CN110389233A - 一种用于检测人amh的fn类抗体免疫检测试剂盒 - Google Patents
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Abstract
本发明涉及一种检测人AMH的FN类抗体免疫检测试剂盒。试剂盒包括缪勒氏管激素抗体包被的磁微粒、碱性磷酸酶标记的抗缪勒氏管激素抗体、标准品、校准品、磷酸盐溶液。本试剂盒可用以检测来自人生物学样品中的AMH水平，并可以用作诊断/预后指标。
Description
技术领域
本发明属于生物医药技术领域，涉及一种检测人抗缪勒氏管激素(AMH)的检测试剂盒，可以用作于控制性超***(COH)效果的预测、卵巢过度刺激(OHSS)的预测、***(PCOS)的诊断和预后、卵巢颗粒细胞瘤的诊断和管理、男性性早熟和***延迟的诊断和鉴别诊断、男童的隐睾症和无睾症的诊断和儿童双性状态的鉴别诊断。
背景技术
随着现代生活方式的改变，普遍的晚婚晚育现象、不良的现代生活方式(如过度节食减肥、吸烟饮酒、熬夜)、巨大的工作精神压力，随着国家生育政策的全面放开，我国大龄产妇人数大幅增长，难孕、***人群比重增加，越来越多的女性受到生育能力问题的困扰。目前临床上国内各诊疗单位评估卵巢功能主要包括年龄、窦卵泡计数(AFC)和基础性激素水平(FSH，FSH/LH、E2、抑制素B等)。但是这些指标都存在一定的局限性：AFC会受到B超操作者的主观因素以及不同的女性盆腔环境的影响，导致超声图像结果的偏差。基础性激素水平(包括FSH、FSH/LH比值、E2和抑制素B)的变化受到***的影响，且变化发生得较晚。相比较而言，近年来在欧美国家，临床上逐渐开始使用抗缪勒管激素(Anti-Mullerianhormone:AMH)作为卵巢功能评估的一项指标。
抗苗勒氏管激素简称AMH，是β-转化因子(TGF-β)家族的成员之一，由ProfessAlfred Jost于1947年首先发现，也被称为苗勒氏管抑制因子(MIF)、苗勒氏管抑制激素(MIH)以及苗勒氏管抑制物(MIS)。AMH蛋白由AMH基因编码，AMH基因是一种位于19号染色体短臂上2.4-2.8Kb的小基因，有5个外显子。AMH在分泌前，是由2个70kDa单体通过二硫键联接形成一个140kDa的二聚体。在穿过细胞膜的过程中，AMH前体一个特异位点被蛋白原转化酶剪切成N端前区和C端成熟区，这两部分仍然非共价结合在一起，C端为活性作用端。
纤维连接蛋白(Fibronectin，FN)，具有传统单克隆抗体类似的3个互补决定区(complementarity determining region,CDR)，即抗体和抗原的结合区，因此FN类抗体具有抗体类似的特异抗原结合性。但其分子量仅为10Kd，为传统单克隆抗体的15分之一，且无二硫键和糖基化修饰，具有更强的结构稳定性，且可用廉价的大肠杆菌表达***替代昂贵的动物细胞表达，大大降低了生产成本。此外由于不含有抗体的FC区，检测过程中不受补体干扰。FN类抗体在开发免疫检测试剂中具有巨大优势。
发明内容
本发明涉及一种检测人AMH的FN类抗体免疫检测试剂盒，可以用作于控制性超***(COH)效果的预测、卵巢过度刺激(OHSS)的预测、***(PCOS)的诊断和预后、卵巢颗粒细胞瘤的诊断和管理、男性性早熟和***延迟的诊断和鉴别诊断、男童的隐睾症和无睾症的诊断和儿童双性状态的鉴别诊断。
本发明提供了免疫化学发光法法检测生物样本中AMH的FN类抗体和重组人AMH蛋白，本发明的试剂盒还包括：缪勒氏管激素抗体包被的磁微粒、碱性磷酸酶标记的抗缪勒氏管激素抗体、标准品、校准品、磷酸盐溶液。进一步，本发明提供的抗人AMH的FN类抗体是采用大肠杆菌表达***获得。
本发明提供的上述试剂盒的开发过程如下：
(a)重组人AMH蛋白的制备
重组人AMH引物设计→重组人AMH菌株构建→大肠杆菌高表达表达体系建立→亲和离子层析→得到纯度＞95％重组人AMH蛋白
(b)重组人AMH检测试剂盒抗体的制备
mRNA体外展示技术建立抗人AMH抗体库→筛选高特异性、高亲和力抗体片段→构建表达FN候选抗体的菌株→FN抗体的亲合力、特异性、理化特性(熔点、聚合性)检测→大肠杆菌表达体系制备FN抗体的放大研究。
具体实施方式
实施例1：本发明的试剂盒可以是下列物质：
(1)缪勒氏管激素抗体包被的磁微粒，3.5mL/瓶，含防腐剂；
(2)抗缪勒氏管激素抗体碱性磷酸酶标记结合物，4.0mL/瓶，含防腐剂；
(3)校准品，1瓶；
(4)磷酸盐溶液，含蛋白稳定剂；
(5)含抗缪勒氏管激素蛋白、蛋白稳定剂、防腐剂的冻干品；
实施例2：样本处理
血清处理：室温血液自然凝固10分钟，2000转/分离心20分钟左右。仔细收集上清，保存过程中如出现沉淀，应再次离心。
尿液处理：用无菌管收集，3000转/分离心20分钟左右。仔细收集上清，保存过程中如有沉淀形成，应再次离心。
组织标本处理：切割标本后，称取重量。按重量体积比1:1加入pH7.4PBS，用液氮迅速冷冻保存备用。标本融化后仍然保持2-8℃的温度。加入同体积的PBS(pH7.4)，用手工或匀浆器将标本匀浆充分。3000转/分离心20分钟左右，仔细收集上清。分装后一份待检测，其余冷冻备用。标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围。
Claims (4)
1.一种检测人AMH的FN类抗体免疫检测试剂盒，其组成为：
抗缪勒氏管激素抗体包被的磁微粒、碱性磷酸酶标记的抗缪勒氏管激素抗体、标准品、校准品、磷酸盐溶液；
其中所述抗缪勒氏管激素抗体包被的磁微粒中的抗体形式为FN类抗体。
其中所述碱性磷酸酶标记的抗缪勒氏管激素抗体形式为FN类抗体。
2.根据权利1所述的检测试剂盒，其特征在于：
(1)AMH为抗缪勒氏管激素，是β-转化因子(TGF-β)家族的成员之一；
(2)FN类抗体为纤维连接蛋白(Fibronectin，FN) 。
3.根据权利要求2所述FN类抗体，其特征在于分子量仅为10Kd，为传统单克隆抗体的15分之一，且无二硫键和糖基化修饰。
4.权利要求1-3任一项中所述试剂盒、使用方法。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110027235A1 (en) * | 2009-04-09 | 2011-02-03 | Sangamo Biosciences, Inc. | Targeted integration into stem cells |
| CN103703370A (zh) * | 2011-05-27 | 2014-04-02 | 北欧生物科技公司 | 诊断性肽的检测 |
| CN105891490A (zh) * | 2016-04-05 | 2016-08-24 | 付国亮 | 用于定量检测抗缪勒氏管激素的试纸条及其制备方法和抗缪勒氏管激素浓度的测定方法 |
| CN106053791A (zh) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | 抗缪勒氏管激素化学发光免疫检测试剂盒及其制备方法和应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110027235A1 (en) * | 2009-04-09 | 2011-02-03 | Sangamo Biosciences, Inc. | Targeted integration into stem cells |
| CN103703370A (zh) * | 2011-05-27 | 2014-04-02 | 北欧生物科技公司 | 诊断性肽的检测 |
| CN105891490A (zh) * | 2016-04-05 | 2016-08-24 | 付国亮 | 用于定量检测抗缪勒氏管激素的试纸条及其制备方法和抗缪勒氏管激素浓度的测定方法 |
| CN106053791A (zh) * | 2016-06-30 | 2016-10-26 | 深圳市亚辉龙生物科技股份有限公司 | 抗缪勒氏管激素化学发光免疫检测试剂盒及其制备方法和应用 |
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