CN110672856A - 一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒 - Google Patents
一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒,属于免疫检测领域。本发明的基于多重微阵列的食物不耐受特异性IgG检测试剂盒包括:包被有食物抗原蛋白的微孔板、清洗缓冲液、封闭液、酶标抗体、人IgG标准品、显色液。本发明的检测试剂盒可以同时检测几十种食物过敏原,具有精确度高、灵敏度高的优点,同时也解决了单一指标的检测费事费力、成本高的问题,大大提高了检测效率,具有很好的临床应用价值。
Description
技术领域
本发明属于免疫检测领域,特别涉及一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒。
背景技术
变态反应性疾病是一种常见的疾病,它是由广泛存在的变应原引发的,大约有5-10%的人受变应原的侵扰。引发过敏反应的物质称为变应原,其中食物是重要的变应原之一。人体对食物敏感可引起食物变态反应,目前认为主要有两种类型:由IgE介导的速发型过敏反应,病发迅速,病症明显;由IgG介导的迟发型变态反应,反应通常在接触食物几小时或几天后才会出现反应症状,如消化道症状(腹痛、腹泻、口臭、口腔溃疡、胃肠胀气等肠胃功能失调)、皮肤症状(湿疹、荨麻疹)、呼吸症状(哮喘、慢性咳嗽、慢性鼻炎、鼻窦炎)、神经方面(头晕、头痛、偏头痛、睡眠障碍)、精神方面(焦虑、犹豫、注意力涣散、暴躁易怒、坐立不安)及其它慢性症状。
通常情况下,由食物引起的IgG介导的异常反应称之为食物不耐受,这是一种复杂的免疫反应性疾病。当肠胃分泌的消化酶不足,胃肠道动力不足时,食物无法被完全消化。一些食物被分解成多肽类的中分子,这些中分子容易被吸收进入血液循环。免疫系统将进入血液的某种或多种中分子物质当成有害物质,从而产生过度的保护性免疫反应,产生食物特异性IgG抗体,IgG抗体与食物颗粒形成免疫复合物(Ⅲ型变态反应)。这些IgG免疫复合物随着血液循环,在身体内不同部位沉积,进而引起一系列相关部位的病症。因此,通过检测血清中食物特异性IgG,对于确定不耐受食物与过敏性疾病轻重程度的诊断具有重要价值。
目前,临床上食物不耐受检测最成熟的是酶联免疫吸附试验(ELISA法),主要通过ELISA法定量检测食物特异性IgG抗体水平来反应食物不耐受状况,可针对性检测不同食物的IgG抗体浓度。但传统酶联免疫吸附法(ELISA)的局限是单个反应器中只能检测一种生物指标。对于多指标筛查,传统ELISA就需要进行多项独立检测,增加了时间成本和费用。而在食物不耐受检测领域中,筛查的食物种类多达几十种,甚至上百种。传统的ELISA很难降低食物不耐受筛查的时间和制造成本,不能满足现场快速检测和普及推广的要求。
多重微阵列是上世纪九十年代的前沿技术,是一种“微型化”技术。微阵列是一种装置,将大量样本(核酸或者蛋白质等生物分子)附着或固定于适合的未反应基底表面(如聚丙烯、聚苯乙烯、环烯等塑料、硅树脂和玻璃)。如果基底表面相对平直,生物分子可以被“印刷”到表面上,使用含有生物分子作为“测定点位”的缓冲液印刷。将固定于基底表面上的已知位置的生物分子暴露于生物化学/化学试剂,用于检测、定性和定量分析。
针对现有问题,目前急需开发一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒。
发明内容
本发明的首要目的提供一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒。
为达到上述目的,本发明所采取的技术方案是:
将微阵列和ELISA结合成为“微型化”ELISA技术,用于实施ELISA型免疫分析。固定于基底的生物分子为抗体(或抗原),基底表面被暴露于含有抗原(或抗体)的测试溶液。然后使用缓冲溶液布满基底表面并使其暴露于第二抗体,该第二抗体选择与可检测标签(如放射性标签或荧光染料)配对,选择与酶(该酶对反应进行催化,对该反应的反应产物进行染色且因此可被检测到)配对。
本发明第一个方面,提出了一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒。根据本发明的实施例,所述试剂盒包括:包被有食物抗原蛋白的微孔板、清洗缓冲液、封闭液、酶标抗体、人IgG标准品、显色液;所述食物抗原蛋白包括肉禽类、海鲜水产类、坚果类、蛋奶类、水果类、蔬菜类、豆类、谷物类、调味品及饮料类的一种或多种。根据本发明的实施例,试剂盒能够同时检测多种食物抗原蛋白,大大提高了检测效率。
根据本发明的实施例,所述包被有食物抗原蛋白的微孔板材质为聚苯乙烯,包含可拆卸式的96个微孔,所述每个微孔含有一个10×10的矩阵微阵列,包含食物抗原蛋白,6个校准点,2个阳性控制,2个阴性控制。
根据本发明的实施例,
所述肉禽类选自:羊肉、牛肉、猪肉、鸡肉、鸭肉的至少一种;
所述海鲜水产类选自:鳕鱼、墨鱼、龙虾、蟹肉、生蚝、鲤鱼、虾、扇贝的至少一种;
所述坚果类选自:板栗、芝麻、花生、榛子、杏仁、腰果、核桃的至少一种;
所述蛋奶类选自:蛋白、蛋黄、牛奶、羊奶、酸奶、酪蛋白的至少一种;
所述水果类选自:香蕉、芒果、桃子、奇异果、苹果、梨、樱桃、李子、葡萄、草莓、木瓜、柚子、橙子、柠檬、菠萝、哈密瓜、西瓜、甜瓜、椰子、橄榄的至少一种;
所述蔬菜类选自:西兰花、茄子、苦瓜、黄瓜、甜椒、芦笋、菠菜、西红柿、西葫芦、胡萝卜、土豆、大蒜、红薯、萝卜、芹菜、洋葱、芋头、南瓜、蘑菇、卷心菜、竹笋、茴香、薄荷、香菜、绿菜椒的至少一种;
所述豆类选自:扁豆、豌豆、腰豆、四季豆、黄豆、绿豆的至少一种;
所述谷物类选自:荞麦、麦麸、玉米、大米、燕麦、小麦的至少一种;
所述调味品及饮料类选自:蜂蜜、咖啡、可可、绿茶、红茶、酵母、胡椒的至少一种。
根据本发明的实施例,所述6个校准点的IgG浓度依次为:校准点1为62.5μg/mL、校准点2为15.6μg/mL、校准点3为31.3μg/mL、校准点4为62.5μg/mL、校准点5为125μg/mL、校准点6为125μg/mL;所述阳性控制点的IgG浓度为7.8μg/mL。
根据本发明的实施例,提出了所述包被有食物抗原蛋白的微孔板制备方法为:将食物粉碎、脱脂、提取蛋白,并SDS-PAGE电泳,洗脱干燥得食物抗原蛋白,溶解,冲匀,点样到微孔板的聚苯乙烯表面,点样的直径为150~200微米。
根据本发明的实施例,所述封闭液为1%牛血清白蛋白-PBS;所述显色液为1-StepTMB;所述清洗缓冲液为含0.05%吐温20的TBST溶液;酶标抗体为HRP抗人IgG、浓度为0.4~0.8mg/L。
本发明第二个方面,提出了一种如前面所述食物不耐受特异性IgG检测试剂盒的检测方法,包括以下步骤:
1)量取血清稀释于封闭液;
2)将稀释的血清加入至前面所述试剂盒中包被有食物抗原蛋白微孔板的微孔内孵育,静置;
3)弃去反应液体;
4)洗涤;
5)加入封闭液稀释的酶标抗体,孵育,静置;
6)弃去孔内反应液,洗涤;
7)加入TMB显色液后,避光孵育,静置;
8)分析仪读数判定。
根据本发明的实施例,步骤(2)22~25℃孵育1小时;步骤(5)孵育30~60分钟;步骤(7)孵育15~20分钟。
根据本发明的实施例,所述分析仪读数判定标准为:浓度小于10U/mL为阴性(0级),浓度10-30U/mL为阳性(1级),浓度30-60U/mL为阳性(2级),浓度大于60U/ml为阳性(3级)。
本发明的有益效果是:
1、本发明的食物抗原蛋白是直接打印在聚苯乙烯材质的表面,也可以用芯片取代,芯片可以选用玻璃载体,硅晶片载体和膜载体等材料。
2、本发明将微阵列的“微型化”技术和ELISA联合使用,通过10×10的微阵列矩阵可以同时处理几十种食物过敏原的检测,提高了精确度。
3、微型化试剂盒的灵敏度更高,检测用的样品量比常规ELISA试剂盒少,减少了取样量。
4、本发明采取可拆卸式条形96孔板设计,可根据实际样品数量,灵活拆取所需要的测试孔数量,不用整板使用。而且与常规ELISA试剂盒相比,最多可同时检测96份样本;同时也解决了单一指标的检测费事费力、成本高的问题,大大提高了检测效率,具有很好的临床应用价值。
附图说明
图1是10×10微阵列分布图。
具体实施方式
下面结合实施例对本发明中的技术方案进行清楚、完整的说明,但并不局限于此。
实施例1多重微阵列食物不耐受特异性IgG检测试剂盒
1.多重微阵列食物不耐受特异性IgG检测试剂盒
多重微阵列的食物不耐受特异性IgG检测试剂盒,包括:包被有食物抗原蛋白的微孔板、清洗缓冲液、封闭液、酶标抗体、人IgG标准品、显色液。
包被有食物抗原蛋白的微孔板材质为聚苯乙烯,包含可拆卸式的96个微孔。每个微孔含有一个10×10的矩阵微阵列。微阵列包含90项食物抗原,6个校准点,2个阳性控制,2个阴性控制,如图1。
90项食物抗原涉及到肉禽类、海鲜水产类、坚果类、蛋奶类、水果类、蔬菜类、豆类、谷物类、调味品及饮料类食物。
肉禽类:羊肉、牛肉、猪肉、鸡肉、鸭肉;
海鲜水产类:鳕鱼、墨鱼、龙虾、蟹肉、生蚝、鲤鱼、虾、扇贝;
坚果类:板栗、芝麻、花生、榛子、杏仁、腰果、核桃;
蛋奶类:蛋白、蛋黄、牛奶、羊奶、酸奶、酪蛋白;
水果类:香蕉、芒果、桃子、奇异果、苹果、梨、樱桃、李子、葡萄、草莓、木瓜、柚子、橙子、柠檬、菠萝、哈密瓜、西瓜、甜瓜、椰子、橄榄;
蔬菜类:西兰花、茄子、苦瓜、黄瓜、甜椒、芦笋、菠菜、西红柿、西葫芦、胡萝卜、土豆、大蒜、红薯、萝卜、芹菜、洋葱、芋头、南瓜、蘑菇、卷心菜、竹笋、茴香、薄荷、香菜、绿菜椒;
豆类:扁豆、豌豆、腰豆、四季豆、黄豆、绿豆;
谷物类:荞麦、麦麸、玉米、大米、燕麦、小麦;
调味品及饮料类:蜂蜜、咖啡、可可、绿茶、红茶、酵母、胡椒。
具体可以参考表1中的食物。
表1 10×10微阵列分布图
A10 | 校准点1 | 鳕鱼 | 墨鱼 | 龙虾 | 蟹肉 | 生蚝 | 鲤鱼 | 虾 | 扇贝 | 校准点5 |
A9 | 羊肉 | 香蕉 | 芒果 | 桃子 | 奇异果 | 苹果 | 梨 | 樱桃 | 李子 | 蛋白 |
A8 | 牛肉 | 葡萄 | 草莓 | 木瓜 | 柚子 | 橙子 | 柠檬 | 菠萝 | 哈密瓜 | 蛋黄 |
A7 | 猪肉 | 西兰花 | 茄子 | 西瓜 | 甜瓜 | 苦瓜 | 阳性控制 | 橄榄 | 茴香 | 牛奶 |
A6 | 鸡肉 | 黄瓜 | 甜椒 | 芦笋 | 菠菜 | 西红柿 | 西葫芦 | 胡萝卜 | 土豆 | 羊奶 |
A5 | 鸭肉 | 大蒜 | 红薯 | 萝卜 | 芹菜 | 洋葱 | 芋头 | 南瓜 | 蘑菇 | 酸奶 |
A4 | 校准点2 | 卷心菜 | 竹笋 | 扁豆 | 豌豆 | 腰豆 | 四季豆 | 黄豆 | 绿豆 | 酪蛋白 |
A3 | 阴性控制 | 薄荷 | 香菜 | 胡椒 | 绿菜椒 | 蜂蜜 | 咖啡 | 可可 | 绿茶 | 红茶 |
A2 | 校准点3 | 酵母 | 荞麦 | 麦麸 | 玉米 | 大米 | 燕麦 | 小麦 | 阴性控制 | 阳性控制 |
A1 | 校准点4 | 板栗 | 芝麻 | 花生 | 榛子 | 椰子 | 杏仁 | 腰果 | 核桃 | 校准点6 |
A | B | C | D | E | F | G | H | I | J |
其中,表1中的校准点1:IgG 62.5μg/mL;校准点2:IgG 15.6μg/mL;校准点3:IgG31.3μg/mL;校准点4:IgG 62.5μg/mL;校准点5:IgG 125μg/mL;校准点6:IgG 125μg/mL;阳性控制:IgG 7.8μg/mL。
2.食物抗原蛋白的制备
将特定食物样品在4℃条件下研磨粉碎,使用缓冲液和试剂(如0.55%NaHCO3、1%NaCl,0.1M磷酸盐缓冲液pH 7.4,70%乙醇以及冷丙酮)进行提取;
将食物分别混合于上述四种溶液中,并在室温下搅拌2小时,2000g离心15分钟后,分别取每种溶液的沉淀,并使用截断值为6,000的透析袋针对0.01M PBS进行透析。将冷丙酮沉淀溶于0.1M PBS缓冲液中并进行透析,重复三次,以确保除去所有小分子。透析后,将透析得到的抗原合并,得到食物抗原蛋白。每种食物的抗原蛋白处理方法如上。使用Bio-Rad(Hercules,CA)试剂盒测量蛋白质浓度。为了对食物中分离抗原进行质量控制,使用SDS丙烯酰胺凝胶(梯度为7~15%)和Western blot验证。
3.包被有食物抗原蛋白的微孔板
通过NanoprintTM2 LM60PRO Microarray,将食物抗原蛋白溶于pH为7.4的磷酸盐缓冲液中,使终浓度为100μg/mL,冲匀,点样到微孔板的聚苯乙烯表面,点样的直径为150~200微米。
实施例2多重微阵列食物不耐受特异性IgG检测试剂盒的检测步骤
分析试剂和仪器:
清洗缓冲液:含0.1%吐温20的TBST溶液(1×),调pH为7.4;
封闭液:1%牛血清白蛋白(V)-PBS,pH 7.4;
酶标抗体:HRP抗人IgG(Fc-特异性)购自Southern Biotech公司,货号6140-05;用含1%小牛血清的pH 7.40、1M PBS稀释,稀释比例为1:1000;浓度为0.5mg/L;
人IgG标准品:将IgG(Abcam,ab97225)使用含1%小牛血清的pH 7.4、1M PBS配制成五个不同浓度作为标准品,标准品浓度分别是0U/mL、50U/mL、100U/mL、200U/mL、400U/mL,标准品经国际质控血清校准;
显色液:含0.48g/L TMB的柠檬酸缓冲液,(Thermo Scientific,货号是37574);
分析仪:基于比色的CCD或CMOS分析仪。
分析样品:血浆或血清;
分析的生物指标:血液中的总IgG(IgG1,IgG2,IgG3,IgG4)。
实验前,将所有试剂平衡至室温。
检测方法:
(1)稀释样品,量取1μl血清以1:100(v/v)的比例加入封闭液稀释,轻柔混合,避免产生泡沫;
(2)取100μl稀释的血清样品加入微孔板的微孔内,22~25℃孵育1小时,静置;
(3)弃去孔内反应液体;
(4)使用100μl清洗缓冲液(1×)洗涤微孔,重复5次,吸干残存液体;
(5)每个孔加入100μl封闭液稀释的酶标抗体,孵育30~60分钟,静置;
(6)弃去孔内反应液,洗涤重复步骤5;
(7)加入TMB显色液100μl/孔,室温、避光条件下孵育15分钟,静置;
(8)分析仪读数判定。
分析仪读数判定标准为:浓度小于10U/mL为阴性0级,浓度10-30U/mL为阳性1级,浓度30-60U/mL为阳性2级,浓度大于60U/ml为阳性3级。
实施例3多重微阵列食物不耐受特异性IgG检测试剂盒性能检测
1.精密度测定
以小麦,牛奶为例,从临床样品中分别选取阴性血清、弱阳性血清和中等阳性血清,分别进行10次批内和批间测定,结果如表2(小麦)和表3(牛奶)所示。
表2小麦特异性IgG精密度测定
表3牛奶特异性IgG精密度测定
从表2和表3中结果可知,本发明的试剂盒具有良好的精密度,符合达到免疫诊断试剂的基本要求,能够满足中国临床检测的技术需要。
2.与参比试剂对比结果
以中国江苏浩欧博生物医药股份有限公司的食物特异性IgG抗体检测试剂盒(14项)作为参比试剂,与本发明的试剂盒分别测定小麦特异性IgG和牛奶特异性IgG。
结果如表4、表5所示,小麦特异性IgG测定结果:敏感度为87.5%(35/40),特异度为91.4%(32/35),准确度为89.3%(67/75);阳性预测值为92.1%(35/38),阴性预测值为86.5%(32/37)。
同时,牛奶特异性IgG测定结果:敏感度为92.5%(37/40),特异度为95%(38/40),准确度为93.8%(75/80);阳性预测值为94.9%(37/39),阴性预测值为92.7%(38/41)。
表4本发明试剂盒与参比试剂对比结果(小麦)
表5本发明试剂盒与参比试剂对比结果(牛奶)
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
1.一种基于多重微阵列的食物不耐受特异性IgG检测试剂盒,其特征在于,包括:包被有食物抗原蛋白的微孔板、清洗缓冲液、封闭液、酶标抗体、人IgG标准品、显色液;所述食物抗原蛋白包括肉禽类、海鲜水产类、坚果类、蛋奶类、水果类、蔬菜类、豆类、谷物类、调味品及饮料类的一种或多种。
2.根据权利要求1所述的试剂盒,其特征在于,所述包被有食物抗原蛋白的微孔板材质为聚苯乙烯,包含可拆卸式的96个微孔;所述微孔各含有一个10×10的矩阵微阵列;所述矩阵微阵列包括食物抗原蛋白,6个校准点,2个阳性控制,2个阴性控制。
3.根据权利要求1所述的试剂盒,其特征在于,
所述肉禽类选自:羊肉、牛肉、猪肉、鸡肉、鸭肉的至少一种;
所述海鲜水产类选自:鳕鱼、墨鱼、龙虾、蟹肉、生蚝、鲤鱼、虾、扇贝的至少一种;
所述坚果类选自:板栗、芝麻、花生、榛子、杏仁、腰果、核桃的至少一种;
所述蛋奶类选自:蛋白、蛋黄、牛奶、羊奶、酸奶、酪蛋白的至少一种;
所述水果类选自:香蕉、芒果、桃子、奇异果、苹果、梨、樱桃、李子、葡萄、草莓、木瓜、柚子、橙子、柠檬、菠萝、哈密瓜、西瓜、甜瓜、椰子、橄榄的至少一种;
所述蔬菜类选自:西兰花、茄子、苦瓜、黄瓜、甜椒、芦笋、菠菜、西红柿、西葫芦、胡萝卜、土豆、大蒜、红薯、萝卜、芹菜、洋葱、芋头、南瓜、蘑菇、卷心菜、竹笋、茴香、薄荷、香菜、绿菜椒的至少一种;
所述豆类选自:扁豆、豌豆、腰豆、四季豆、黄豆、绿豆的至少一种;
所述谷物类选自:荞麦、麦麸、玉米、大米、燕麦、小麦的至少一种;
所述调味品及饮料类选自:蜂蜜、咖啡、可可、绿茶、红茶、酵母、胡椒的至少一种。
4.根据权利要求2所述的试剂盒,其特征在于,所述6个校准点的IgG浓度依次为:校准点1为62.5μg/mL、校准点2为15.6μg/mL、校准点3为31.3μg/mL、校准点4为62.5μg/mL、校准点5为125μg/mL、校准点6为125μg/mL;所述阳性控制点的IgG浓度为7.8μg/mL。
5.根据权利要求1或2所述的试剂盒,其特征在于,所述包被有食物抗原蛋白的微孔板制备方法为:将食物粉碎、脱脂、提取蛋白,并SDS-PAGE电泳,洗脱干燥得食物抗原蛋白,溶解,冲匀,点样到微孔板的聚苯乙烯表面,点样的直径为150~200微米。
6.根据权利要求1所述的试剂盒,其特征在于,所述封闭液为1%牛血清白蛋白-PBS;所述显色液为1-Step TMB;所述清洗缓冲液为含0.05%吐温20的TBST溶液;所述酶标抗体为HRP抗人IgG,浓度为0.4~0.8mg/L。
7.一种如权利要求1所述试剂盒的检测方法,其特征在于,包括以下步骤:
1)量取血清稀释于封闭液;
2)将稀释的血清加入至权利要求1试剂盒中包被有食物抗原蛋白微孔板的微孔内孵育,静置;
3)弃去反应液体;
4)洗涤;
5)加入封闭液稀释的酶标抗体,孵育,静置;
6)弃去孔内反应液,洗涤;
7)加入TMB显色液后,避光孵育,静置;
8)分析仪读数判定。
8.根据权利要求7所述的检测方法,其特征在于,步骤(2)22~25℃孵育1小时;步骤(5)孵育30~60分钟;步骤(7)孵育15~20分钟。
9.根据权利要求7所述的检测方法,其特征在于,所述分析仪读数判定标准为:浓度小于10U/mL为阴性或0级,浓度10-30U/mL为阳性或1级,浓度30-60U/mL为阳性或2级,浓度大于60U/ml为阳性或3级。
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