CN111458301B - 菜豆凝集素检测试剂盒 - Google Patents
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Abstract
本发明公开了一种菜豆凝集素检测试剂盒，所述的试剂盒含有菜豆凝集素卵黄抗体，该抗体可以特异性识别菜豆凝集素PHA‑E及PHA‑L两种亚基。本发明利用酶联免疫法定量检测菜豆凝集素的含量，克服了现有检测方法存在的菜豆凝集素的漏检、检测结果容易受凝血或抗凝血成分影响的不足。
Description
技术领域
本发明属于植物活性成分检测技术领域，具体涉及一种用于定量检测菜豆凝集素的检测试剂盒。
背景技术
菜豆又叫四季豆、扁豆、芸豆等，是我国主要蔬菜，多数地区春秋两季栽培。由于进食贮存过久或烹调不当的四季豆，如水焯后做凉菜，或炒食时不够熟透而未能充分灭活凝集素造成中毒，中毒事件多发生在集体食堂，据报道，菜豆中毒主要是由菜豆凝集素导致，菜豆凝集素是由4个分子量约30kD的亚基组成，分子量大小在128KD左右。菜豆凝集素亚基分别为红细胞凝集素(PHA-E)和白细胞凝集素(PHA-L)，红细胞凝集素有强红细胞凝集活性，白细胞凝集素具有强白细胞凝集活性。菜豆中毒最重要的因素是由于菜豆凝集素中的红细胞凝集素可与红细胞表面特异糖基识别并专一性结合，使红细胞发生凝集，白细胞凝集素也会特异性结合白细胞，使其发生凝聚，进而引起机体的整体反应。不同菜豆品种中，由于品种、产地、种植季节的不同，使不同菜豆中凝集素的含量不尽相同，因此快速检测菜豆凝集素的活性，对低毒品种菜豆的选育提供指导。
现有菜豆凝集素检测方法都是通过凝集素亚基中红细胞凝集素(PHA-E)的红细胞凝集实验来检测的。该方法一般利用兔红细胞，配制成一定浓度的红细胞悬液，将凝集素溶液经过稀释后加入红细胞悬液中，静置一段时间后，利用肉眼观察血细胞凝集效果的定性分析；或者以分光光度计及酶标仪测定的特定波长下吸光度的半定量分析方法。这些分析方法存在两个重要的问题：第一，菜豆凝集素是由PHA-E、PHA-L亚基形成的四聚体，主要有5种形式：L4、L3E1、L2E2、L1E3、E4，而红细胞聚集实验时，只能检测含PHA-E亚基的四聚体，L4不含可以聚集红细胞的PHA-E亚基，不参与血细胞凝集反应，另外，血细胞聚合只有两个或以上PHA-E亚基结合红细胞才能聚合，E1L3四聚体对血细胞凝集反应也不敏感，因此现有检测方法无法检测L4、E1L3四聚体，检测结果只反应E4、E3L1、E2L2三种四聚体含量，不能反应L4、E1L3两种四聚体真实含量，因此，并不能真实反应几种四聚体的真实含量；第二，红细胞凝聚检测方法中的红细胞每次需要新鲜取兔子血，血液在冰箱保存最多只能两周，因此要长期饲养兔子，而且每次使用前都需要把破裂的红细胞洗去，并在显微镜下调整细胞浓度，因此使用起来非常麻烦；第三，红细胞凝集实验是非特异性的凝血实验，因此，实验过程中还会受到其它凝血物质或抗凝血物质的干扰；第四，红细胞聚集实验肉眼定性实验中结果判断主观性强，分光光度计及酶标仪方法需要专业实验室和人员才能操作。
针对现有菜豆凝集素检测存在的缺陷，我们制备了针对菜豆凝集素的卵黄抗体，抗体可特异性识别菜豆凝集素PHA-E及PHA-L两种亚基，因此5种形式L4、L3E1、L2E2、L1E3、E4均可以被特异性识别。此外，检测过程不需要用到红细胞，试剂可长期保存，检测质量不会随红细胞质量改变或者其它而受到影响。
发明内容
为了解决上述技术问题，本发明提供了一种菜豆凝集素定量检测试剂盒，利用菜豆凝集素卵黄抗体特异性识别菜豆凝集素，特异性强，准确度高。
为了实现上述目的，本发明采用以下技术方案：
一种菜豆凝集素检测试剂盒，所述的试剂盒含有菜豆凝集素卵黄抗体和酶标记的兔抗鸡IgY，还包括酶标板、菜豆凝集素标准样品溶液、封闭液、洗涤液、底物显色液和终止液。
进一步地，所述的菜豆凝集素卵黄抗体的制备方法如下：
(1)菜豆凝集素标准品免疫鸡后，收集鸡蛋，去蛋清留蛋黄；
(2)用硫酸铵沉淀法粗提菜豆凝集素卵黄抗体；
(3)用离子交换柱层析法纯化卵黄抗体。
菜豆凝集素检测试剂盒的应用，其方法如下：
(1)取菜豆豆荚或豆籽，加入缓冲液室温研磨，离心取上清；
(2)在酶标板上设空白孔、标准品孔、待测样品孔，将菜豆凝集素标准品溶液和待测样品分别加于酶标板孔底部，37℃孵育2小时后，洗涤液充分洗涤；
(3)加入封闭液(含5％脱脂牛奶的磷酸盐缓冲液)，37℃孵育2小时后，洗涤液充分洗涤；
(4)加入菜豆凝集素卵黄抗体溶液，使其与菜豆凝集素结合，37℃的孵育1小时后充分洗涤；
(5)加入酶标记的兔抗鸡IgY，37℃孵育45分钟后充分洗涤；
(6)加入显色液，避光显色反应，再加入终止液终止显色反应；
(7)采用酶标仪于450nm波长处检测吸光值，根据标准品的浓度与OD450nm值计算出标准曲线的直线回归方程式，将样品的OD450nm值代入方程式，计算出样品浓度。
与现有技术相比，本发明具有以下优点及有益效果：
1、同时检测菜豆凝集素L4、L3E1、L2E2、L1E3、E4五种四聚体，不会造成漏检的情况。
2、凝集素抗体特异性识别凝集素，避免了由于检测环境中或检测条件及操作中存在的抗凝血或凝血剂的影响，特异性强，准确度高
3、检测过程不需要用到兔血细胞，因此避免了饲养兔子及每次检测需要采兔子新鲜血的麻烦。
附图说明
图1为菜豆凝集素卵黄抗体火箭电泳结果。孔1为磷酸缓冲液，孔2为菜豆粗提液，孔3为菜豆凝集素标准品，孔4为刀豆粗提液。
图2为菜豆凝集素标准曲线。
具体实施方式
实施例1
菜豆凝集素卵黄抗体IgY的制备方法，包括以下步骤：
1、注射样本的制备
取菜豆凝集素标准品200μg，用200μL灭菌的生理盐水溶解，加入等体积的弗氏完全佐剂FCA(第一针)或者弗氏不完全佐剂FICA(第二，三针)，然后用注射器双推法混匀佐剂与免疫抗原，使其充分混匀成油包水乳状物，用于免疫蛋鸡，注射时间分别为1、7、14天。
2、鸡蛋的收集
蛋鸡免疫后，40天后开始收集鸡蛋。
3、卵黄抗体的粗提取
收集的鸡蛋去蛋清，留蛋黄。按蛋黄重量用蒸馏水稀释9倍，辛酸调pH5.2-5.6，4℃过夜，9000r/min离心20min，上清过滤，以50％硫酸铵进行第一次沉淀，4℃过夜，9000r/min离心30min。去除上清，再次以50％硫酸铵进行第二次沉淀，4℃过夜，9000r/min离心30min，去除上清后得到白色沉淀即为粗提的IgY。
4、卵黄抗体的精制
离子交换柱层析法：先用3倍的Buffer1试剂平衡层析柱(DEAE-SFF)，(Buffer1溶液为：20mmol/L Tris，20mmol/LNaCl，总体积为150mL)，平衡时间为30分钟。粗提取的IgY用Buffer1溶液溶解，最终体积为80mL，进行IgY样品上柱，时间为20分钟。IgY样品的洗脱分为六个盐浓度阶梯：①20mmol/L Tris，100mmol/L NaCl；②20mmol/L Tris，200mmol/L NaCl；③20mmol/L Tris，400mmol/L NaCl；④20mmol/L Tris，600mmol/L NaCl；⑤20mmol/LTris，800mmol/L NaCl；⑥20mmol/L Tris，1000mmol/L NaCl，总体积为60mL，洗脱时间为30min。最后分别收集洗脱液，用终浓度为60％的硫酸铵进行沉淀，4℃冰箱过夜。第二天进行离心，9000r/min 30min，即可得到精纯化后的IgY。
5、抗体溶液的制备
纯化的IgY抗体用pH值为7.2的PBS溶液配置浓度为1mg/mL，并添加1％BSA、0.01％庆大霉素。
菜豆凝集素卵黄抗体IgY的特异性试验：
1、1.5g琼脂糖加50ml蒸馏水置水中煮沸溶解或用微波炉加热溶解，然后再加入50ml巴比妥缓冲液混匀，置4℃保存备用。
2、200μg菜豆凝集素卵黄抗体与6ml 56℃的1.5％巴比妥缓冲琼脂混合，浇制抗血清琼脂板。琼脂厚度为1～2mm，5×7.5cm大小玻板。
3、琼脂凝固后，于距玻板一长边10mm处，以3mm孔径打孔器打孔一排，孔距5mm，于各孔中加入抗原液(磷酸缓冲液作为对照，抗原液包括菜豆粗提液、菜豆凝集素标准品、刀豆粗提液)，每孔20μg。
4、将琼脂板置于电泳槽横梁上，加样端位于负极，以双层滤纸联接琼脂板与电泳槽内缓冲液(槽内缓冲液为0.06M pH8.6巴比妥缓冲液)。接通电源，板端电压3v/cm，电流强度3mA/cm，电泳5小时，氨基黑溶液染色1小时后，蒸馏水浸泡脱色2小时。
电泳结果如图1所示，菜豆凝集素卵黄抗体特异性识别菜豆凝集素。
实施例2
菜豆凝集素定量检测方法：
1、菜豆待测样本的预处理
取菜豆豆荚或豆籽1g，加入1mL 0.1M碳酸盐缓冲液室温研磨，12000×g离心10分钟，取上清，上清再次15000×g离心10分钟，取上清，4℃保存备用。
2、菜豆待测样品液孵育
菜豆待测样品利用0.05M碳酸盐缓冲液液(pH＝9.6)稀释10倍，菜豆凝集素标准品梯度稀释，浓度为0.2mg/mL、0.4mg/mL、0.6mg/mL、0.8mg/mL。96孔聚苯乙烯凹孔板每孔分别添加100μL菜豆碾磨液及标准品梯度稀释样品，37℃孵育2小时。
倒去96孔聚苯乙烯凹孔板中溶液，每孔中加入磷酸盐缓冲液300μL，浸泡5分钟后弃去缓冲液，重复3次。
3、封闭
每孔加入300μL含5％脱脂牛奶的磷酸盐缓冲液，37℃孵育2小时；倒出溶液，每孔加入磷酸盐缓冲液300μL，浸泡5分钟后弃去缓冲液，重复3次。
4、孵育第一抗体
将纯化好的菜豆凝集素卵黄抗体溶液利用磷酸盐缓冲液稀释至0.5mg/mL，每个孔内加入100μL稀释后的卵黄抗体溶液，于37℃的温箱孵育1小时，倾去孔内溶液，得到结合了抗原的第一抗体。用三乙醇胺吐温缓冲盐水溶液浸泡残余第一抗体1分钟，倾去三乙醇胺吐温缓冲盐水溶液。残留第一抗体洗脱步骤可重复5次，每次都把聚苯乙烯板中的水分拍干净。
5、孵育第二抗体
将辣根过氧化酶(HRP)标记的兔抗鸡IgY作为第二抗体，按照1：500的比例用磷酸盐缓冲溶液进行稀释，并向孔内加入100μL，于37℃的温箱孵育45分钟，倾去孔内溶液，得到了与第一抗体结合的第二抗体。每孔加入磷酸盐缓冲液300μL，浸泡5分钟后弃去缓冲液，重复6次。
6、显色
每孔加入TMB(3,3',5,5'-四甲基联苯胺)显色液50μL，在避光条件下，于37℃显色20分钟后，向每个孔中加入50μL 0.5M的硫酸用于终止显色反应。
7、测定菜豆凝集素含量
采用酶标仪于450nm波长处检测吸光值，根据标准品的浓度与与OD450nm值计算出标准曲线的直线回归方程式y＝0.905x+0.49(R2＝0.984)，将样品的OD450nm值代入方程式，计算出样品浓度，即为样品的实际浓度。
Claims (3)
1.一种菜豆凝集素检测试剂盒，其特征在于，所述的试剂盒含有菜豆凝集素卵黄抗体和酶标记的兔抗鸡IgY，所述的菜豆凝集素卵黄抗体的制备方法如下：
（1）菜豆凝集素标准品免疫鸡后，收集鸡蛋，去蛋清留蛋黄；
（2）用硫酸铵沉淀法粗提菜豆凝集素卵黄抗体；
（3）用离子交换柱层析法纯化卵黄抗体；
用于检测的菜豆凝集素为L4、L3E1、L2E2、L1E3、E4五种四聚体。
2.根据权利要求1所述的菜豆凝集素检测试剂盒，其特征在于，所述的试剂盒还包括酶标板、菜豆凝集素标准样品溶液、封闭液、洗涤液、底物显色液和终止液。
3.权利要求1或2所述的试剂盒在定量检测菜豆凝集素中的应用。
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