CN110940810A - 一种检测沙门氏菌毒素的塑封胶体金检测卡 - Google Patents
一种检测沙门氏菌毒素的塑封胶体金检测卡 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种检测沙门氏菌毒素的塑封胶体金检测卡及其制备方法。所述检测卡包括试纸条,所述试纸条包括背板、样品垫、胶体金垫、硝酸纤维素膜和吸水垫,所述样品垫、胶体金垫、硝酸纤维素膜和吸水垫在背板上按顺序依次固定,所述胶体金垫内含有胶体金标记抗沙门氏菌毒素抗原的单克隆抗体;所述硝酸纤维素膜上设有检测线和质控线。通过本工艺的实施,开发出沙门氏菌毒素快速免疫检测卡的快速生产制备技术。
Description
技术领域
本发明属于生物技术领域,具体涉及一种检测沙门氏菌毒素的塑封胶体金检测卡及其制备方法。
背景技术
根据WHO估计,发达国家食源性疾病的漏报率在90%以上,而发展中国家则为95%以上。以此推论,我国目前掌握的食物中毒数据仅为我国实际发生的食源性疾病的“冰山一角”。而如此高的漏报率,除管理上的问题外,致病微生物的检测和溯源手段的限制也是一个重要因素。当前细菌性食物中毒主要有感染型中毒和毒素型中毒两种。沙门氏菌及其毒素是一种常见的食源性致病原。
目前我国食品安全研究队伍、设备和经费都十分缺乏,与国外相比,仍具有较大差距,主要表现在检测项目少、质量不稳定,不能满足食品检测工作的要求。严峻的现实向微生物检测提出了更高的要求―更准确、更快速地检出与监测病原菌。
传统上我国食源性疾病的病原检测、鉴定手段仍停留在传统的病原菌培养,此检验法的最大弱点是慢,难以适应诊断与治疗,主要包括两种方法:一是PCR分子鉴定技术检测病原菌,虽然灵敏度高、快速,可直接检测,但对实验室和试剂的要求比较高,否则结果十分不可靠;二是酶联免疫法(ELISA)检测虽是一项比较成熟的免疫分析技术,但该法操作比较繁琐,检测时间长,并需在实验室进行,不能满足现场快速检测的需求。因此,目前病原微生物检测方法的落后性在相当大的程度上限制了对引起食源性疾病的病原菌的溯源及鉴定能力。而当发生食物中毒事件时,由于中毒潜伏期短,来势急剧,短时间内可能有多数人发病,发病曲线呈突然上升的趋势,只有在最短时间内掌握食物中毒发生情况,确定中毒原因,才能控制食物中毒的蔓延和事态的扩大,保障人民身体健康。因此,开发应用于现场的快速、灵敏的食品安全检测技术平台是今后食品安全检测的趋势。
发明内容
本发明提供了一种检测沙门氏菌毒素的塑封胶体金检测卡及其制备方法,通过本工艺的实施,开发出沙门氏菌毒素快速免疫检测卡的快速生产制备技术。
本发明通过以下技术方案来实现:
一种检测沙门氏菌毒素的塑封胶体金检测卡,所述检测卡包括试纸条,所述试纸条包括背板、样品垫、胶体金垫、硝酸纤维素膜和吸水垫,所述样品垫、胶体金垫、硝酸纤维素膜和吸水垫在背板上按顺序依次固定,所述胶体金垫内含有胶体金标记抗沙门氏菌毒素抗原的单克隆抗体;所述硝酸纤维素膜上设有检测线和质控线。
进一步的,所述试纸条通过冷裱塑封。
进一步的,所述检测卡上设置有样本孔,所述样本孔位于样品垫正上方。
一种检测沙门氏菌毒素的塑封胶体金检测卡制备方法,包括以下步骤:
沙门氏菌毒素抗原提纯;
利用提纯后的抗原进行沙门氏菌毒素的单克隆抗体制备及筛选;
胶体金标记抗体制备;
组装检测卡。
进一步的,所述胶体金标记抗体制备方法包括:
将抗体置于透析袋中,透析掉多余的盐离子透析掉;从透析袋中取出抗体,离心后除去多余的抗体聚合物,取上清液和胶体金连接进行标记;
加入碳酸钾溶液调节胶体金溶液的pH值,将抗体溶液逐滴地加入到胶体金溶液中,快速混匀,将混合后的溶液静置;
加入BSA溶液和PEG20000溶液,以封闭金颗粒的表面的残留表位使颗粒聚集,继续静置;
离心后吸取上清液,移入新的离心管中,除去标记过程中未连接的金粒子;
将吸取的上清液离心,弃去上清,除去未标记成功的抗体及胶体金粒子,得到胶体金标记抗体。
进一步的,胶体金标记抗体的质量鉴定方法为:将羊抗鼠二抗用PB稀释后在NC膜上点样,包被完全后,再将稀释过的金标抗体溶液滴在二抗位置上,直接与二抗反应作用,观察结果。
进一步的,组装检测卡的步骤包括:
将硝酸纤维素膜贴到背板上,在硝酸纤维素膜上用划膜仪以包被羊抗鼠二抗和靶标捕获抗体作为C线和T线,胶体金垫上喷涂稀释好的金标抗体,干燥后贴在背板上,然后依次贴上样品垫和吸水纸,使用切条机将组装好的试纸条切条,进一步采用压塑工艺制成产品。
进一步的,沙门氏菌毒素抗原提纯的方法包括:沙门氏菌毒素培养液裂解并除盐后进行弱阴离子交换层析。
进一步的,沙门氏菌毒素的单克隆抗体制备方法包括:取制备所得沙门氏菌毒素抗原与弗氏完全佐剂等体积混合均匀,取混合液皮下注射小鼠;之后用弗氏不完全佐剂与贝类毒素-BSA混匀重复免疫;加强免疫后对小鼠眼眶取血,用酶联免疫吸附测定间接法测定其效价;将免疫小鼠脾细胞与骨髓瘤细胞混合,用PEG快速融合,加入含有次黄嘌呤、氨基喋呤和胸腺嘧啶核苷的培养基进行培养和筛选。
进一步的,沙门氏菌毒素的单克隆抗体制备方法包括:用降植烷进行小鼠腹腔注射,将杂交瘤细胞接种到小鼠腹腔中去;采集小鼠腹水,离心收集上清,再过玻璃纤维柱,收集到澄清的腹水;经过辛酸-硫酸铵沉淀法粗提纯小鼠腹水,用葡萄球菌A蛋白与葡聚糖交联,制备亲和层析柱将粗提抗体上样结合后洗脱后得到沙门氏菌毒素的单克隆抗体。
有益效果:
通过本工艺的实施,开发出沙门氏菌毒素快速免疫检测卡的快速生产制备技术,推进本领域的技术进步。
通过项目产品的推广,方便检测卡的保存,延长了检测卡的保质期。
项目实施后将提供一个新产品系列,预计扩大销售额100万元以上。
附图说明
图1为检测卡示意图:1、样品垫;2、胶体金垫;3、检测线;4、质控线;5、吸水垫。
图2为免疫胶体金试纸条结构示意图;
图3为制作的塑封检测卡。采用多联试纸条塑封,撕开后可开始检测;
图4为检测结果判定图示。
具体实施方式
实施例1
1)沙门氏菌毒素抗原提纯
(1)沙门氏菌毒素培养液裂解及除盐。
(2)弱阴离子交换层析:start buffer:20mmol/L PBS,HCl调pH值至6.0。elutionbuffer:20mmol/L PBS,1mol/L NaCl,HCl调pH值至6.0。平衡:将Hiprep.16/10 DEAE FF弱阴离子交换层析柱接入AKTA Explorer,以elution buffer冲洗100ml,再以start buffer平衡100ml。流速5ml/min。UV 280nm、254nm、215nm同时监测,至3条曲线呈水平。上样:将除盐产物注入50ml上样环,上样流速5ml/min。平衡:待流出峰流尽后,以start buffer平衡至监测曲线呈水平。洗脱:采用盐度梯度洗脱,洗脱液为elution buffer。盐梯度为使NaCl的浓度由0mol/L升到1mol/L,加入的elution buffer的量在200ml以内,流速为5ml/min。收集:用收集管收集洗脱下的蛋白峰,2ml/管,冻干后4℃保存备用。
2)沙门氏菌毒素的单克隆抗体制备及筛选
取制备所得TD抗原与弗氏完全佐剂等体积混合均匀。取0.3mL混合液皮下注射7周龄的BALB/c小鼠。之后每隔2周用弗氏不完全佐剂与贝类毒素-BSA混匀重复免疫。加强免疫7天后眼眶取血,用酶联免疫吸附测定间接法(ELISA,Enzyme-Linked ImmunosorbnentAssay)测定其效价。将免疫小鼠脾细胞与骨髓瘤细胞SP2/0按5:1~10:1的比例混合,用PEG快速融合,加入含有次黄嘌呤(hypoxanthine,H)、氨基喋呤(aminopterin,A)和胸腺嘧啶核苷(thymidine,T)的培养基(HAT)进行细胞筛选并铺于有准备好的饲养细胞的96孔细胞培养板,置37℃,6%CO2培养箱内培养。
用含20%血清的HAT培养基稀释筛选出来的杂交瘤细胞至每毫升含2.5、15、和50个细胞三种不同的稀释度,分别分装到96孔板,每个稀释度32孔,每孔0.1mL,然后在每孔中加入0.1mL的饲养细胞,置37℃,5%CO2培养箱内培养。3次克隆化后的结果均为阳性的为能稳定分泌抗贝类毒素抗体的杂交瘤细胞,采用小鼠腹腔接种法生产单克隆抗体腹水。选用标准BALB/c小鼠,先用降植烷进行小鼠腹腔注射,一周后每只老鼠按照5X106杂交瘤细胞接种到小鼠腹腔中去。1周后采集腹水,可以连续采集2-3次。将腹水置于37℃静置2小时后,13000rpm离心10min,除去细胞成分和其他的沉淀物,收集上清,再过玻璃纤维柱,收集到澄清的腹水。经过辛酸-硫酸铵沉淀法粗提纯小鼠腹水,可除去白蛋白等绝大部分杂蛋白,然后用葡萄球菌A蛋白与萄聚糖(Sepharose CL-4B)交联,制备亲和层析柱将粗提抗体上样结合后洗脱。葡萄球菌A蛋白可与IgG1、IgG2a、IgG2b和IgG3结合,以达到纯化的目的。洗脱液中的抗体浓度可用紫外光吸收法粗测,贝类毒素单克隆抗体溶液在A280nm时,1.44(吸光单位)相当于1mg/ml。经低pH洗脱后,在收集管内预置中和液可保持纯化抗体的活性。亲和层析法具有特异性高、纯度高的特点。通过上述整套工艺,腹水纯化后的单抗抗体纯度超过95%,且具有稳定效价。
3)胶体金标记抗体制备
(1)蛋白处理:胶体金标记蛋白前需要彻底去除多余的盐,将抗体置于透析袋中,用PB缓冲液在4℃条件下透析过夜,将多余的盐离子透析掉。从透析袋中取出抗体,4℃下以10000r/min速度离心5min,除去多余的抗体聚合物,取上清液和胶体金连接进行标记。
(2)加入适量的碳酸钾溶液调节胶体金溶液至最适pH值,将抗体溶液逐滴地加入到胶体金溶液中,快速混匀,将混合后的溶液置于4℃条件下静置1小时
(3)加入浓度为20%的BSA溶液和20%的PEG20000溶液,以封闭金颗粒的表面的残留表位使颗粒聚集,继续静置30min;
(4)在4℃条件下,以2000r/min速度离心15min,吸取1ml上清液,移入新的离心管中,除去标记过程中未连接的金粒子;
(5)将吸取的上清液在4℃下以10000r/min速度离心30min,弃去上清,除去未标记成功的抗体及胶体金粒子;
(6)制备好的金标抗体使用时,用金标工作液重悬到一定的浓度,4℃条件下保存备用。
(7)胶体金标记抗体的质量鉴定:将羊抗鼠二抗用PB稀释100倍后以0.6μl/条在NC膜上点样,包被完全后,再将稀释过的金标抗体溶液滴在二抗位置上,直接与二抗反应作用,观察结果。
4)组装检测卡
根据优化的检测参数,按比例混合胶体金标记抗体,保证各标记抗体的终浓度为最优检测浓度。胶体金免疫层析试纸条由硝酸纤维素膜、金标垫、样品垫、吸水纸、背板四部分组成(图1)。将硝酸纤维素膜贴到PVC背板上,在NC膜上用划膜仪以1μl/cm包被羊抗鼠二抗和靶标捕获抗体作为C线和T线,金标垫采用SB08玻璃纤维素膜喷涂稀释好的金标抗体,干燥后贴在PVC背板上,然后依次贴上样品垫(SB06玻璃纤维素膜)和吸水纸,使用切条机将组装好的试纸条切成60mm×3.7mm的成条,进一步采用压塑工艺制成产品,每10/50/100个作为一个包装,真空低温干燥保存。
实施例2
一种检测沙门氏菌毒素的胶体金塑封检测卡,所述试纸条包括背板、样品垫、胶体金垫、硝酸纤维素膜和吸水垫,所述样品垫、胶体金垫、硝酸纤维素膜和吸水垫在背板上按顺序依次固定(图1),所述胶体金垫内含有胶体金标记抗沙门氏菌毒素抗原的单克隆抗体;所述硝酸纤维素膜上设有检测线和质控线。试纸条通过冷裱塑封,所述样本孔位于样品垫正上方,通过撕开盖膜或塑封撕口露出加样区(图2)。检测后直接观察(图3)。
实施例3
采用上述方法,试制了一批产品并进行了样品测试。
从养殖场采集了土样、污水样、鸡粪样品共50个样本。分别取1克样品,加2ml去离子水搅匀,沉淀5分钟后吸取上清液,加5滴到4ml去离子水中作为待检液。吸取待检液滴加4滴到检测卡样品垫处,反应5分钟左右观测结果。结果测得阳性样本数9个(18%),与国标方法检测结果符合率为100%。
Claims (10)
1.一种检测沙门氏菌毒素的塑封胶体金检测卡,所述检测卡包括试纸条,其特征在于,所述试纸条包括背板、样品垫、胶体金垫、硝酸纤维素膜和吸水垫,所述样品垫、胶体金垫、硝酸纤维素膜和吸水垫在背板上按顺序依次固定,所述胶体金垫内含有胶体金标记抗沙门氏菌毒素抗原的单克隆抗体;所述硝酸纤维素膜上设有检测线和质控线。
2.根据权利要求1所述的检测沙门氏菌毒素的塑封胶体金检测卡,其特征在于,所述试纸条通过冷裱塑封。
3.根据权利要求2所述的检测沙门氏菌毒素的塑封胶体金检测卡,其特征在于,所述检测卡上设置有样本孔,所述样本孔位于样品垫正上方。
4.一种检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,包括以下步骤:
沙门氏菌毒素抗原提纯;
利用提纯后的抗原进行沙门氏菌毒素的单克隆抗体制备及筛选;
胶体金标记抗体制备;
组装检测卡。
5.根据权利要求4所述的检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,
所述胶体金标记抗体制备方法包括:
将抗体置于透析袋中,透析掉多余的盐离子透析掉;从透析袋中取出抗体,离心后除去多余的抗体聚合物,取上清液和胶体金连接进行标记;
加入碳酸钾溶液调节胶体金溶液的pH值,将抗体溶液逐滴地加入到胶体金溶液中,混匀后静置;
加入BSA溶液和PEG20000溶液,以封闭金颗粒的表面的残留表位使颗粒聚集,继续静置;
离心后吸取上清液,移入新的离心管中,除去标记过程中未连接的金粒子;
将吸取的上清液离心,弃去上清,除去未标记成功的抗体及胶体金粒子,得到胶体金标记抗体。
6.根据权利要求5所述的检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,胶体金标记抗体的质量鉴定方法为:将羊抗鼠二抗用PB稀释后在NC膜上点样,包被完全后,再将稀释过的金标抗体溶液滴在二抗位置上,直接与二抗反应作用,观察结果。
7.根据权利要求4所述的检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,组装检测卡的步骤包括:将硝酸纤维素膜贴到背板上,在硝酸纤维素膜上用划膜仪以包被羊抗鼠二抗和靶标捕获抗体作为C线和T线,胶体金垫上喷涂稀释好的金标抗体,干燥后贴在背板上,然后依次贴上样品垫和吸水纸,使用切条机将组装好的试纸条切条,进一步采用压塑工艺制成产品。
8.根据权利要求4所述的检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,沙门氏菌毒素抗原提纯的方法包括:沙门氏菌毒素培养液裂解并除盐后进行弱阴离子交换层析。
9.根据权利要求4所述的检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,沙门氏菌毒素的单克隆抗体制备方法包括:取制备所得沙门氏菌毒素抗原与弗氏完全佐剂等体积混合均匀,取混合液皮下注射小鼠;之后用弗氏不完全佐剂与贝类毒素-BSA混匀重复免疫;加强免疫后对小鼠眼眶取血,用酶联免疫吸附测定间接法测定其效价;将免疫小鼠脾细胞与骨髓瘤细胞混合,用PEG快速融合,加入含有次黄嘌呤、氨基喋呤和胸腺嘧啶核苷的培养基进行培养和筛选。
10.根据权利要求4所述的检测沙门氏菌毒素的塑封胶体金检测卡制备方法,其特征在于,沙门氏菌毒素的单克隆抗体制备方法包括:用降植烷进行小鼠腹腔注射,将杂交瘤细胞接种到小鼠腹腔中去;采集小鼠腹水,离心收集上清,再过玻璃纤维柱,收集到澄清的腹水;经过辛酸-硫酸铵沉淀法粗提纯小鼠腹水,用葡萄球菌A蛋白与萄聚糖交联,制备亲和层析柱将粗提抗体上样结合后洗脱后得到沙门氏菌毒素的单克隆抗体。
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CN112630421A (zh) * | 2021-03-05 | 2021-04-09 | 山东康华生物医疗科技股份有限公司 | 胶体金标记伤寒沙门菌重组抗原的方法、试剂条及试剂盒 |
CN112630421B (zh) * | 2021-03-05 | 2021-06-01 | 山东康华生物医疗科技股份有限公司 | 胶体金标记伤寒沙门菌重组抗原的方法、试剂条及试剂盒 |
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