胰蛋白酶(Trypsin)是一种来自 PA 族超家族的丝氨酸蛋白酶，在许多脊椎动物的消化系统中发现，它能水解蛋白质。Trypsin 在小肠中形成，当它的原酶形式，即由胰腺产生的胰蛋白酶原被激活后，就会形成胰蛋白酶。它主要在氨基酸赖氨酸或精氨酸的羧基侧切割肽链。Trypsin 被用于许多生物技术过程。这个过程通常被称为胰蛋白酶蛋白分解或胰蛋白酶化，用胰蛋白酶消化/处理过的蛋白质被称为胰蛋白酶化。
实验原理：
本试剂盒采用双抗体夹心法酶联免疫吸附试验（ELISA）。往预先包被有人胰蛋白酶(trypsin)捕获抗体的微孔中，依次加入样本、标准品、生物素标记的检测抗体，HRP酶结合物，中间经过温育和洗涤，用底物TMB显色，TMB在过氧化物酶(HRP)的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样本中的人胰蛋白酶(trypsin)呈正相关。用酶标仪在 450nm 波长下测定吸光度（OD值），计算样本浓度。
注意事项：
1. 严格按照规定的时间和温度进行温育以保证准确结果。所有试剂都必须在使用前达到室温20-25℃。使用后立即冷藏试剂。
2. 洗板不正确可能导致不准确的结果。在加入底物前确保尽量吸干孔内液体。整个过程中不要让微孔干燥时间过长。
3. 清洁板底残留的液体和手指印，否则影响OD值。
4. 底物显色液应呈无色的颜色，已经变蓝的底物液不能使用。
5. 避免试剂和样本的交叉污染以免造成错误结果。
6. 在储存和温育时避免强光直接照射。
7. 任何反应试剂不能接触漂白溶剂或漂白溶剂所散发的强烈气体。任何漂白成分都会破坏试剂盒中试剂的生物活性。
8. 不能使用过期产品，不同货号和批号组分不得混用。
9. 试剂盒以外来源的重组蛋白可能会出现与本试剂盒抗体不匹配而不被识别的情况。
10. 如果可能传播疾病，所有的样本都应管理好，按照规定的程序处理样本和检测装置。
试剂盒组成：
| 项目号 | 物理外观 | 组件 | 48T | 96T | 储存 | | EJ1514725A | — | 预包被酶标板 | 48孔 | 96孔 | 2-8℃. | | EJ1514725B | 固体 | 标准品 | 1支 | 2支 | 2-8℃. | | EJ1514725C | 液体 | 通用稀释液 | 1×20 mL | 2×20 mL | 2-8℃. | | EJ1514725D | 液体 | 浓缩Biotin-抗体 (100×) | 60 μL | 120 μL | 2-8℃. | | EJ1514725E | 液体 | 浓缩酶结合物 (100×) | 60 μL | 120 μL | 2-8℃. | | EJ1514725F | 液体 | 洗涤液 (20×) | 1×10 mL | 2×10 mL | 2-8℃. | | EJ1514725G | 液体 | 底物 (TMB) | 5 mL | 10 mL | 2-8℃. | | EJ1514725H | 液体 | 终止液 | 3 mL | 6 mL | 2-8℃. | | EJ1514725I | — | 封板膜 | 4张 | 4张 | 2-8℃. |
备注： EJ1514725B、EJ1514725D、EJ1514725E、EJ1514725F需按说明书进行稀释 |
样本处理及要求：
1. 试剂盒检测范围不等同于样本中待测物的浓度范围，建议实验前通过相关文献预估样本中待测物的浓度并通过预实验确定样本的实际浓度。如果样本中待测物浓度过高或过低，请对样本做适当的稀释或浓缩。
2. 若所检样本不在说明书所列样本类型之中，建议做预实验验证其检测有效性。
3. 血清：将收集于血清分离管的全血样本在室温放置2小时或2-8℃过夜，然后1000× g离心20分钟，取上清即可，或将上清置于-20℃或-80℃保存，但应避免反复冻融。
4. 血浆：用EDTA或肝素作为抗凝剂采集样本，并将样本在采集后的30分钟内于2-8℃ 1000× g离心15分钟，取上清即可检测，或将上清置于-20℃或-80℃保存，但应避免反复冻融。
5. 组织匀浆：用预冷的PBS (0.01M, pH=7.4)冲洗组织，去除残留血液 (匀浆中裂解的红细胞会影响检测结果)，称重后将组织剪碎。将剪碎的组织与对应体积的PBS (一般按1:9的重量体积比，比如1g的组织样本对应9mL的PBS，具体体积可根据实验需要适当调整，并做好记录。推荐在PBS中加入蛋白酶抑制剂)加入玻璃匀浆器中，于冰上充分研磨或匀浆机研磨。为了进一步裂解组织细胞，可以对匀浆液进行超声破碎，或反复冻融。最后将匀浆液于5000× g离心5~10分钟，取上清检测。
6. 细胞培养物上清：请1000× g离心20分钟，取上清即可检测，或将上清置于-20℃或-80℃保存，但应避免反复冻融。
7. 细胞裂解液：贴壁细胞用预冷PBS轻轻清洗，然后用胰蛋白酶消化，1000× g离心5分钟后收集细胞；悬浮细胞可直接离心收集。收集的细胞用预冷PBS清洗3次，每1×10⁶个细胞中加入150-200 μL PBS重悬 (推荐在PBS中加入蛋白酶抑制剂；若含量很低可适当减少PBS体积)并通过反复冻融或超声使细胞破碎。将提取液于2-8℃，1500× g离心10分钟，取上清检测。
8. 其它生物样本：1000× g离心20分钟，取上清即可检测。
9. 样本外观：样本应清澈透明，悬浮物应离心去除。
10. 样本保存：样本收集后若在1周内进行检测的可保存于4℃，若不能及时检测，请按一次使用量分装，冻存于-20℃ (1个月内检测)，或-80℃ (6个月内检测)，避免反复冻融，样本溶血会影响最后检测结果，因此溶血样本不宜进行此项检测。
样本稀释方案：
请提前预估样本的浓度范围，如果您的检测样本需要稀释，参考稀释方案如下：
稀释100倍：一步稀释。取5μL样本到495μL通用稀释液内，做100倍稀释；
稀释1000倍：两步稀释。取5μL样本到95μL通用稀释液内，做20倍稀释，再取5μL20倍稀释样本到245μL通用稀释液内，做50倍稀释，总共稀释1000 倍；
稀释100000 倍：三步稀释。取5μL样本到195μL通用稀释液内，做40倍稀释，再取5μL40倍稀释样本到245μL通用稀释液内，做50倍稀释，最后取5μL 2000 倍稀释样本到245μL通用稀释液内，做50倍稀释，总共稀释100000倍；
每步稀释时取液量不少于3μL，稀释倍数不超过100倍。每步稀释都需混合均匀，避免起泡。
实验所需自备试验器材：
1. 酶标仪 (450 nm)
2. 高精度移液器及吸头：0.5-10 μL、5-50 μL、20-200 μL、200-1000 μL
3. 37 ℃恒温箱
4. 蒸馏水或去离子水
检测前准备工作：
1. 请提前 10 分钟从冰箱中取出试剂盒，平衡至室温。
2. 标准品梯度工作液配制：加入 1mL 通用稀释液至冻干标准品中，静置15分钟待其完全溶解后轻轻混匀(浓度为20ng/mL)，然后按照以下浓度：
20ng/mL、10ng/mL、5ng/mL、2.5ng/mL、1.25ng/mL、0.625ng/mL、0.312ng/mL、0ng/mL进行稀释。
倍比稀释方法：取7支EP管，每管中加入500μL通用稀释液,20ng/mL 的标准品工作液中吸取500μL到第一支EP管中混匀配成10ng/mL 的标准品工作液，按此步骤往后依次吸取混匀。最后一管直接作为空白孔，不需要再从倒数第二管中吸取液体，具体如下图。
3. 生物素化检抗工作液配制：使用前15分钟将100×浓缩生物素化检抗于1000×g离心1分钟，以通用稀释液将100×浓缩生物素化检抗稀释成1×工作浓度(例：10μL浓缩液+990μL通用稀释液)，现配现用。
4. 酶结合物工作液配制：使用前15分钟将100×浓缩酶结合物于1000×g离心1分钟，以通用稀释液将100×浓缩酶结合物稀释成1×工作浓度(例：10μL浓缩液+990μL通用稀释液)，现配现用。
5. 1×洗涤液配制：取10mL20×洗涤液到190mL蒸馏水中（从冰箱中取出的浓缩洗涤液可能有结晶，属于正常现象，可放置室温，待结晶完全溶解后再配制)。
操作步骤：
1. 从室温平衡10分钟后的铝箔袋中取出所需板条，剩余板条用自封袋密封放回4℃。
2. 加样：分别将样本或阳性、阴性对照品按照100 μL每孔加入相应孔中，空白孔加入100 μL通用稀释液。盖上封板膜后37℃温育60分钟。（建议：将待测样本用通用稀释液最低稀释1倍后再加入酶标板内测试。从而减少基质效应对测试结果的影响，最后计算样本浓度时需乘以对应的稀释倍数。所有的待测样本和阳性、阴性对照品在检测中建议设立复孔）。
3. 加生物素化抗原：取出酶标板，弃去液体，不用洗涤。每孔直接加入100 μL生物素化抗原工作液，盖上封板膜后37℃温育60分钟。
4. 洗板：弃去液体，每孔加入300 μL 1x洗涤液，静置1分钟，甩去洗涤液，吸水纸上拍干，如此重复洗板3次 (也可用洗板机洗板)。
5. 加酶结合物工作液：每孔加入100 μL酶结合物工作液，盖上封板膜后37℃温育30分钟。
6. 洗板：弃去液体按步骤4洗涤方法，洗板5次。
7. 加底物：每孔加入90 μL底物 (TMB)，盖上封板膜，37℃避光温育15分钟。
8. 加终止液：取出酶标板，每孔直接加入50 μL终止液，立即在450 nm波长处测定各孔的OD值。
实验结果计算：
结果判断：
1. 计算标准品和样本复孔的平均 OD 值并减去空白孔的 OD 值作为校正值。以浓度为横坐标，OD 值为纵坐标，在双对数坐标纸上绘出四参数逻辑函数的标准曲线。
2. 若样本 OD 值高于标准曲线上限，应适当稀释后重测并在计算样本浓度时乘以相应的稀释倍数。
典型数据和参考曲线：
以下数据和曲线仅供参考，实验者需根据自己的实验建立标准曲线。
浓度(ng/mL)
| 20
| 10 | 5 | 2.5 | 1.25 | 0.625 | 0.312 | 0 | OD 值
| 2.22 | 1.56 | 0.92 | 0.63 | 0.42 | 0.25 | 0.18 | 0.08 | 校正 OD 值
| 2.14 | 1.48 | 0.84 | 0.55 | 0.34 | 0.17 | 0.1 | —
|
|
注意：本图仅供参考，应以每次实验数据所绘制标准曲线计算样本含量。
试剂盒性能：
1. 重复性：板内变异系数小于10%，板间变异系数小于10%。
2. 回收率：在选取的健康人血清、血浆和细胞培养上清中加入3个不同浓度水平的人trypsin，计算回收率。
样本类型
| 范围（%）
| 平均回收率（%）
|
血清 (n=8)
| 84-101
| 96
|
血浆 (n=8)
| 93-104
| 102
|
细胞培养上清 (n=8)
| 96-108
| 105
|
3. 线性稀释：分别在选取的4份健康人血清、血浆和细胞培养上清中加入高浓度人trypsin，在标准曲线动力学范围内进行稀释，评估线性。
| 稀释比例 | 回收率（%） | 血清 | 血浆 | 细胞培养上清 | | 1:2 | 范围 | 84-95 | 88-97 | 90-110 | | 1:2 | 平均回收率 | 91 | 94 | 96 | | 1:4 | 范围 | 89-103 | 87-108 | 105-115 | | 1:4 | 平均回收率 | 94 | 98 | 108 |
|
问题分析：
若实验效果不好，请及时对显色结果拍照，保存实验数据，保留所用板条及未使用试剂，然后联系我公司技术支持为您解决问题。同时您也可以参考以下资料（问题描述、可能原因及相应对策）：
1. 标曲线性差：
①标准品稀释不正确：确保标准品按照推荐方法溶解和稀释。
②移液不准确：定期校准移液器并检查枪头密封性。
③反应液蒸发：用封板膜密封酶标板。
④洗板不彻底：足够的洗涤次数和加入足量洗涤液。
⑤孔底有异物：读数前清洁板底。
2. 显色弱或无色：
①孵育时间不够：确保孵育时间。
②孵育温度不正确：按推荐温度孵育。
③试剂体积添加不足：检查移液器并严格按照操作步骤操作。
④稀释不正确：检查试剂稀释步骤。
⑤酶结合物失活：混合酶结合物和底物，通过显色反应检查。
3. OD值低：
①酶标仪设置不正确：检查仪器波长
②没加终止液：加入适量终止液
③读板时等待时间太长：及时读板
④样本含量过高：通过预实验确定合适的稀释倍数
⑤样本含量过低：通过预实验确定合适的稀释倍数
4. 背景高：
①显色液被污染：更换显色液
②显色时间太长：控制显色时间
③检抗或酶结合物稀释错误：使用推荐稀释方法
④洗板不彻底：足够的洗涤次数和加入足量洗涤液
Trypsin is a serine protease belonging to the PA superfamily, which exists in the digestive system of numerous vertebrates and functions to hydrolyze proteins. It is generated in the small intestine when trypsinogen secreted by the pancreas is activated. This enzyme primarily cleaves peptide chains at the carboxyl side of lysine and arginine residues. Trypsin is widely applied in diverse biotechnological procedures. The degradation process mediated by trypsin is commonly termed tryptic digestion, and proteins processed with trypsin are defined as trypsinized proteins.
Experimental Principle:
This kit adopts double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotinylated detection antibodies and HRP conjugates are sequentially added into microplates pre-coated with human trypsin capture antibodies. Following incubation and washing steps, chromogenic substrate TMB is applied. Catalyzed by horseradish peroxidase (HRP), TMB turns blue and subsequently shifts to yellow under acidic conditions. The color intensity is positively correlated with the concentration of human trypsin in samples. The optical density (OD value) is measured at 450 nm via a microplate reader to calculate sample concentration.
Precautions:
1. Incubate strictly at the specified time and temperature for accurate results. All reagents must reach room temperature (20–25°C) before use. Refrigerate reagents immediately after use.
2. Improper washing may cause inaccurate results. Ensure wells are completely aspirated before adding substrate. Do not let microwells dry out for extended periods.
3. Wipe residual liquid and fingerprints from the plate bottom, as they affect OD readings.
4. TMB substrate should be colorless; do not use if it turns blue.
5. Avoid cross-contamination of reagents and samples to prevent false results.
6. Protect from direct strong light during storage and incubation.
7. Do not expose reagents to bleach or its fumes, as bleach inactivates kit bioactivity.
8. Do not use expired products. Do not mix components from different catalog numbers/lots.
9. Recombinant proteins from external sources may not be recognized by kit antibodies due to mismatch.
10. Handle all samples as potentially infectious. Dispose of samples and test devices per regulations.
Kit Components
| Item No. | Appearance | Components | 48T | 96T | Storage | | EJ1514725A | — | Pre-coated Assay Plate | 48 wells | 96 wells | 2-8℃. | | EJ1514725B | Solid | Standard | 1 vial | 2 vials | 2-8℃. | | EJ1514725C | Liquid | Universal Diluent | 1×20 mL | 2×20 mL | 2-8℃. | | EJ1514725D | Liquid | Biotin-antibody (100×) | 60 μL | 120 μL | 2-8℃. | | EJ1514725E | Liquid | Streptavidin-HRP (100×) | 60 μL | 120 μL | 2-8℃. | | EJ1514725F | Liquid | Wash Buffer (20×) | 1×10 mL | 2×10 mL | 2-8℃. | | EJ1514725G | Liquid | TMB Substrate | 5 mL | 10 mL | 2-8℃. | | EJ1514725H | Liquid | Stop Solution | 3 mL | 6 mL | 2-8℃. | | EJ1514725I | — | Plate Sealer | 4 pieces | 4 pieces | 2-8℃. |
Note: EJ1514725B、EJ1514725D、EJ1514725E and EJ1514725F shall be diluted in accordance with the instructions. |
Sample Handling and Requirements
1. The detection range of the kit is not equivalent to the concentration range of the analyte in samples. It is recommended to estimate the analyte concentration in samples by referring to relevant literature before the experiment, and confirm the actual concentration through pre-experiments. Appropriately dilute or concentrate samples if the analyte concentration is too high or too low.
2. If the test sample type is not listed in the instructions, a pre-experiment is recommended to verify the detection applicability.
3. Serum: Place whole blood collected in serum separation tubes at room temperature for 2 hours or at 2–8 °C overnight. Centrifuge at 1000× g for 20 minutes and collect the supernatant. The supernatant can be stored at -20 °C or -80 °C; repeated freeze-thaw cycles must be avoided.
4. Plasma: Collect samples using EDTA or heparin as anticoagulants. Within 30 minutes after collection, centrifuge the sample at 1000× g and 2–8 °C for 15 minutes, then collect the supernatant for detection. The supernatant can be stored at -20 °C or -80 °C with avoidance of repeated freeze-thawing.
5. Tissue homogenate: Rinse tissues with pre-cooled PBS (0.01 M, pH=7.4) to remove residual blood (lysed red blood cells in homogenate will interfere with test results). Weigh and mince the tissues. Transfer minced tissues into a glass homogenizer with an appropriate volume of PBS (generally at a weight-to-volume ratio of 1:9, e.g., 1 g tissue with 9 mL PBS; the volume may be adjusted properly with experimental records. Addition of protease inhibitor to PBS is recommended). Fully grind on ice or use a homogenizer. For thorough cell lysis, perform ultrasonication or repeated freeze-thaw treatment on the homogenate. Finally, centrifuge the homogenate at 5000× g for 5–10 minutes and collect the supernatant for testing.
6. Cell culture supernatant: Centrifuge at 1000× g for 20 minutes and collect the supernatant for detection. Store the supernatant at -20 °C or -80 °C and avoid repeated freeze-thaw cycles.
7. Cell lysate: Gently wash adherent cells with pre-cooled PBS, digest with trypsin, and collect cells after centrifugation at 1000× g for 5 minutes. Suspension cells can be collected by direct centrifugation. Wash harvested cells three times with pre-cooled PBS. Resuspend every 1×10⁶ cells in 150–200 μL PBS (protease inhibitor addition is recommended; reduce PBS volume appropriately if the target content is low). Lyse cells by repeated freeze-thawing or ultrasonication. Centrifuge the extract at 1500× g and 2–8 °C for 10 minutes, and collect the supernatant for detection.
8. Other biological samples: Centrifuge at 1000× g for 20 minutes and collect the supernatant for testing.
9. Sample appearance: Samples shall be clear and transparent; suspended substances must be removed by centrifugation.
10. Sample storage: Samples to be tested within 1 week can be stored at 4 °C. For delayed detection, aliquot samples into single-use portions and store frozen at -20 °C (within 1 month) or -80 °C (within 6 months). Repeated freeze-thawing is prohibited. Hemolysis will affect test results, so hemolyzed samples are not suitable for this assay.
Sample Dilution Protocol:
Estimate the sample concentration range in advance. Refer to the following scheme if dilution is required.
100-fold dilution: Single-step dilution. Add 5 μL sample into 495 μL universal diluent.
1000-fold dilution: Two-step dilution. First, add 5 μL sample into 95 μL universal diluent for 20-fold dilution. Then take 5 μL of the diluted solution and add into 245 μL diluent for 50-fold dilution, reaching a total 1000-fold dilution.
100000-fold dilution: Three-step dilution. First, add 5 μL sample into 195 μL diluent for 40-fold dilution. Second, take 5 μL of the above solution into 245 μL diluent for 50-fold dilution. Finally, take 5 μL of the 2000-fold diluted solution into 245 μL diluent for another 50-fold dilution, achieving total 100000-fold dilution.
The volume taken for each dilution shall not be less than 3 μL, and single dilution fold shall not exceed 100 times. Mix thoroughly without foaming after each dilution step.
Self-supplied laboratory equipment required for the experiment:
1. Microplate reader (450 nm)
2. High-precision pipettes & tips: 0.5–10 μL, 5–50 μL, 20–200 μL, 200–1000 μL
3. 37°C incubator
4. Distilled or deionized water
Pre-test Preparation:
1. Take the kit out of the refrigerator 10 minutes in advance and equilibrate it to room temperature.
2. Preparation of serial standard working solutions
Add 1 mL universal diluent into the lyophilized standard. Let it stand for 15 minutes until fully dissolved, then mix gently to obtain the stock solution at 20 ng/mL. Prepare serial dilutions at concentrations of 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL and 0 ng/mL.
Double dilution method: Prepare 7 EP tubes and add 500 μL universal diluent into each tube. Pipette 500 μL of 20 ng/mL standard solution into the first tube and mix well to make 10 ng/mL solution. Repeat the operation sequentially for subsequent concentrations. The last tube serves as blank control without liquid transfer from the penultimate tube. Refer to the figure below for details.
3. Preparation of biotinylated detection antibody working solution
Centrifuge 100× concentrated biotinylated detection antibody at 1000×g for 1 minute 15 minutes prior to use. Dilute the concentrate to 1× working concentration with universal diluent (e.g. 10 μL concentrate mixed with 990 μL universal diluent). Prepare freshly before use.
4. Preparation of enzyme conjugate working solution
Centrifuge 100× concentrated enzyme conjugate at 1000×g for 1 minute 15 minutes prior to use. Dilute the concentrate to 1× working concentration with universal diluent (e.g. 10 μL concentrate mixed with 990 μL universal diluent). Prepare freshly before use.
5. Preparation of 1× washing solution
Add 10 mL 20× washing solution into 190 mL distilled water. Crystallization may occur in refrigerated concentrated washing solution, which is normal. Place it at room temperature until crystals dissolve completely before preparation.
Assay Procedure
1. Remove the required plate strips from the aluminum foil pouch (equilibrated at room temperature for 10 minutes). Seal the remaining strips in a zip-lock bag and return to 4°C.
2. Sample Loading: Add 100 μL of sample/positive control/negative control to the respective wells. Add 100 μL of Universal Diluent to blank wells. Cover with a plate sealer and incubate at 37°C for 60 minutes.
Recommendation: Dilute the test sample at least 1-fold with Universal Diluent before adding to the ELISA plate to reduce matrix effects on test results. Multiply the final sample concentration by the corresponding dilution factor. It is recommended to set up duplicate wells for all test samples and positive/negative controls during the test.
3. Add Biotin-Antigen: Remove the ELISA plate, discard the liquid (do not wash). Add 100 μL of Biotin-Antigen Working Solution to each well directly. Cover with a plate sealer and incubate at 37°C for 60 minutes.
4. Wash Plate: Discard the liquid. Add 300 μL of 1× Wash Solution to each well, let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat the washing process 3 times (a plate washer can also be used).
5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a plate sealer and incubate at 37°C for 30 minutes.
6. Wash Plate: Discard the liquid and wash 5 times according to the washing method in Step 4.
7. Add Substrate: Add 90 μL of Substrate (TMB) to each well. Cover with a plate sealer and incubate at 37°C in the dark for 15 minutes.
8. Add Stop Solution: Remove the ELISA plate, add 50 μL of Stop Solution to each well directly, and immediately measure the OD value of each well at a wavelength of 450 nm.
Calculation of Experimental Results:
Result Evaluation:
1. Calculate the average optical density (OD) value of duplicate wells for standards and samples, then subtract the OD value of blank wells to obtain corrected values. Plot the four-parameter logistic standard curve on log-log graph paper with concentration as the horizontal axis and OD value as the vertical axis.
2. Retest the sample after proper dilution if its OD value exceeds the upper limit of the standard curve. Multiply the corresponding dilution factor when calculating sample concentration.
Typical Data and Reference Curve:
The following data and curve are for reference only. Establish the standard curve based on actual experimental results.
Concentration (ng/mL)
| 20
| 10 | 5 | 2.5 | 1.25 | 0.625 | 0.312 | 0 | OD
| 2.22 | 1.56 | 0.92 | 0.63 | 0.42 | 0.25 | 0.18 | 0.08 | Corrected OD
| 2.14 | 1.48 | 0.84 | 0.55 | 0.34 | 0.17 | 0.1 | —
|
|
Note: This figure is for reference only. Sample content shall be calculated according to the standard curve plotted from experimental data of each test.
Kit Performance:
1. Reproducibility: Intra-assay coefficient of variation is less than 10%, inter-assay coefficient of variation is less than 10%.
2. Recovery rate: Three concentrations of human trypsin were spiked into healthy human serum, plasma and cell culture supernatant respectively, and the recovery rate was calculated.
Sample Type
| Range (%)
| Average Recovery Rate (%)
|
Serum (n=8)
| 84-101
| 96
|
Plasma (n=8)
| 93-104
| 102
|
Cell culture supernatant (n=8)
| 96-108
| 105
|
3. Linear dilution: High-concentration human trypsin was added into 4 batches of selected healthy human serum, plasma and cell culture supernatant. Samples were diluted within the dynamic range of standard curve to evaluate linearity.
| Dilution Ratio | Recovery Rate (%) | Serum | Plasma | Cell Culture Supernatant | | 1:2 | Range | 84-95 | 88-97 | 90-110 | | 1:2 | Average Recovery Rate | 91 | 94 | 96 | | 1:4 | Range | 89-103 | 87-108 | 105-115 | | 1:4 | Average Recovery Rate | 94 | 98 | 108 |
|
Troubleshooting:
If unsatisfactory experimental results occur, take photos of color reaction immediately, save experimental data, reserve used plate strips and unused reagents, then contact our technical support for solutions. You can also refer to the following contents including problem description, potential causes and corresponding solutions:
1. Poor standard curve linearity
① Incorrect standard dilution: Dissolve and dilute standards strictly in accordance with recommended protocols.
② Inaccurate pipetting: Calibrate pipettes regularly and check airtightness of pipette tips.
③ Evaporation of reaction solution: Seal the microplate with sealing film.
④ Inadequate washing: Ensure sufficient washing cycles and adequate volume of washing solution.
⑤ Foreign matters at well bottom: Clean the plate bottom before detection.
2. Weak or absent color development
① Insufficient incubation duration: Follow specified incubation time.
② Improper incubation temperature: Incubate at recommended temperature.
③ Insufficient reagent dosage: Verify pipettes and operate strictly as instructed.
④ Wrong dilution procedure: Recheck reagent dilution steps.
⑤ Inactivated enzyme conjugate: Mix enzyme conjugate with substrate and verify activity via color reaction.
3. Low OD value
① Incorrect microplate reader setting: Check detection wavelength.
② Termination solution not added: Add adequate termination solution.
③ Excess waiting time before reading: Measure absorbance promptly.
④ Excessively high sample concentration: Determine proper dilution ratio via preliminary test.
⑤ Excessively low sample concentration: Determine proper dilution ratio via preliminary test.
4. High background signal
① Contaminated chromogenic solution: Replace with fresh chromogenic solution.
② Excessive color developing time: Control reaction duration strictly.
③ Wrong dilution of detection antibody or enzyme conjugate: Adopt recommended dilution method.
④ Incomplete plate washing: Perform sufficient washing times and use enough washing buffer.
ZJ26F0636200
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