人核因子κB(NF-κB) ELISA试剂盒


ELISA是酶联接免疫吸附剂测定（Enzyme-Linked Immunosorbnent Assay ）的简称。它是继免疫荧光和放射免疫技术之后发展起来的一种免疫酶技术。此项技术自70年代初问世以来，发展十分迅速，目前已被广泛用于生物学和医学科学的许多领域。　
（一）　原理

人核因子κB(NF-κB) ELISA试剂盒人核因子κB(NF-κB) ELISA试剂盒


ELISA是酶联接免疫吸附剂测定（Enzyme-Linked Immunosorbnent Assay ）的简称。它是继免疫荧光和放射免疫技术之后发展起来的一种免疫酶技术。此项技术自70年代初问世以来，发展十分迅速，目前已被广泛用于生物学和医学科学的许多领域。　
（一）　原理

ELISA是以免疫学反应为基础，将抗原、牽9体的特异性反应与酶对底物的高效催化作用相结合起来的一种敏感性很高的试验技术。由于抗原、抗体的反应在一种固相载体──聚苯乙烯微量滴定板的孔中进行，每加入一种试剂孵育后，可通过洗涤除去多余的游离反应物，从而保证试验结果的特异性与稳定性。在实际应用中，通过不同的设计，具体的方法步骤可有多种。即:用于检测抗体的间接法（图a）、用于检测抗原的双抗体夹心法（图b）以及用于检测小分子抗原或半抗原的抗原竞争法等等。比较常用的是ELISA双抗体夹心法及ELISA间接法。

ELISA是以免疫学反应为基础，将抗原、牽9体的特异性反应与酶对底物的高效催化作用相结合起来的一种敏感性很高的试验技术。由于抗原、抗体的反应在一种固相载体──聚苯乙烯微量滴定板的孔中进行，每加入一种试剂孵育后，可通过洗涤除去多余的游离反应物，从而保证试验结果的特异性与稳定性。在实际应用中，通过不同的设计，具体的方法步骤可有多种。即:用于检测抗体的间接法（图a）、用于检测抗原的双抗体夹心法（图b）以及用于检测小分子抗原或半抗原的抗原竞争法等等。比较常用的是ELISA双抗体夹心法及ELISA间接法。

ELISA检测试剂盒组成:

ELISA检测试剂盒组成:

1:浓缩洗涤液（50ml）20x

1:浓缩洗涤液（50ml）20x

2.标本稀释液（12ml）

2.标本稀释液（12ml）

3.终止液（12ml）

3.终止液（12ml）

4.标准品（2管）

4.标准品（2管）

5.酶标抗体稀释液体（12ml）

5.酶标抗体稀释液体（12ml）

6.第一抗体稀释液（12ml）

6.第一抗体稀释液（12ml）

7.包被酶标板一块

7.包被酶标板一块

8.坐标纸一张

8.坐标纸一张

9.封板纸一张

9.封板纸一张

样品类型（SAMPLE TYPES）：

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。

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。

标本处理:

标本处理:

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。

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。

◇血清：

◇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。

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：

◇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。

应根据试剂盒试剂盒的要求选择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。

◇尿液、胸腹水、脑脊液：

◇尿液、胸腹水、脑脊液：

用无菌管收集。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。

用无菌管收集。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。

◇细胞培养上清：

◇细胞培养上清：

检测分泌性的成份时,用无菌管收集。离心20 分钟左右（2000-3000 转/ 分）。仔细收集上清。检测细胞内的成份时，用PBS （PH7.2-7.4 ）稀释细胞悬液，细胞浓度达到100 万/ml 左右。通过反复冻融，以使细胞破坏并放出细胞内成份。离心20 分钟左右（2000-3000 转/ 分）。仔细收集上清。保存过程中如有沉淀形成，应再次离心。

检测分泌性的成份时,用无菌管收集。离心20 分钟左右（2000-3000 转/ 分）。仔细收集上清。检测细胞内的成份时，用PBS （PH7.2-7.4 ）稀释细胞悬液，细胞浓度达到100 万/ml 左右。通过反复冻融，以使细胞破坏并放出细胞内成份。离心20 分钟左右（2000-3000 转/ 分）。仔细收集上清。保存过程中如有沉淀形成，应再次离心。

◇组织标本：

◇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。

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。

检测原理：

检测原理：

采用双抗体夹心ABC-ELISA法

采用双抗体夹心ABC-ELISA法

保存温度：2-8℃保存6个月.

保存温度：2-8℃保存6个月.

注意事项：

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。

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。

备注:以上为ELISA试剂盒通用说明书,不包括特别的试剂盒,具体的请参照每个产品的说明书.

备注:以上为ELISA试剂盒通用说明书,不包括特别的试剂盒,具体的请参照每个产品的说明书.
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 suppliedSamplecollection 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 suppliedSamplecollection and storagesSamplecollection and storagesSSSSSampleampleampleampleamplecollection and storagescollection and storagescollection and storagescollection and storagescollection 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 cyclesSerum - 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 cyclesSerum - 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 cyclesSerumSerumSerumSerumSerumSerum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for  - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for  - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 1110 minutes at approximately 0 minutes at approximately 0 minutes at approximately 333000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles000×g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.Avoid repeated freeze-thaw cycles000×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.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.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.PlasmaPlasmaPlasmaPlasmaPlasmaPlasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 303030 minutes at  minutes at  minutes at 333000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.000×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.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.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.Cell culture supernates and other biological fluidsCell culture supernates and other biological fluidsCell culture supernates and other biological fluidsCell culture supernates and other biological fluidsCell culture supernates and other biological fluidsCell 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. Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles. 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.Note: The samples should be centrifugated adequately and no hemolysis or granule was allowed.Note: The samples should be centrifugated adequately and no hemolysis or granule was allowed.Note: Note: Note: Note: Note: Note: The samples shoulThe samples shoulThe samples shoulddd be centrifugated  be centrifugated  be centrifugated aaadequately and no hemolysis or granule was allowed.dequately and no hemolysis or granule was allowed.dequately and no hemolysis or granule was allowed.
Materials required but not suppliedMaterials required but not suppliedMaterials required but not suppliedMaterials required but not suppliedMaterials required but not suppliedMaterials required but not suppliedMaterials required but not supplied
1. Standard microplate reader(450nm)1. Standard microplate reader(450nm)1. Standard microplate reader(450nm)1. 1. 1. Standard microplate readerStandard microplate readerStandard microplate reader(450nm)(450nm)(450nm)
2. Precision pipettes and Disposable pipette tips.2. Precision pipettes and Disposable pipette tips.2. Precision pipettes and Disposable pipette tips.2. 2. 2. Precision pipettes and Disposable pipette tipsPrecision pipettes and Disposable pipette tipsPrecision pipettes and Disposable pipette tips...
3. 37 ℃ incubator3. 37 ℃ incubator3. 37 ℃ incubator3. 3. 3. 37 ℃ incubator37 ℃ incubator37 ℃ incubator
PrecautionsPrecautionsPrecautionsPPPPPPrecautionsrecautionsrecautionsrecautionsrecautionsrecautions
1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer.1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer.1. Donot substitutereagentsfromone kit to another.Standard, conjugateandmicroplates are matchedfor optimal performance. Useonly thereagentssuppliedby manufacturer.1. 1. 1. 1. DDDDDoooo notnotnotnot s s s s sububububstitstitstitstitstituuuuuttttteeee rrrrreageeageeageeagennnnntttttssss frfrfrfrfromomomom one kone kone kone kiiiiitttt t t t t to anoo anoo anoo anotttttheheheherrrrr.... SSSStttttandandandandaaaaarrrrrd, cod, cod, cod, connnnnjjjjjugugugugaaaaattttteeee andandandand mmmmmiiiiiccccrrrrropopopoplllllaaaaatttttes aes aes aes arrrrre e e e mmmmmaaaaatttttchedchedchedched fffffor opor opor opor optititititimmmmmal peal peal peal perfrfrfrfrfoooorrrrrmmmmmance.ance.ance.ance. U U U U Ussssseeee ononononllllly y y y ttttthehehehe rrrrreageeageeageeagennnnntttttssss sssssuppuppuppupplililililiedededed by by by by mmmmmanuanuanuanufffffacacacactttttuuuurrrrreeeerrrrr....
2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided.2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided.2. Donot removemicroplatefrom the storage baguntilneeded. Unusedstripsshouldbe stored at2-8°Cin their pouchwith the desiccantprovided.2. 2. 2. 2. DDDDDoooo notnotnotnot r r r r reeeemmmmmoooovvvvveeee mmmmmiiiiiccccrrrrropopopoplllllaaaaattttteeee frfrfrfrfromomomom t t t t the he he he stststststoooorrrrrage bagage bagage bagage bag uuuunnnnntttttiiiiillll needed.needed.needed.needed. U U U U Ununununusssssedededed stristristristristripspspsps ssssshouhouhouhoullllldddd be be be be stststststoooorrrrred ated ated ated at 2222-----8°C8°C8°C8°C iiiiin n n n tttttheheheheiiiiir pouchr pouchr pouchr pouch witwitwitwitwithhhh t t t t the dehe dehe dehe desisisisisiccaccaccaccannnnntttt pppprrrrroooovvvvviiiiided.ded.ded.ded.
3.  Mix all reagents before using.3.  Mix all reagents before using.3.  Mix all reagents before using.3333. . . .  Mix all reagents before using. Mix all reagents before using. Mix all reagents before using. Mix all reagents before using.
Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature( 20-25°C)Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature( 20-25°C)Remove allkit reagentsfrom refrigerator and allow them to reachroom temperature( 20-25°C)RRRRReeeemmmmmoooovvvvve ae ae ae alllllllll kkkkiiiiitttt r r r r reageeageeageeagennnnntttttssss frfrfrfrfromomomom r r r r reeeefrifrifrifrifrigegegegerrrrraaaaatttttor and aor and aor and aor and allllllllllowowowow t t t t themhemhemhem t t t t to o o o rrrrreacheacheacheach rrrrroomoomoomoom t t t t teeeemmmmmpepepeperrrrraaaaatttttuuuurrrrreeee ( 20( 20( 20( 20-----222255555°°°°CCCCC)))))
Materials suppliedMaterials suppliedMaterials suppliedMaterials suppliedMaterials suppliedMaterials suppliedMaterials 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

Name96determinations48determinationsMicroelisa stripplate12*8strips12*4stripsStandard0.3ml0.3mlSample diluent6.0ml3.0mlHRP-Conjugate reagent10.0ml5.0ml20X Wash solution25ml15mlChromogen Solution A6.0ml3.0mlChromogen Solution B6.0ml3.0mlStop Solution6.0ml3.0mlClosure plate membrane22User manual11Sealed bags11Name96determinations48determinationsNameNameNameNameNameNameNameName96determinations96determinations96determinations96determinations96determinations969696 determinationsdeterminationsdeterminations48determinations48determinations48determinations48determinations48determinations484848 determinationsdeterminationsdeterminationsMicroelisa stripplate12*8strips12*4stripsMicroelisa stripplateMicroelisa stripplateMicroelisa stripplateMicroelisa stripplateMicroelisa stripplateMicroelisa stripplateMicroelisa stripplateMicroelisa stripplate12*8strips12*8strips12*8strips12*8strips12*8strips12*8strips12*8strips12*8strips12*4strips12*4strips12*4strips12*4strips12*4strips1112*4strips2*4strips2*4stripsStandard0.3ml0.3mlStandardStandardStandardStandardStandardStandardStandardStandard0.3ml0.3ml0.3ml0.3ml0.3ml0.0.0.333mlmlml0.3ml0.3ml0.3ml0.3ml0.3ml0.0.0.333mlmlmlSample diluent6.0ml3.0mlSample diluentSample diluentSample diluentSample diluentSample diluentSample diluentSample diluentSample diluent6.0ml6.0ml6.0ml6.0ml6.0ml6.06.06.0mlmlml3.0ml3.0ml3.0ml3.0ml3.0ml3.0ml3.0ml3.0mlHRP-Conjugate reagent10.0ml5.0mlHRP-Conjugate reagentHRP-Conjugate reagentHRP-Conjugate reagentHRP-Conjugate reagentHRP-Conjugate reagentHRP-Conjugate reagentHRP-Conjugate reagentHRP-Conjugate reagent10.0ml10.0ml10.0ml10.0ml10.0ml10.010.010.0mlmlml5.0ml5.0ml5.0ml5.0ml5.0ml5.0ml5.0ml5.0ml20X Wash solution25ml15ml20X Wash solution20X Wash solution20X Wash solution20X Wash solution20X Wash solution20X W20X W20X Wash solutionash solutionash solution25ml25ml25ml25ml25ml252525mlmlml15ml15ml15ml15ml15ml15ml15ml15mlChromogen Solution A6.0ml3.0mlChromogen Solution AChromogen Solution AChromogen Solution AChromogen Solution AChromogen Solution AChromogen Solution AChromogen Solution AChromogen Solution A6.0ml6.0ml6.0ml6.0ml6.0ml666.0.0.0mlmlml3.0ml3.0ml3.0ml3.0ml3.0ml333.0ml.0ml.0mlChromogen Solution B6.0ml3.0mlChromogen Solution BChromogen Solution BChromogen Solution BChromogen Solution BChromogen Solution BChromogen Solution BChromogen Solution BChromogen Solution B6.0ml6.0ml6.0ml6.0ml6.0ml666.0.0.0mlmlml3.0ml3.0ml3.0ml3.0ml3.0ml333.0ml.0ml.0mlStop Solution6.0ml3.0mlStop SolutionStop SolutionStop SolutionStop SolutionStop SolutionStop SolutionStop SolutionStop Solution6.0ml6.0ml6.0ml6.0ml6.0ml666.0.0.0mlmlml3.0ml3.0ml3.0ml3.0ml3.0ml333.0ml.0ml.0mlClosure plate membrane22Closure plate membraneClosure plate membraneClosure plate membraneClosure plate membraneClosure plate membraneClosure plate membraneClosure plate membraneClosure plate membrane2222222222222222User manual11User manualUser manualUser manualUser manualUser manualUser manualUser manualUser manual1111111111111111Sealed bags11Sealed bagsSealed bagsSealed bagsSealed bagsSealed bagsSealed bagsSealed bagsSealed bags1111111111111111 Note: Standard concentration was followed by:
500、250、125、62.5、31.25、0 ng/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 notNote: Standard concentration was followed by:
500、250、125、62.5、31.25、0 ng/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 notNote: Standard concentration was followed by: Note: Standard concentration was followed by: Note: Note: Note: Note: Note: Standard concentration was followed by: Standard concentration was followed by: Standard concentration was followed by:
500、250、125、62.5、31.25、0 ng/mL.500、250、125、62.5、31.25、0 ng/mL.500500500、250、125、62.5、31.25、0 、250、125、62.5、31.25、0 、、250、、125125、、62.562.5、、31.2531.25、、0 0 ng/mLng/mLng/mL...
Reagent preparationReagent preparationReagent preparationReagent preparationReagent preparationReagent preparationReagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.20×wash solution:Dilute with Distilled or deionized water 1:20.20×wash solution:Dilute with Distilled or deionized water 1:20.20×wash solution:Dilute with Distilled or deionized water 1:20.202020×××wash solution:Dilute with wash solution:Dilute with wash solution:Dilute with Distilled or deionized waterDistilled or deionized waterDistilled or deionized water 1:20. 1:20. 1:20.
Assay procedureAssay procedureAssay procedureAAAAAAssay proceduressay proceduressay proceduressay proceduressay proceduressay procedure
1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.1. Prepare allreagentsbeforestartingassayprocedure. ItisrecommendedthatallStandardsand Samplesbe addedin duplicateto the MicroelisaStripplate.1. 1. 1. 1. PPPPrrrrrepepepepaaaaarrrrre ae ae ae alllllllll reagentreagentreagentreagentreagentssss bebebebefffffoooorrrrreeee stststststaaaartirtirtirtirtingngngng aaaassssssssssaaaaayyyy pppprrrrroceduoceduoceduocedurrrrre.e.e.e. I I I I Itttt iiiiissss rrrrrecoecoecoecommmmmmmmmmendedendedendedended ttttthhhhaaaaatttt aaaalllllllll SSSStttttandandandandaaaaarrrrrdsdsdsds and Sand Sand Sand Saaaaammmmmppppllllleseseses be addedbe addedbe addedbe added iiiiin dupn dupn dupn duplililililiccccaaaaattttteeee ttttto o o o ttttthe he he he MiMiMiMiMiccccrrrrrooooelisaelisaelisaelisa StrippStrippStrippStrippStripplllllaaaaattttte.e.e.e.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.2. 2. 2. 2. Add Add Add standardstandardstandard: : : Set Standard wellsSet Standard wellsSet Standard wells, , , testing sample welltesting sample welltesting sample wells. s. s. Add standard 50μl to standard wellAdd standard 50μl to standard wellAdd 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.3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.3. Add Sample: Add testing sample 10μl Then add sample diluent 40μl to testing sample well; Blank well doesn’t add anyting.3. 3. 3. 3. Add Add Add SampleSampleSample: Add testing s: Add testing s: Add testing sampleampleample 1 1 10μl 0μl 0μl Then aThen aThen add dd dd sssample diluent ample diluent ample diluent 4440μl to testing sample well0μl to testing sample well0μl to testing sample well; B; B; Blank welllank welllank well doesn doesn doesn’’’t add anyting.t add anyting.t add anyting.
4. Add100μlofHRP-conjugate reagent to each well,cover with an adhesive stripandincubatefor60 minutes at37°C.4. Add100μlofHRP-conjugate reagent to each well,cover with an adhesive stripandincubatefor60 minutes at37°C.4. Add100μlofHRP-conjugate reagent to each well,cover with an adhesive stripandincubatefor60 minutes at37°C.4. 4. 4. 4. 4. AAAAAdddddddd 101010100μ0μ0μ0μllll ofofofof HRP-conjugate reagentHRP-conjugate reagentHRP-conjugate reagent to each well to each well to each well,,, cccover with aover with aover with annn adhesive strip adhesive strip adhesive strip andandandand iiiiincubncubncubncubaaaaattttteeee ffffforororor 66660 minutes0 minutes0 minutes0 minutes at at at at 333377777°°°°CCCCC.... 
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.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.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.5555. . . . Aspirate each well and wash, repeating the process Aspirate each well and wash, repeating the process Aspirate each well and wash, repeating the process fourfourfour times for a total of f times for a total of f times for a total of five ive ive washes.washes.washes. Wash by filling each well with Wash Wash by filling each well with Wash Wash by filling each well with Wash SolutionSolutionSolution (400 (400 (400μμμμllll) using a squirt bottle, manifold) using a squirt bottle, manifold) using a squirt bottle, manifold dispenserdispenserdispenser or autowasher. Complete removal of liquid at each step is essential to gooor autowasher. Complete removal of liquid at each step is essential to gooor autowasher. Complete removal of liquid at each step is essential to good d d performance. After the last wash, remove any remaining Wash performance. After the last wash, remove any remaining Wash performance. After the last wash, remove any remaining Wash SolutionSolutionSolution by aspirating or by aspirating or by aspirating or decanting. Invert the plate and blot it against clean paper towels.decanting. Invert the plate and blot it against clean paper towels.decanting. 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.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.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.6666. . . . Add chromogen solution A 50μl and chromogen solution B 50μl to each well.Add chromogen solution A 50μl and chromogen solution B 50μl to each well.Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix  Gently mix  Gently mix and and and incubate for 15 minutes at incubate for 15 minutes at incubate for 15 minutes at 373737°C°C°C°C. . . Protect from lightProtect from lightProtect from lightProtect from lightProtect 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 not7. 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 not7. 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 not7. 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 not7777. . . . Add 50Add 50Add 50μμμμl l l l Stop Solution to each well. The color in the wells should change from blue toStop Solution to each well. The color in the wells should change from blue toStop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does notyellow. If the color in the wells is green or the color change does notyellow. If the color in the wells is green or the color change does not

appear uniform,gently tap the plate to ensure thorough mixing.appear uniform,gently tap the plate to ensure thorough mixing.appear uniform,gently tap the plate to ensure thorough mixing.appear uniform,gently tap the plate to ensure thorough mixing.appear uniform,appear uniform,appear uniform, gently tap the plate to ensure thorough mixinggently tap the plate to ensure thorough mixinggently tap the plate to ensure thorough mixing...
8. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes.8. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes.8. ReadtheOpticalDensity(O.D.)at450nmusinga microtiterplatereaderwithin15minutes.8888. . . . RRRRRRReadeadeadeadeadead ttttttthehehehehehe OOOOOOOpppppptititititititicalcalcalcalcalcal DDDDDDDenenenenenensitsitsitsitsitsitsityyyyyy (((((((OOOOOOO......DDDDDDD.).).).).).) atatatatatat 450450450450450450 nmnmnmnmnmnm uuuuuusisisisisisisingngngngngng a a a a a a mmmmmmmiiiiiiiccccccrrrrrrrooooootittittittittittittiterererererer pppppplllllllaaaaaaattttttteeeeee rrrrrrreadereadereadereadereadereader witwitwitwitwitwitwithhhhhhiiiiiiinnnnnn 15151515151515 mmmmmmmiiiiiiinunununununuttttttteeeeeesssssss......
Calculation of resultsCalculation of resultsCalculation of resultsCCCCCCCalculation of resultsalculation of resultsalculation of resultsalculation of resultsalculation of resultsalculation of resultsalculation of results

1. 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.
2. 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.
3. 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.
4. 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.
5. The sensitivity by this assay is 1.0 ng/mL.
6. Standard curve

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.

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. 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. 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. 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. 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. 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. 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.

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. 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. 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. 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. 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. 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. 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.

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. 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. 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. 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. 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. 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. 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.

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.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.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.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.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.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.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 ng/mL.

The sensitivity by this assay is 1.0 ng/mL.The sensitivity by this assay is 1.0 ng/mL.The sensitivity by this assay is 1.0 ng/mL.The sensitivity by this assay is 1.0 ng/mL.The sensitivity by this assay isThe sensitivity by this assay isThe sensitivity by this assay is 1.0 ng/mL. 1.0 ng/mL. 1.0 ng/mL.

Standard curve

Standard curveStandard curveStandard curveStandard curveStandard curveStandard curveStandard curve

Storage： 2-8℃.
validity： six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!

Storage： 2-8℃.Storage： 2-8℃.SSStorage： torage： torage： ： 2-82-82-8℃.℃.℃..
validity： six months.validity： six months.validityvalidityvalidity： six months.： six months.：： six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!