动物（兔、鼠）-人抗-弓形虫 IgM嵌合抗体作为特异IgM类抗体检测质控替代品的研究

The immunoglobulin M (IgM) antibody assay is common used immunoassays to detect Toxoplasma gondii infection. In order to ensure the accuracy of results, a positive control can be included in each run to identify the analytical problems. Traditionally, a positive control is a complex of seropositive plasma or serum with known quantities of anti-TOXO IgM and the negative control reagent. However, the use of plasma or serum as positive control has several significant drawbacks, including difficulty in economically viable large-scale production, high cost, and potential infection etc. Therefore, it's necessary to find out nonserological control as surrogate. The preliminary experiments found that rabbit-human anti-virus chimeric antibody could be used as control surrogate in IgM detection.This study will investigate the feasibility of replacing the conventional positive controls with animals (rabbit, mice )-human anti-TOXO IgM chimeric antibody conjugates constructed by technologies of chemical synthetic method , engineered method and bacterial periplasmic display. Then these three kinds of chimeric antibody conjugates would been compared with conventional positive controls. This study provides new methods can be also used to construct a series of positive controls that are applicable to all immunoassays.

目前特异IgM抗体检测是应用最广泛的弓形虫感染免疫血清学检测，在检测中，为保证检测结果准确可靠，需要阳性质控品进行室内质量控制。通常用于抗特定病原体IgM检测的质控品，为来自于病人或者献血员的含有已知量抗病原体 IgM的血清或血浆与正常血浆或血清的混合物，最大的问题是来源有限，价格昂贵及有潜在的传染危险性等。为此，需寻找非血清来源的质控品作为其替代品。我们前期的研究发现，兔-人抗病原体IgM嵌合抗体可作为抗病原体 IgM抗体检测的质控替代品。因此，本项目拟采用化学交联、基因工程和细菌展示库技术研究制备动物（兔、鼠）-人抗-弓形虫 IgM嵌合抗体，作为抗-弓形虫 IgM免疫测定的质控替代品，并与人源性质控品进行比较研究，以期为病原体特异IgM类抗体质控替代品的研究提供一个新的思路和方法。

本项研究采用动物免疫、亲和层析、化学交联、分子克隆、真核表达技术等方法构建兔-人嵌合型抗-弓形虫IgM抗体和基因重组鼠-人嵌合型抗-弓形虫 IgM抗体，经聚丙烯酰胺凝胶电泳分析鉴定及不同试剂厂家ELISA试剂盒检测，结果表明已成功构建此两种嵌合型抗体，并且嵌合型抗体的稳定性可满足质控品的要求。此外，本研究成功构建了重组兔-人嵌合型抗-TOXO IgM抗体真核表达载体库，为重组兔-人嵌合型抗-TOXO IgM抗体库的研究奠定了基础。本研究为动物（兔、鼠）-人抗-弓形虫 IgM化学交联和基因工程嵌合抗体作为特异抗-弓形虫IgM类抗体检测质控替代品的研究提供了一种新的思路，本研究建立的方法不仅可以基本解决抗弓形虫IgM检测质控品的制备问题，也为其他病原体特异性IgM、IgG质控替代品的研制提供了有价值的参考依据，从而为克服传统IgM抗体质控品有生物传染危险性、来源有限及获取病人阳性样本时可能遇到的伦理问题等缺点提供了有益的探索。