实验方法：
酶联免疫法/酶免法（ELISA）
检测原理：
采用双抗体夹心ABC-ELISA法
保存：2-8℃
样品类型（SAMPLE TYPES）：
标本必须为液体，不含沉淀。包括血清、血浆、尿液、胸腹水、脑脊液、细胞培养上清、组织匀浆等。1ml的全血可得到0.5ml的血清
或血浆。每个标本量收集体积＝100ul×检测种类。取材前须向销售人员索要说明书。
样品采集：
收集标本前必须清楚要检测的成份是否足够稳定。对收集后当天进行检测的标本，储存在4℃备用，如有特殊原因需要周期收集标本，将标本及时分装后放在-20℃或-70℃条件下保存。避免反复冻融。标本2-8℃可保存48小时，-20℃可保存1个月。-70度可保存6个月。部分激素类标本需添加抑肽酶。
注意事项：
收集标本前必须清楚要检测的成份是否足够稳定。对收集后当天进行检测的标本，储存在4 ℃备用，如有特殊原因需要周期收集标本，
将标本及时分装后放在-20 ℃或-70 ℃条件下保存。避免反复冻融。标本2 -8 ℃可保存48 小时，-20 ℃可保存1 个月。-70 度可保存6 个月。部分激素类标本需添加抑肽酶。
*液体类标本*
标本必须为液体，不含沉淀。包括血清、血浆、尿液、胸腹水、脑脊液、细胞培养上清、组织匀浆等。1ml 的全血可得到0.5ml 的血清或血浆。每个标本量收集体积＝100ul ×检测种类。取材前须向销售人员索要说明书。
*血清*
室温血液自然凝固10-20 分钟后，离心20 分钟左右（2000-3000 转/ 分）。收集上清。如有沉淀形成，应再次离心。
*血浆*
应根据
试剂盒的要求选择EDTA 、柠檬酸钠或肝素作为抗凝剂，加入10 ％（v/v ）抗凝剂（0.1M 柠檬酸钠或1% heparin 或2.0%EDTA.Na2）混合10-20 分钟后，离心20 分钟左右（2000-3000 转/ 分）。仔细收集上清。如有沉淀形成，应再次离心。
*组织标本*
切割标本后，称取重量。加入一定量的PBS ，缓冲液中可加入1 μg/L 蛋白酶抑制剂或50U/ml 的Aprotinin （抑肽酶）。用手工或匀浆器将标本匀
浆充分。离心20 分钟左右（2000-3000 转/ 分）。仔细收集上清置于-20 度或- 70 度保存，如有必要，可以将样品浓缩干燥。分装后一份待检测，其余冷冻备用。
操作步骤：
参照说明书（具体说明书请发邮件）
灵敏度：
具体请查看说明书.
检测范围（RANGN）：
具体请查看说明书.
参考值：
产品用途:
科研ELISA检测试剂盒.提供实验代测服务.
产品范围:
人,小鼠,大鼠,猪,兔,其它动物细胞因子;细胞凋亡,活性多肽、自身抗体,血栓与止血,
骨代谢,肝纤维化,肿瘤,激素内分泌、自身抗体科研ELISA检测
试剂盒提供实验代测服务
具体详情请来电咨询。
Samplecollection and storages
Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 10 minutes at approximately 3000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles
Plasma- Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Cell culture supernates and other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.
Note: The samples should be centrifugated adequately and no hemolysis or granule was allowed.
Materials required but not supplied
1. Standard microplate reader(450nm)
2. Precision pipettes and Disposable pipette tips.
3. 37 ℃ incubator
Precautions
1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer.
2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided.
3. Mix all reagents before using.
Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature( 20-25°C)
Materials supplied| Name | 96determinations | 48determinations |
| Microelisa stripplate | 12*8strips | 12*4strips |
| Standard | 0.3ml | 0.3ml |
| Sample diluent | 6.0ml | 3.0ml |
| HRP-Conjugate reagent | 10.0ml | 5.0ml |
| 20X Wash solution | 25ml | 15ml |
| Chromogen Solution A | 6.0ml | 3.0ml |
| Chromogen Solution B | 6.0ml | 3.0ml |
| Stop Solution | 6.0ml | 3.0ml |
| Closure plate membrane | 2 | 2 |
| User manual | 1 | 1 |
| Sealed bags | 1 | 1 |
Note: Standard concentration was followed by:
800、400、200、100、50、0 μg/mL.
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.
4. Add100μlofHRP-conjugate reagent to each well,cover with an adhesive stripandincubatefor60 minutes at37°C.
5. Aspirate each well and wash, repeating the process four times for a total of five washes.Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifolddispenseror autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating ordecanting. Invert the plate and blot it against clean paper towels.
6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
7. Add 50μl Stop Solution to each well. The color in the wells should change from blue toyellow. If the color in the wells is green or the color change does not appear uniform,gently tap the plate to ensure thorough mixing.8. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes.Calculation of results- This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
- First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
- To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
- Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
- The sensitivity by this assay is 1.0 μg/mL.
- Standard curve
Storage： 2-8℃.validity： six months.FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!
