
Cardiolipin (CL) is a unique phospholipid with a very interesting chemical and specific ultrastructural characteristics. It is a highly acid dimer of phosphatidylglycerol (PG) and phosphatidic acid (PA), containing four acyl chains; three glycerols and two phosphate headgroups. Due to deprotonation of one of these phosphate groups, cardiolipin is negatively charged in physiological pH [1,2].
Cardiolipin (CL) is known as mitochondria-specific phospholipid since it is almost exclusively biosynthesized and located in the inner mitochondrial membranes. The name “cardiolipin” is derived from fact that it was first found and isolated from animal heart. Cardiolipin is considered to be intimately linked to mitochondrial bioenergetic process. It plays a functional role in mitochondrial membrane stability and dynamics, interacts with a number of inner mitochondrial membrane metabolite carriers, enzymes and proteins. During apoptosis in presence of H2O2, CL-bound Cytochrome c catalyzes the peroxidation of cardiolipin, releasing Cytochrome c, which is a death-inducing protein. CL peroxidation and depletion have important implications to age-related mitochondrial dysfunction, resulting in a number of pathophysiological conditions, such as hypo/hyperthyroid states [3-7], heart ischemia–reperfusion [8-12], nonalcoholic fatty liver disease [13], diabetes [14,15], Barth syndrome [16,17] and aging [18-21]. According Birk et al. [22], the main functions of cardiolipin are: “(i) to support spatial organization of mitochondrial cristae; (ii) to create the proton trap necessary for sustaining the proton gradient and ATP synthesis by the F0F1 ATP synthase; (iii) to act as a scaffold for assembly of respiratory complexes and super-complexes to facilitate optimal electron transfer among the redox partners.”
Extensive studies [23-29] of pharmacological, toxicological, and therapeutic effects have shown that the incorporation of doxorubicin in cardiolipin liposomes improved the antitumor activity of doxorubicin, while the histopathologic lesions in cardiac tissue of mice significantly decreased. It has been reported that cardiolipin-containing liposomes have lower (at least 2-fold lower than that observed with conventional doxorubicin) cardiotoxicity associated with doxorubicin by altering the pharmacokinetics and tissue distribution of the drug in mice [29]. Also, it has been indicated that cardiolipin provides two types of binding possibility for drugs; one mostly exposed at the surface, and the other deeply buried in the membrane [30,31]. Hence, the cardiolipin-liposomes has been suggested as a convenient carrier for doxorubicin delivery to increase the therapeutic index of the drug [23].
Cardiolipin is a negatively charged lipid. Cellsome® made from cardiolipin lipid catalog containing many different types of saturated and unsaturated cardiolipin based liposomes made from 0.5 up to 100 percent of cardiolipin.


ebiomall.com






>
>
>
>
>
>
>
>
>
>
>
>
现在国内最差也是用3代试剂,有些地方会用4代试剂。
4代试剂(检查抗原+抗体)——窗口期为4周。因为抗原于3-4周达到复制的峰值,此时通过4代试剂检查,如果感染了HIV,抗原/抗体至少有一个为阳性,如果都是阴就排除了。
3代试剂(只查抗体)——窗口期为6周。
以上为理论分析+临床经验的结果,可以说是99.9%的准确度。
但是目前FDA、CDC和试剂生产商统一达成的共识,也就是针对普通人,最保守的窗口期是3个月。无论什么试剂,3个月都100%排除。
需要的仪器有:酶标仪、恒温箱、枪、枪头、烧杯、滤纸.当然有自动洗板机最好.
复习了很多文献在测定方法里面均提到了CV
例如:
Theintra-assaycoefficientofvariationfortheassaywas4-6%.
Theanalyticcoefficientofvariationwas10.2%
问题是:
我是否可以直接使用说明书里的CV值?
如果不可以,我该如何操作进行计算
ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性,酶标记的抗原或抗体既保留其免疫学活性,又保留酶的活性。在测定时,受检标本(测定其中的抗体或抗原)与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体,也通过反应而结合在固相载体上。此时固相上的酶量与标本中受检物质的量呈一定的比例。加入酶反应的底物后,底物被酶催化成为有色产物,产物的量与标本中受检物质的量直接相关,故可根据呈色的深浅进行定性或定量分析。由于酶的催化效率很高,间接地放大了免疫反应的结果,使测定方法达到很高的敏感度。
1.取出试剂盒室温平衡30min,取出血样放至室温。
2.配标准品:取150uL标准品加入150uL标准品稀释液稀释,依次稀释5次。
3.加样:分别于各反应孔中加入标准品50uL,样品40UL,标准品做复孔,样品做3孔。
4.分别于样品孔中加入10UL抗体。
5.标准品和样品孔中分别加入50uL链酶亲和素-HRP,盖上封板膜,轻轻震荡混匀,37℃温育60min。
6.配洗涤液:将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。
7.洗涤:小心揭开封板膜,弃去液体,甩干,每孔加200uL洗涤液,静置30s后弃去,如此重复5次,拍干。
8.显色:每孔先加入显色剂A 50uL,再加显色剂B 50uL,轻轻震荡混匀,37℃避光显色6min。
9. 终止:每孔加入终止液50uL,终止反应(此时蓝色立即转为黄色)。
10.测定:以空白孔调零,450nm波长依序测量各孔的吸光度。测定应在加终止液10min之内进行。
11.保存结果,收拾桌面。
12.分析处理数据
注意事项
1. 取出板条前恢复到室温后再打开外包装袋,实验中不用的板条立即放回包装中,密闭封口,其余不用试剂应盖好。
2. 实验操作中请使用一次性的吸头,避免交叉污染。
3.实验板孔加入试剂的顺序应一致,以保证所有反应孔的孵育时间一致。
4.洗涤过程中反应孔中残留的洗涤液应在滤纸上充分拍干,勿将滤纸直接放入反应孔中吸水。
5.试剂盒内试剂请在保质期内使用,不同批号试剂不要混用。
6.1000pg/ml以上的结果为非线性的,根据此标准曲线无法得到精确的结果。大于1000pg/ml 的样品应以标准稀释缓冲液稀释后重做。在结果分析时,结合考虑相应的稀释度。