CN114397293A - 一种胶原基生物材料内毒素提取检测方法 - Google Patents
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Abstract
本发明公开了一种胶原基生物材料内毒素提取检测方法，包括内毒素提取和内毒素检测；所述内毒素提取包括：研磨、离心、稀释；其中，所述生物材料在研磨时加入浸提介质；所述内毒素检测包括：向稀释后的样本中加入检测介质，获得待检测溶液。本发明提供的内毒素检测方法专用于胶原基生物材料。
Description
技术领域
本发明属于生物材料生物学检测领域，尤其涉及一种胶原基生物材料内毒素提取检测方法。
背景技术
细菌内毒素是革兰氏阴性菌细胞壁外层上特有的结构，其活性成分主要为脂多糖(LPS)。内毒素进入人体，可能引起发热、微循环障碍、内毒素血症、脓毒性休克和弥散性血管内凝血等严重后果。因此在GB/T14233.2-2005 《医用输液、输血、注射器具检验方法第2部分：生物试验方法》中明确规定在输液、输血、注射器具内毒素每件不超过20EU，与脑脊液接触和胸腔内应用的医疗器械每件不超过2.15EU。
对于胶原基植入材料而言，低剂量内毒素残留是导致植入医疗器械材料发生炎症相关并发症的重要原因之一。内毒素极易与胶原纤维的三维螺旋结构结合，藏匿于结构内，导致无法检出。且内毒素存在的屏蔽作用(Masking effect)可能导致低剂量内毒素无法检出，但存在屏蔽作用的低剂量内毒素同样可以导致全身性炎症反应，影响植入材料的生物安全性。因此，应当开发能够最大程度提取胶原基生物材料内毒素残留的检测手段，优化检出率，保证医疗器械植入材料的安全性。经检索，目前尚无经过论证的针对膜片状或固体状的胶原基医用材料的有效提取、检测方法。
发明内容
本发明针对现有技术的不足，提供一种胶原基生物材料内毒素提取检测方法，优化检测结构，提高检出率。
为实现上述目的，本发明的技术方案为：
一种胶原基生物材料内毒素提取检测方法，包括内毒素提取和内毒素检测；
所述内毒素提取包括：研磨、离心、稀释；其中，所述生物材料在研磨时加入浸提介质；
所述内毒素检测包括：向稀释后的样本中加入检测介质，获得待检测溶液。
所述研磨为：将生物材料与浸提介质混合，优选将生物材料剪碎成 1mm*1mm以下的小片，充分研磨或匀浆5-10分钟，在37℃静置2h，重复多次，获得混合溶液。
所述浸提介质为含有表面活性剂的溶液。
优选的，所述浸提介质为含有磷酸酯类表面活性剂的溶液，包括单酯、二酯及混合物及其盐。
所述磷酸酯类表面活性剂含量比例为0.01％-0.5％(w/t)。
进一步的，所述磷酸酯类表面活性剂优选磷酸酯盐类表面活性剂，包括醇磷酸酯盐、醇醚磷酸酯盐；如甘油磷酸酯盐、仲辛醇磷酸酯盐、乙二醇单丁醚磷酸酯钠盐、脂肪醇醚磷酸酯盐、脂肪醇聚氧乙烯磷酸酯盐、脂肪醇磷酸酯三乙醇胺盐等。
优选地，所述浸提介质包括多种表面活性剂的配方组合，以模拟天然细胞膜表面磷脂成分但较天然磷脂更易与内毒素分子结合。
所述离心为：将研磨后获得的混合溶液8000-14000rpm/min离心5min，取上清液2-10℃冷藏保存，冷藏保存时间小于2h。
所述稀释为：以内毒素检查用水适当稀释离心获得的上清液，根据检测需求，可保留多个稀释梯度的样本用于后续检测。
所述检测介质为含有金属离子的阴离子表面活性剂的溶液。
所述检测介质的添加使得含金属离子的阴离子表面活性剂在待检测溶液中的终浓度为0.005-5％(w/t)。
所述金属离子在待检测溶液中的终浓度为20-500mM，优选的，所述金属离子在待检测溶液中的终浓度为20-200mM。
所述金属离子为二价金属离子，包括钙离子、镁离子。
所述检测介质中含有的阴离子表面活性剂包括但不限于脱氧胆酸、十二烷基硫酸钠、十二烷基磺酸钠、辛基硫酸钠、仲烷基硫酸钠、油醇硫酸钠、烷基苯磺酸盐、烷基萘磺酸盐、烷基甘油醚磺酸钠等。
上述的浸提介质和检测介质可使用含有相同或相似成分的商业化产品替代或部分替代。
所述内毒素检测方法包括但不限于鲎试剂的凝胶法或鲎试剂的光度测定法。
优选地，所述鲎试剂的光度测定法包括浊度法和显色基质法，优选采用显色基质法。
若采用鲎试剂的光度测定法检测内毒素，还需进行标准曲线可靠性试验和干扰试验，必要时还需对待检测的溶液进行梯度浓度稀释，以排出可能存在的表面活性剂干扰，确定浸提介质和检测介质的加入比例，稀释倍数不应大于8倍。
所述浸提介质和检测介质是基于内毒素检查用水配置的pH缓冲溶液，溶液中包括Tris、磷酸盐缓冲液。
所述胶原基生物材料为天然组织经过脱细胞处理制成的材料，其主要成分为胶原，包括经过小肠粘膜下层、膀胱基底膜、心包、真皮、腹膜、眼角膜、羊膜等。
所述胶原基生物材料经过脱细胞、交联、免疫原性清除中的一种或多种处理，或所述胶原基生物材料包括使用提纯胶原蛋白制成的支架材料。
本发明由于采用以上技术方案，使其与现有技术相比具有以下的优点和积极效果：
本发明提供的内毒素检测方法专用于胶原基生物材料，特别是在制备过程中可能产生内毒素污染的材料，如猪小肠粘膜下层。浸提介质作用为分离抽提出材料中含有的内毒素，破坏内毒素的脂质A与脱细胞基质类产品中可能存在的脂质、蛋白结合，使之从脱细胞基质三维网状结构中脱离。检测介质的作用为在检测前将内毒素与溶液中可能存在的游离生物蛋白分离，解除屏蔽作用，提高检出率。
具体实施方式
以下结合具体实施例对本发明提出的一种胶原基生物材料内毒素提取检测方法作进一步详细说明。根据下面说明，本发明的优点和特征将更清楚。
实施例1
本实施例采用鲎试剂的光度测定法检测内毒素来阐述本发明的内容。
分别称取0.02g经过常规脱细胞方法处理获得的猪小肠粘膜下层膜片(实验组1)和经过过度脱细胞方法处理获得的猪小肠粘膜下层膜片(实验组2)，剪碎后置于研磨容器内，加入浸提介质(甘油磷酸酯盐，终浓度为0.02％w/t)，混匀静置2分钟，充分研磨5分钟，12000rpm离心5min，取上清液以内毒素检查用水梯度稀释为待测样本。实验组3、4分别为实验组1、2样品常规内毒素检测方法测试组，仅使用内毒素检查用水浸提。取待测样本，加入检测介质(十二烷基硫酸钠，终浓度为0.01％w/t；Mg2+，终浓度为100mM) 混匀后立即上机检测。
考虑到排除胶原基生物材料可能存在的溶出物可能对试验存在干扰，设立对照组。阳性对照1为含有定量内毒素标准品的浸提介质溶液，添加实验组1补片进行研磨、离心等相同处理后制备为供试品进行检测。阳性对照2 为含有定量内毒素标准品的浸提介质溶液，不添加补片进行研磨、离心等处理后制备为供试品进行检测。
考虑到排除浸提介质和检测介质可能对试验存在干扰，设立阴性对照为不含有内毒素标准品的浸提介质溶液，不添加补片进行研磨、离心等处理后制备为供试品进行检测。空白对照为浸提介质溶液与检测介质的混合液。
标准曲线的可靠性试验：使用内毒素标准品制成溶液，制成5个浓度梯度的稀释液，浓度范围在检测试剂盒的测定范围内，本试验中分别为50，5， 0.5，0.05，0.005。每个浓度设置3个平行，另设2个阴性对照，阴性对照吸光度应小于标准曲线最低点的检测值。根据线性回归分析，标准曲线的相关系数的绝对值大于或等于0.980，试验方为有效。本试验条件下标准曲线R 值为0.98214。
检测结果：
表1上述的实验组1、2、3、4和阳性对照组1、2以及阴性对照、空白对照组的成分总结
采用动态光度法对上述样本的内毒素含量进行检测，检测结果如表2。
表2检测结果
| 内毒素含量(EU/mL) | |
| 实验组1 | 0.77 |
| 实验组2 | 0.02 |
| 实验组3 | 0.26 |
| 实验组4 | 0.20 |
| 阳性对照1 | 1.73 |
| 阳性对照2 | 0.74 |
| 阴性对照 | 未检出 |
| 空白对照 | 未检出 |
考虑到表面活性剂可能对结果产生假阳性干扰，采用巨噬细胞活化法对内毒素含量结果进行复核。使用的实验材料是实验组1、实验组2、实验组5 (添加符合国家标准的15EU/件的聚丙烯对照品)。取生长对数期的THP-1 单核巨噬细胞按照1×104个细胞/孔的密度接种至24孔细胞培养板，37℃培养过夜。过夜培养后的细胞更换新鲜培养基。将补片裁剪至1×1cm2，放置于transwell上室，将transwell置入培养板孔中，上室加入0.5mL细胞培养基，共培养24hr后，吸取细胞培养上清，离心后ELISA法检测TNF-α含量，结果如表3所示。细胞加入Alamar Blue试剂，进行活细胞染色计数。以 100ng/mL LPS为细胞阳性对照，常规完全培养基为细胞阴性对照。
表3 TNF-α含量测量结果
| TNF-α含量(pg/50000个细胞) | |
| 实验组1 | 204.5 |
| 实验组2 | 32.1 |
| 实验组5 | 156.3 |
| 细胞阳性对照 | 1466.7 |
| 细胞阴性对照 | 未检出 |
结果表明：实验回收率为74％，符合药典细菌内毒素检测法的要求。浸提介质和检测介质的加入对检测数据结果的干扰可接受。本实施例方法提高了检测数据。且经过巨噬细胞活化半定量试验结果证实实验组1的内毒素含量确实高于实验组2。方法专属性结果可接受。
干扰试验：选择标准曲线中点内毒素浓度设定为λm。按照本例内毒素含量测定中规定的方法制备SIS浸提液。按照表4制备溶液A、B、C、D。
表4干扰实验溶液制备
其中，A：不超过最大有效稀释倍数的SIS浸提液；
B：加入已知浓度内毒素标准品的，且与A溶液有相同稀释倍数的SIS 浸提液；
C：用于制备标准曲线的内毒素标准品溶液；
D：阴性对照。
按照标准曲线计算出溶液A和B的内毒素浓度分别为ct、cs，按照下式计算该实验条件下的回收率R，
R＝(cs-ct)/λm×100％
当内毒素的回收率在50％-200％，则认为在此试验条件下供试品溶液对检测不存在干扰作用。本实验条件下R值为65％-78％。
实施例2
对于不同组织来源的材料，其含有的各项成分区别较大，如蛋白含量、脂肪含量、处理试剂残留等因素可能对测试结果产生影响。本实施例以真皮基质和猪小肠粘膜下层为例，对适用于本发明的常用表面活性剂的测试浓度范围进行小结。
测试方法：分别称取一定量的经过常规脱细胞方法处理获得的猪小肠粘膜下层、猪真皮基质，剪碎后置于研磨容器内，加入浸提介质，混匀静置2 分钟。充分研磨5分钟，12000rpm离心5min，取上清液以内毒素检查用水梯度稀释样本。取待测样本，加入检测介质充分混匀后立即上机检测。经过巨噬细胞活化法复核，确认介质使用范围。
测试结果：
表1真皮基质测试结果
表2猪小肠粘膜下层测试结果
| 浸提介质 | 添加浓度范围 |
| 甘油磷酸酯钠盐 | 0.02-0.3％ |
| 乙二醇单丁醚磷酸酯钠盐 | 0.01-0.2％ |
| 分散剂(charles river) | 0.1-0.5％ |
| 分散剂(湛江安度斯) | 0.3-0.5％ |
| 检测介质 | 添加浓度范围 |
| 脱氧胆酸 | 0.01-0.1％ |
| 十二烷基硫酸钠 | 0.01-0.2％ |
| Pyrosperse(Lonza) | 0.5-5％ |
| Ca2+ | 20-100mM |
| Mg2+ | 100-500mM |
上面对本发明的实施方式作了详细说明，但是本发明并不限于上述实施方式。即使对本发明做出各种变化，倘若这些变化属于本发明权利要求及其等同技术的范围之内，则仍落入在本发明的保护范围之中。
Claims (10)
1.一种胶原基生物材料内毒素提取检测方法，其特征在于，包括内毒素提取和内毒素检测；
所述内毒素提取包括：研磨、离心、稀释；其中，所述生物材料在研磨时加入浸提介质；
所述内毒素检测包括：向稀释后的内毒素提取样本中加入检测介质，获得待检测溶液；然后对待检测溶液进行内毒素检测。
2.根据权利要求1所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述浸提介质为含有磷酸酯类表面活性剂的溶液。
3.根据权利要求2所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述磷酸酯类表面活性剂含量比例为0.01％-0.5％(w/t)。
4.根据权利要求1所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述检测介质为含有金属离子的阴离子表面活性剂的溶液。
5.根据权利要求4所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述检测介质的添加使得阴离子表面活性剂在待检测溶液中的终浓度为0.005-5％(w/t)。
6.根据权利要求4所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述金属离子含量在待检测溶液中的终浓度为20-500mM。
7.根据权利要求4所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述金属离子为二价金属离子，包括钙离子、镁离子。
8.根据权利要求1所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述内毒素检测方法包括基于鲎试剂的光度测定法。
9.根据权利要求1所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述浸提介质和检测介质是内毒素检测用水或基于内毒素检查用水配置的pH缓冲溶液，pH缓冲溶液中包括Tris、磷酸盐缓冲液等。
10.根据权利要求1-9任意一项所述的胶原基生物材料内毒素提取检测方法，其特征在于，所述胶原基生物材料为脱细胞基质等纯化胶原制备成的软组织填充修复材料。
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