形式由三抗体夹心组成，其中生物素化的捕获抗体与商用p53蛋白结合在一起。加入检测抗体以在另一个位点结合p53蛋白。然后，加入用三联吡啶钌（II）标记的二抗，当与微珠免疫复合物结合时，在过量的三丙胺存在下发光。用我们制造的分析仪检测和测量光。我们的实验结果表明，该测定的灵敏度为每毫升参考血清（正常人血清）10 pg p53抗体。建立了宽动态范围的稳定校准曲线。校正曲线在0.01至1000 ng / ml之间呈线性，因此可以进行定量分析。每毫升血清1000 ng p53抗体以上可观察到免疫前​​带作用。肺和鼻咽癌患者的血清样本使用IM-ECL分析进行了测试。 p53抗体在肺癌中的阳性率为28.6％，在鼻咽癌中为8.33％。从校准曲线中量化了人类食道癌中p53抗体的浓度。在肺癌的情况下，发现趋势是血清中较高的p53抗体浓度可能与癌症的较高分期有关。在鼻咽癌中未发现这种趋势。该分析每次测试仅使用50微升样品，除了需要50 s的采集时间外，还需要30分钟的孵育时间。在灵敏度，线性范围和测定时间方面，该测定法与常用的ELISA方法相比具有多个优点。研究结果表明，IM-ECL是一种快速，灵敏地检测人血清中p53抗体的可行方法。

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