Product Specifications:
Item#: 106
Description: HIV-1 Reverse Transcriptase (p66) Capture ELISA.
Storage: Store Components as specified.
Stability: See Kit Label
Note: Read instructions and familiarize yourself with kit components before performing experiments with this kit.
Some components of this kit are light sensitive. Do not expose to direct sunlight.
Component List:
A) Plate holder with solid phase capture ligand (Anti-p66 Monoclonal Antibody) strips, store at 4oC
B) Wash Buffer 10 x, store at 4oC
C) Blocking/Diluent Buffer, store at 4oC
D) Reverser Transcriptase (rp66) Reference, store at 4oC
E) Detector Reagent, store at 4oC
F) Substrate, store at 4oC
G) Stop Solution, store at 4oC
Section-1
Pre-Test Preparation
1. Bring components A, B, and C to room temperature.
2. Dilute wash buffer (Component B) 1 : 10 with deionized water for use.
3. Remove plate holder with strips from shielded bag (Use scissors to cut the sealed end of bag).
4. Add 200µl of wash buffer to each well and leave at romm temperature until ready for the next step.
Section-2
Capture Assay
1. Prepare several dilutions (minimum 200µl) of positive reference rp66 (Component D,1ug/ml ) in diluent buffer (Component C) in Eppendorf or comparable tubes - labeled accordingly: 500ng/ml to 0.5ng/ml in 4-fold serial dilutions. Note: low rp66 detection sensitivity in 100-200pg/ml or 10-20pg/test range.
2. Prepare test samples using diluent buffer (Component C) in 10-100ng/ml range. Unknown samples may be used in 1:2 to 1:10 dilution range with Diluent Buffer (Component C).
3. Continue from Section-1, Step 4: Dump contents of wells and pat dry. Set up strip well holder on a grid.
4. Pipette 100µl of positive reference (Step 1 above) and test samples into wells. Note position on the grid. Tap holder gently against table to dislodge air bubbles in wells. Cover plate and leave at RT for 1 hour.
5. Dump Contents of wells and wash 3 times with 1 x wash buffer (Component B, 300µl/well). Leave last wash until ready to add detector reagent. Pat dry wells.
6. Dilute detector reagent (Component E) 1:100 in diluent buffer (Component C). Add 100µl of detector reagent to each well. Leave plate at RT for 1 hour in dark.
7. Repeat washing as in Step 5 above, leave last wash in wells until ready to add substrate.
8. Dump contents of wells from Step 7 above.
9. Add 100µl of TMB substrate (Component F) to each well. Blue color will develop over 10 minutes at RT.
10. Stop color development (Step 9) after 10 minutes by adding 100µl of Stop Solution (Component G) to each well. Color will change to yellow. Read plate at 450nm as soon as possible (no more than 10 minutes).
Caution: Steps 6 and 10 above, plate must be left in a dark place.
STRICTLY FOR NON-HUMAN RESEARCH AND DIAGNOSTICS
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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。
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。
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
首先要什么有什么的，你得好好考虑一下。
2、看生产地址
根本没有生产地址，我们知道做实验做产品需要很多的仪器、试剂、耗材，没有人相信一间简单的屋子可以生产各种样的试剂盒。
3、看产品包装
没有任何的生产地址、联系方式等信息，这种产品有问题了连个投诉的地方都没有。
4、看公司网站
有些打着国外原装旗号，整个公司网站为英文页面，实际注册IP地址在中国。如果写着国外的地址，让你国外的朋友实地去看一下！
5、做交叉验证
拿对方提供的几个种类的试剂盒，把里面的关键组份相互替换做做实验，如果交叉严重，只能说明是一种原料生产的试剂盒贴了不同的标签。
6、看价格
价格低得离谱，却打着进口大公司原料分装，核算成本，这种低得离谱的价格是连原料都买不起的。
