CN106093401B - 一种检测胸苷激酶1的诊断试剂盒 - Google Patents

一种检测胸苷激酶1的诊断试剂盒 Download PDF

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CN106093401B
CN106093401B CN201610611223.0A CN201610611223A CN106093401B CN 106093401 B CN106093401 B CN 106093401B CN 201610611223 A CN201610611223 A CN 201610611223A CN 106093401 B CN106093401 B CN 106093401B
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杨永芳
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Zhejiang Jukang Bioengineering Co Ltd
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Abstract

本发明涉及一种检测胸苷激酶1的诊断试剂盒，该试剂盒包含药剂和辅助剂，其中所述的药剂由包被缓冲液、稀释缓冲液、细胞核染色体和磷酸化胸苷激酶1抗体组成，所述的辅助剂是由正常人血清、过氧化脲、冰醋酸和生物防腐剂组成，所述的药剂和辅助剂的体积之比为3∶5‑8，其中包被缓冲液为pH为9.6时0.08M的碳酸盐缓冲液，稀释缓冲液为pH7.4时0.15M的(NH4)2SO4。对于预测乳腺癌具有重要的临床意义和社会经济效益。

Description

一种检测胸苷激酶1的诊断试剂盒
技术领域
本发明属于多肽化学和免疫学领域，具体涉及一种根据TK1方向写诊断试剂盒。
背景技术
TK1在早期乳腺癌患者的血清含量显著提高，而炎症与正常对照组没有明显差异。血清TK1含量的多寡被认为能够反应乳腺癌病理进程，因而他提出TK1可以做为一个乳腺癌早期诊断依据。
胸腺嘧啶核苷激酶是一种DNA合成的关键酶。TK1与细胞分裂密切相关，在细胞分裂G1期含量比较低，而到S期后逐渐升高，至G2期达到最高。因此编码TK1的mRNA及其表达的蛋白质也就成为细胞增生的标志物。
乳腺癌患者中血清中，胸腺嘧啶核苷激酶蛋白量及酶活性显著增强，并随着肿瘤恶化程度提高而进一步升高。因此胸腺激酶被认为是一新型的肿瘤诊断标志物，分析其蛋白以及活性的数据变化能够为乳腺癌早期诊断提供有力依据。正常人血清样本中检出TK1表达异常，提示存在乳腺癌潜在性；良性乳腺肿瘤患者若检出TK1表达异常，提示有恶变的可能；乳腺癌肿瘤TK1表达高低，能够显示出其恶化程度。
发明内容
为了解决上述技术问题，本发明提供了一种检测胸苷激酶1的诊断试剂盒，其特征在于，该试剂盒包含药剂和辅助剂，其中所述的药剂由包被缓冲液、稀释缓冲液、细胞核染色剂和磷酸化胸苷激酶1抗体组成，所述的辅助剂是由正常人血清、过氧化脲、冰醋酸和生物防腐剂组成，所述的药剂和辅助剂的体积之比为3∶5-8。
所述包被缓冲液优选为pH为9.6时0.08M的碳酸盐缓冲液，稀释缓冲液优选为pH7.4时0.15M的(NH4)2SO4。
所述生物防腐剂优选为50wt％ε-聚赖氨酸的酒精溶液。
所述细胞核染色剂优选为4′，6-二脒基-2-苯基吲哚和或溴化丙啶，进一步优选细胞核染色剂由重量比1∶1的4′，6-二脒基-2-苯基吲哚和溴化丙啶组成。
所述药剂中包被缓冲液、稀释缓冲液的体积比优选为1∶1，其中细胞核染色体占所述药剂总体积的20-30％。
所述的辅助剂中正常人血清与过氧化脲的体积比优选为5∶2-3，而冰醋酸和生物防腐剂之比为5g/ml。
所述生物防腐剂优选为50wt％ε-聚赖氨酸的酒精溶液。
有益效果
可根据对上述方案的叙述得知，乳腺癌风险诊断试剂盒利用特异和高度灵敏免疫检测法(ELISA)来定量检测人血清或血浆中的胸腺嘧啶核苷激酶的含量，进而用于预测乳腺癌的风险。对于病情的预测、预防具有重要的临床意义和社会经济效益。
用本发明制备的诊断试剂盒中的磷酸化胸苷激酶1抗体能够高度特异地与血液样本中的胸腺嘧啶核苷激酶结合，可以有效地监测胸腺嘧啶核苷激酶，极大地保证了检测结果的准确性、真实性和稳定性。
本发明的诊断试剂盒对于样品浓度为90-897ng/ml的胸腺嘧啶核苷激酶，回收率＞97％。对低浓度(200ng/ml)，中浓度(500ng/ml)和高浓度(780ng/ml)，其组内变异系数分别为3.7％，5.0％和4.1％(N＝30)，而组间的变异系数分别为4.9％，7.8％和7.6％(N＝10)；试剂盒真阳性率：95.8％；特异度(真阴性率)：99.1％左右；假阳性率：2.6％；假阴性率：16.2％。
具体实施方式
下面结合具体的实施例对本发明进行进一步的详细描述。
实施例中所使用的磷酸化胸苷激酶1抗体购自上海微蒙生物科技有限公司，其性质如下：
靶点：25；浓度为：1mg/ml；宿主为：兔子；保存温度：-20℃；亚型：IgG。
实施例1
一种检测胸苷激酶1的诊断试剂盒，该试剂盒包含药剂和辅助剂，其中所述的药剂由包被缓冲液、稀释缓冲液、4′，6-二脒基-2-苯基吲哚和磷酸化胸苷激酶1抗体组成，所述的辅助剂是由正常人血清、过氧化脲、冰醋酸和50wt％ε-聚赖氨酸的酒精溶液组成，所述的药剂和辅助剂的体积之比为3∶8，其中包被缓冲液为pH为9.6时0.08M的碳酸盐缓冲液，稀释缓冲液为pH7.4时0.15M的(NH4)2SO4。进一步的，其中药剂中包被缓冲液、稀释缓冲液的体积比为1∶1，其中4′，6-二脒基-2-苯基吲哚占所述药剂总体积的30％。所述的辅助剂中正常人血清与过氧化脲的体积比为5∶3，而冰醋酸和50wt％ε-聚赖氨酸的酒精溶液之比为5g/ml。
实施例2
一种检测胸苷激酶1的诊断试剂盒，该试剂盒包含药剂和辅助剂，其中所述的药剂由包被缓冲液、稀释缓冲液、溴化丙啶和磷酸化胸苷激酶1抗体组成，所述的辅助剂是由正常人血清、过氧化脲、冰醋酸和50wt％ε-聚赖氨酸的酒精溶液组成，所述的药剂和辅助剂的体积之比为3∶5，其中包被缓冲液为pH为9.6时0.08M的碳酸盐缓冲液，稀释缓冲液为pH7.4时0.15M的(NH4)2SO4。进一步的，其中药剂中包被缓冲液、稀释缓冲液的体积比为1∶1，其中溴化丙啶占所述药剂总体积的20％。所述的辅助剂中正常人血清与过氧化脲的体积比为5∶2，而冰醋酸和50wt％ε-聚赖氨酸的酒精溶液之比为5g/ml。
实施例3
一种检测胸苷激酶1的诊断试剂盒，该试剂盒包含药剂和辅助剂，其中所述的药剂由包被缓冲液、稀释缓冲液、4′，6-二脒基-2-苯基吲哚、溴化丙啶和磷酸化胸苷激酶1抗体组成，所述的辅助剂是由正常人血清、过氧化脲、冰醋酸和50wt％ε-聚赖氨酸的酒精溶液组成，所述的药剂和辅助剂的体积之比为3∶5，其中包被缓冲液为pH为9.6时0.08M的碳酸盐缓冲液，稀释缓冲液为pH7.4时0.15M的(NH4)2SO4，4′，6-二脒基-2-苯基吲哚呈微酸性与溴化丙啶的重量比为1∶1。进一步的，其中药剂中包被缓冲液、稀释缓冲液的体积比为1∶1，其中4′，6-二脒基-2-苯基吲哚和溴化丙啶占所述药剂总体积的20％。所述的辅助剂中正常人血清与过氧化脲的体积比为5∶2.5，而冰醋酸和50wt％ε-聚赖氨酸的酒精溶液之比为5g/ml。
本发明的具体实施方式仅为本创作的较佳实施例，并不用以限制本创作，凡在本创作的精神及原则之内所做的任何修改、等同替换、改进等，均应包含在本创作的保护范围之内。

Claims (1)

1.一种检测胸苷激酶1的诊断试剂盒，其特征在于，该试剂盒包含药剂和辅助剂，其中所述的药剂由包被缓冲液、稀释缓冲液、细胞核染色剂和磷酸化胸苷激酶1抗体组成，所述的辅助剂是由正常人血清、过氧化脲、冰醋酸和生物防腐剂组成，所述的药剂和辅助剂的体积之比为3∶5-8；所述细胞核染色剂由重量比1∶1的4′，6-二脒基-2-苯基吲哚和溴化丙啶组成；所述包被缓冲液为pH为9.6时0.08M的碳酸盐缓冲液，稀释缓冲液为pH7.4时0.15M的(NH4)2SO4；其中药剂中包被缓冲液、稀释缓冲液的体积比为1∶1，其中细胞核染色体占所述药剂总体积的20-30％， 所述的辅助剂中正常人血清与过氧化脲的体积比为5∶2-3，而冰醋酸和生物防腐剂之比为5g/ml；所述生物防腐剂为50wt％ε-聚赖氨酸的酒精溶液。
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