CN109541208A - 一种检测ca24-2的诊断试剂盒及其制备方法 - Google Patents
一种检测ca24-2的诊断试剂盒及其制备方法 Download PDFInfo
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Abstract
本发明的一种检测CA24‑2的诊断试剂盒采用双抗体夹心法的免疫吸附试验原理，结合时间分辨免疫荧光技术定量检测糖类抗原24‑2；可以用于消化道恶性肿瘤的辅助诊断和治疗效果的监测；同时本发明的试剂盒具有特异性强、灵敏度高、重复性好、稳定性优异、可测定范围宽、检测自动化程度高和无环境污染等优点。
Description
技术领域
本发明涉及肿瘤癌抗原免疫分析技术领域，尤其涉及一种检测糖类抗原24-2的诊断试剂盒及其制备方法。
背景技术
糖类抗原24-2(CA24-2)是一种唾液酸化的鞘糖脂类肿瘤相关抗原，CA24-2检测对于消化道恶性肿瘤有较高的诊断价值，特别是对胰腺癌、结直肠癌具有较高阳性率。CA24-2常用于治疗后的跟踪监控，当CA24-2持续升高时，提示肿瘤可能有残留或者转移。某些良性阻塞性黄疸以及肝实质性损害患者也会出现CA24-2的升高。
目前检测CA24-2的方法主要有：酶免ELISA法和化学发光CLIA法。酶免ELISA法的优点是操作简单、不需要复杂的仪器、只需要进行简单的培训即可进行操作，但是由于平台所限，其灵敏度相对较低，测定范围相对较窄，而且一般检测窗口期较长。化学发光CLIA法优点是灵敏度、特异性较高，检测范围较宽；缺点是由于大多数板式CLIA试剂盒仍采用EIA相同的酶促反应，以辣根酶或碱性磷酸酶交联抗体，使用鲁米诺或金刚烷等作为底物，检测的是动态发光，导致检测信号值衰减较快、高浓度和低浓度信号衰减速度不一致，限制了其在免疫定量分析中的低端检测灵敏度、稳定性和重复性。
发明内容
本发明为解决现有技术中的上述问题提出的一种兼具灵敏度高、特异性强、稳定性好、可测定范围宽、试剂有效期长、操作简单等优势的检测CA24-2的时间分辨免疫荧光诊断试剂盒及其制备方法。
为了实现本发明的上述目的，本发明所采取的技术方案为：
一种检测CA24-2的诊断试剂盒，包括包被反应板，还包括镧系元素离子标记的抗CA24-2单抗；所述包被反应板为吸附有抗CA24-2单抗的微孔板。
为了优化上述技术方案，本发明采取的技术措施还包括：
进一步地，本发明的检测CA24-2的诊断试剂盒还包括校准品、缓冲液、洗涤液、荧光增强液。
优选地，本发明中所述镧系元素离子为Eu3+、Tb3+、Sm3+、Dy3+中的至少一种，更优选为为Eu3+。
优选地，本发明使用的荧光增强液含有β-二酮类化合物或芳香胺类化合物。
另一方面，本发明还提供制备上述检测CA24-2的诊断试剂盒的方法，包括下述步骤：
步骤1：制备预处理包被反应板
使用黑色聚苯乙烯制备板架，预留与酶标板相互配合的通孔；酶标板经过钴60辐照30天后，与板架配合组装，获得预处理包被反应板，备用；
步骤2：包被板的获得
用碳酸盐缓冲液将抗CA24-2单抗稀释至1-4μg/mL，以100μL/孔包被过夜，然后洗涤、封闭、干燥，将微孔板真空密封于铝箔袋内，冷藏备用；
步骤3：制备镧系元素离子标记的抗CA24-2单抗
抗CA24-2单抗经碳酸盐缓冲液透析后，与镧系元素离子按2:1比例混合，静置24-48小时，用Sephadex S-200分离柱纯化，得到镧系元素离子标记的抗CA24-2单抗，用保存液将上述镧系元素离子标记的抗CA24-2单抗保存备用；
步骤4：荧光增强液的配制
以无水乙醇溶解β-NTA、TOPO，再加入令苯二甲酸氢钾和三蒸水，40℃溶解后，加入乙酸、Ttiton X-100，调PH至3.2，定容后保存备用；
步骤5：组装各个组分，获得检测CA24-2的诊断试剂盒。
优选地，上述镧系元素离子为Eu3+。
本发明采用上述技术方案，与现有技术相比，具有如下技术效果：
本试剂盒采用的是时间分辨免疫荧光分析法，应用镧系元素作为示踪剂，标记抗体，根据镧系元素螯合物的发光特点，用时间分辨技术测量荧光。检测CA24-2，有助于消化道恶性肿瘤的诊断，特别是对胰腺癌、结直肠癌具有较高阳性率。对恶性肿瘤患者进行动态监测以辅助判断疾病进程或治疗效果，还可以作为预后判断、评价抗病毒治疗效果的依据；利用时间分辨免疫荧光分析法同时检出波长和时间两个参数进行信号分辨，可有效地排除非特异性荧光的干扰，极大的提高了灵敏度；另外，本发明的包被反应板的为完全不透光的板架和相应配合的酶标板组成，使得阴性样本与Blank的荧光本底大大降低，信噪比显著提高，酶标板包被采用特殊辐照，使得包被效果显著提高，进而进一步地提高了试剂盒的灵敏度；所述本发明试剂盒具有特异性强、灵敏度高、重复性好、稳定性优异、可测定范围宽、检测自动化程度高和无环境污染等优点。
附图说明
图1为利用本发明的试剂盒进行CA24-2检测的检测原理示意图；
图2为本发明的试剂盒中包被反应板的板架示意图；
图3为发明的试剂盒中包被反应板的整体示意图。
具体实施方式
本发明提供了一种检测CA24-2的诊断试剂盒，包括包被反应板，还包括镧系元素离子标记的抗CA24-2单抗；所述包被反应板为吸附有抗CA24-2单抗的微孔板。
本发明的一种检测CA24-2的诊断试剂盒采用双抗体夹心法的免疫吸附试验原理，结合时间分辨免疫荧光技术定量检测糖类抗原24-2。测定程序如附图1所示，微孔表面预包被抗CA24-2单抗，配以镧系元素离子标记抗CA24-2单抗、增强液等其他试剂。当待检样本中存在糖类抗原24-2时，在微孔表面反应形成“固相抗CA24-2单抗-糖类抗原24-2-镧系元素离子标记的抗CA24-2单抗”的免疫复合物。然后加入增强液，将固相表面复合物上的镧系元素离子解离至溶液中。镧系元素离子与增强液中的组分形成强荧光络合物，其荧光强度与样本中的糖类抗原24-2的浓度成正相关。
下面通过具体实施例对本发明的试剂盒及其制备方法进行详细和具体的介绍，以使更好的理解本发明，但是下述实施例并不限制本发明范围。
实施例一 检测CA24-2的诊断试剂盒的制备
本实施例中，使用Eu3+作为荧光标记物，具体操作如下：
1.包被板的获得：
本发明的包被反应板和现有技术中的包被反应板均不同，在制备过程中使用特制模具，整体的板架采用完全不透光的黑色聚苯乙烯制作，底部设有和酶标板相应孔径的通孔(如图2所示)；酶标板则采用透光优秀的材料制备，在包被抗体前，经钴60辐照30天，获得高吸附性后，与板架组装，获得仅仅微孔底部透光的高吸附性预处理包被反应板备用，如附图3所示。
用碳酸盐缓冲液将抗CA24-2单抗稀释至1-4μg/mL，100μL/孔，包被过夜后经洗涤、封闭、干燥等程序，将微孔板条真空密封于铝箔袋内，冷藏备用。
2.铕标记物的获得：
铕标记物为铕标记抗CA24-2单抗。抗CA24-2单抗经碳酸盐缓冲液透析后，与铕按2:1比例混合，静置24-48小时，用Sephadex S-200分离柱纯化，得到铕标记抗CA24-2单抗。用保存液将铕标记抗CA24-2单抗进行稀释得到铕标记物。
3.校准品(A-F)的获得：用校准品稀释液将CA24-2重组抗原稀释到不同的浓度，经由上一级校准品赋值得到校准品(A-F)。
4.增强液的配制：以无水乙醇溶解β-NTA、TOPO，再加入令苯二甲酸氢钾和三蒸水，40℃溶解后，加入乙酸、Ttiton X-100，调PH至3.2，定容。
上述组分制备完毕后，加入缓冲液、洗涤液等其他试剂盒组分，制备得到检测CA24-2的时间分辨免疫荧光诊断试剂盒。
实施例二 利用检测试剂盒检测糖类抗原24-2
反应板每板设置校准品(A-F)各2孔，按顺序分别在相应孔中加入25μL校准品及待测样本，然后每孔加入50μL稀释液，室温(18℃～28℃)于振荡器慢档反应1小时；
用洗涤液清洗4遍，每孔加入100μL铕标记物工作液，室温(18℃～28℃)于振荡器慢档反应40分钟；
用洗涤液清洗6遍，每孔加入100μL增强液，室温(18℃～28℃)于振荡器慢档反应5分钟；
将反应板置于荧光检测仪选择相应程序读取数值。
实施例三 本发明的试剂盒与酶免诊断试剂盒的比较实验
利用本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒与普通酶免诊断试剂盒(康乃格)同时检测200份CA24-2样本，若样本初测结果不一致，则用两种检测试剂盒再检测一遍。
检测结果显示：有5份样本的检测结果不一致。以酶免ELISA法的检测结果为参考，则时间分辨免疫荧光法的灵敏度达到98％，特异性达到97％，符合率达到97.5％(表1)。
表1 两种检测方法检测HIV样本结果比较
灵敏度、特异性的比较
对上述5份初测结果不一致的样本用第三方试剂进行检测，结果显示这5份样本检测结果与时间分辨免疫荧光法结果一致。证明本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒在灵敏度和特异性方面比现行酶免ELISA试剂盒高。
时间分辨免疫荧光法的重复性分析
选取低、高2份不同浓度的CA24-2样品，用同一批本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒进行检测，每份样品分别平行检测10孔，进行试剂盒批内重复性分析；用3批不同批次的CA24-2诊断试剂盒进行检测，每份样品平行检测2孔，进行试剂盒批间重复性分析。
检测结果为：本发明的试剂盒的批内平均变异系数为3.14％(表3)；批间平均变异系数为5.69％(表4)，由此可以确定，本发明的试剂盒批内及批间重复性均较好。
表3 时间分辨免疫荧光法诊断试剂盒批内重复性分析
| 样品 | 浓度值均值 | 浓度值SD值 | CV(％) |
| 1 | 19.32 | 0.59 | 3.05％ |
| 2 | 95.48 | 3.09 | 3.24％ |
表4 时间分辨免疫荧光法诊断试剂盒批间重复性分析
| 样品 | 浓度值均值 | 浓度值SD值 | CV(％) |
| 1 | 20.87 | 1.14 | 5.46％ |
| 2 | 99.43 | 5.89 | 5.92％ |
时间分辨免疫荧光法的稳定性分析
将本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒于37℃环境中保存7天后取出，与4℃环境中保存的试剂盒同时检测低/中/高3份不同浓度的CA24-2样品，结果显示：37℃的检测结果与4℃的检测结果相比较没有明显的差异，偏差在10％以内(表5)，说明本发明的试剂盒至少在4℃能稳定保存12个月，其稳定性较好。
表5 时间分辨免疫荧光法诊断试剂盒稳定性分析
从上述对比可知，相较于现行的普通酶免ELISA法试剂盒，本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒的灵敏度更高、特异性更好；相较于现行的化学发光CLIA法，本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒的重复性、稳定性更好，所以本发明的检测CA24-2的时间分辨免疫荧光诊断试剂盒能更好地满足临床检测的要求。
综上所述，本试剂盒采用的是时间分辨免疫荧光分析法，应用镧系元素作为示踪剂，标记抗体，根据镧系元素螯合物的发光特点，用时间分辨技术测量荧光。检测糖类抗原24-2，有助于消化道恶性肿瘤的诊断，特别是对胰腺癌、结直肠癌具有较高阳性率。对恶性肿瘤患者进行动态监测以辅助判断疾病进程或治疗效果，还可以作为预后判断、评价抗病毒治疗效果的依据；利用时间分辨免疫荧光分析法同时检出波长和时间两个参数进行信号分辨，可有效地排除非特异性荧光的干扰，极大的提高了灵敏度；所述本发明试剂盒具有特异性强、灵敏度高、重复性好、稳定性优异、可测定范围宽、检测自动化程度高和无环境污染等优点。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
Claims (7)
1.一种检测CA24-2的诊断试剂盒，包括包被反应板，其特征在于，还包括镧系元素离子标记的抗CA24-2单抗；所述包被反应板为吸附有抗CA24-2单抗的微孔板。
2.根据权利要求1所述的试剂盒，其特征在于，还包括校准品、缓冲液、洗涤液、荧光增强液。
3.根据权利要求1所述的试剂盒，其特征在于，所述镧系元素离子为Eu3+、Tb3+、Sm3+、Dy3+中的至少一种。
4.根据权利要求3所述的试剂盒，其特征在于，所述镧系元素离子为Eu3+。
5.根据权利要求2所述的试剂盒，其特征在于，所述荧光增强液含有β-二酮类化合物或芳香胺类化合物。
6.一种制备如权利要求1所述的试剂盒的方法，其特征在于，包括下述步骤：
步骤1：制备预处理包被反应板
使用黑色聚苯乙烯制备板架，预留与酶标板相互配合的通孔；酶标板经过钴60辐照30天后，与板架配合组装，获得预处理包被反应板，备用；
步骤2：包被板的获得
用碳酸盐缓冲液将抗CA24-2单抗稀释至1-4μg/mL，以100μL/孔包被过夜，然后洗涤、封闭、干燥，将微孔板真空密封于铝箔袋内，冷藏备用；
步骤3：制备镧系元素离子标记的抗CA24-2单抗
抗CA24-2单抗经碳酸盐缓冲液透析后，与镧系元素离子按2:1比例混合，静置24-48小时，用Sephadex S-200分离柱纯化，得到镧系元素离子标记的抗CA24-2单抗，用保存液将上述镧系元素离子标记的抗CA24-2单抗保存备用；
步骤4：荧光增强液的配制
以无水乙醇溶解β-NTA、TOPO，再加入令苯二甲酸氢钾和三蒸水，40℃溶解后，加入乙酸、Ttiton X-100，调PH至3.2，定容后保存备用；
步骤5：组装各个组分，获得检测CA24-2的诊断试剂盒。
7.根据权利要求6所述的方法，其特征在于，所述镧系元素离子为Eu3+。
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