转化生长因子 β（TGF‑β）是一种多功能的细胞因子，属于转化生长因子超家族，包括三种不同的哺乳动物异构体（TGF‑β 1 至 3，HGNC 符号 TGFB1、TGFB2、TGFB3）和许多其他信号蛋白。TGFB 蛋白由所有白血球系产生。被激活的 TGF‑β 与其他因子复合形成丝氨酸 / 苏氨酸激酶复合物，与 TGF‑β 受体结合。TGF‑β 受体由 1 型和 2 型受体亚单位组成。
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）。往预先包被有人转化生长因子 β(TGF‑β) 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转化生长因子 β(TGF‑β) 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：
| 组分货号 | 组分名称 | 物理外观 | 包装1 | 包装2 | 储存 | | EJ1514990A | 预包被酶标板 | — | 48孔 | 96孔 | 2-8℃. | | EJ1514990B | 标准品 | 固体 | 1支 | 2支 | 2-8℃. | | EJ1514990C | 通用稀释液 | 液体 | 1×20mL | 2×20mL | 2-8℃. | | EJ1514990D | 浓缩生物素化检抗100× | 液体 | 60μL | 120μL | 2-8℃. | | EJ1514990E | 浓缩酶结合物100× | 液体 | 60μL | 120μL | 2-8℃. | | EJ1514990F | 活化试剂①(1N HCl) | 液体 | 3mL | 5mL | 2-8℃. | | EJ1514990G | 活化试剂②(1.2N NaOH) | 液体 | 3mL | 5mL | 2-8℃. | | EJ1514990H | 20×洗涤液 | 液体 | 1×10mL | 2×10mL | 2-8℃. | | EJ1514990I | 底物（TMB） | 液体 | 5mL | 10mL | 2-8℃. | | EJ1514990J | 终止液 | 液体 | 3mL | 6mL | 2-8℃. | | EJ1514990K | 封板膜 | — | 4张 | 4张 | 2-8℃. |
注：
1. EJ1514990B、EJ1514990D、EJ1514990E、EJ1514990H需按说明书进行稀释。
2. 试剂盒各组分开封后，若 1 周内使用，可于 2‑8℃条件下保存。如保存时长超过 1 周，EJ1514990A、EJ1514990B、EJ1514990C、EJ1514990D、EJ1514990E 需避光置于‑20℃冻存；其余试剂保存于 2‑8℃。避免反复冻融。 |
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 1×10^6 个细胞中加入 150-200μL 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。
样本活化：
生物样本中的 TGF‑β 通常以无活性的形式存在，在检测 TGF‑β 前须进行活化处理。
活化方法如下：
1. 血清、血浆：每 5μL 体积样品中加入 355μL 通用稀释液，混匀后加入 10μL 活化试剂①(1N HCl)，混匀后室温孵育 10 分钟。再加入 10μL 活化试剂②(1.2N NaOH)，混匀后立即检测。
注意：样本被稀释了若干倍！计算样本浓度时需要乘以相应的倍数。
2. 组织匀浆、细胞培养上清：每 100μL 样品中加入 20μL 活化试剂①(1N HCl)，混匀后室温孵育 10 分钟。再加入 20μL 活化试剂②(1.2N NaOH)，混匀后立即检测。
注意：样本被稀释了若干倍！计算样本浓度时需要乘以相应的倍数。
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 5μL 20 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 5μL 40 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 (450nm)
2. 高精度移液器及吸头：0.5-10μL、5-50μL、20-200μL、200-1000μL
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2000pg/mL)，然后按照以下浓度：
2000pg/mL、1000pg/mL、500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.2pg/mL、0pg/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，2000pg/mL 的标准品工作液中吸取 500μL 到第一支 EP 管中混匀配成 1000pg/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。
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3. 生物素化检抗工作液配制：使用前 15 分钟将 100× 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 10mL 20× 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：分别将样本或不同浓度标准品按照 100μL 每孔加入相应孔中，空白孔加入 100μL 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。所有的待测样本和标准品在检测中建议设立复孔）。
3. 加生物素化检抗：取出酶标板，弃去液体，不用洗涤。每孔直接加入 100μL 生物素化检抗工作液，盖上封板膜后 37℃温育 60 分钟。
4. 洗板：弃去液体，每孔加入 300μL 1× 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 100μL 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 90μL 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 50μL 终止液，立即在 450nm 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 (pg/mL) | 2000 | 1000 | 500 | 250 | 125 | 62.5 | 31.2 | 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 TGF‑β，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) | 92‑105 | 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 TGF‑β，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 | 88‑96 | 90‑110 |
| 1：2 | 平均回收率 |
| 91 | 93 | 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：
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。
Transforming growth factor‑β (TGF‑β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. It comprises three distinct mammalian isoforms (TGF‑β 1‑3, HGNC symbols: TGFB1, TGFB2, TGFB3), along with numerous other signaling proteins. TGFB proteins are produced by all leukocyte lineages. Upon activation, TGF‑β complexes with other factors to form a serine/threonine kinase complex that binds to the TGF‑β receptor. The TGF‑β receptor consists of type 1 and type 2 receptor subunits.
Assay Principle
This kit adopts the double‑antibody sandwich enzyme‑linked immunosorbent assay (ELISA). Add samples, standards, biotin‑labeled detection antibody and HRP conjugate sequentially into micro‑wells pre‑coated with human transforming growth factor‑β (TGF‑β) capture antibody. Incubate and wash between steps, then develop color with TMB substrate. Catalyzed by horseradish peroxidase (HRP), TMB turns blue, and converts to final yellow upon acid addition. The color intensity is positively correlated with the concentration of human TGF‑β in samples. Measure the optical density (OD value) at 450 nm using a microplate reader to calculate sample concentrations.
Precautions
1. Strictly follow the specified incubation time and temperature to guarantee accurate results. All reagents must be equilibrated to room temperature (20–25°C) before use. Refrigerate reagents immediately after use.
2. Improper plate washing may lead to inaccurate readings. Ensure complete removal of residual liquid in wells prior to substrate addition. Avoid prolonged drying of microwells throughout the procedure.
3. Wipe residual liquid and fingerprints from the bottom of the microplate, as contaminants will interfere with OD values.
4. The TMB substrate solution shall remain colorless; discard substrate solution that has turned blue.
5. Prevent cross-contamination between reagents and samples to avoid erroneous test results.
6. Avoid direct intense light exposure during reagent storage and incubation.
7. All reaction reagents must not come into contact with bleach solvents or their volatile fumes; bleach components will destroy the biological activity of kit reagents.
8. Do not use expired products; components from different lot numbers or batch codes cannot be mixed.
9. Recombinant proteins from external sources may fail to be recognized due to mismatched antibody affinity with kit capture antibodies.
10. Handle all potentially infectious samples properly, and dispose of specimens and testing devices in accordance with standardized biosafety protocols.
Kit Components:
| Component No. | Component Name | Appearance | 48T | 96T | Storage | | EJ1514990A | Pre-coated Assay Plate | — | 48wells | 96wells | 2-8℃. | | EJ1514990B | Standard | Solid | 1vial | 2 vials | 2-8℃. | | EJ1514990C | Universal Diluent | Liquid | 1×20mL | 2×20mL | 2-8℃. | | EJ1514990D | Biotin-antibody (100×) | Liquid | 60μL | 120μL | 2-8℃. | | EJ1514990E | Streptavidin-HRP (100×) | Liquid | 60μL | 120μL | 2-8℃. | | EJ1514990F | Activation Reagent① (1N HCl) | Liquid | 3mL | 5mL | 2-8℃. | | EJ1514990G | Activation Reagent② (1.2N NaOH) | Liquid | 3mL | 5mL | 2-8℃. | | EJ1514990H | Wash Buffer (20×) | Liquid | 1×10mL | 2×10mL | 2-8℃. | | EJ1514990I | TMB Substrate | Liquid | 5mL | 10mL | 2-8℃. | | EJ1514990J | Stop Solution | Liquid | 3mL | 6mL | 2-8℃. | | EJ1514990K | Plate Sealer | — | 4 pieces | 4 pieces | 2-8℃. |
Notes:
1. EJ1514990B、EJ1514990D、EJ1514990E and EJ1514990H shall be diluted in accordance with the instructions.
2. After opening, kit components may be stored at 2–8 °C if used within one week. For storage beyond one week, components EJ1514990A, EJ1514990B, EJ1514990C, EJ1514990D, and EJ1514990E should be stored at –20 °C in the dark. All other reagents may be kept at 2–8 °C. Avoid repeated freeze‑thaw cycles.
|
Sample Handling & Requirements
1. The detectable concentration range of the kit does not equal the analyte concentration range in raw samples. It is recommended to estimate sample analyte concentrations via relevant literature and conduct pre-tests to confirm actual concentrations before formal experiments. Dilute or concentrate samples appropriately if analyte levels fall outside the linear range.
2. If your sample matrix is not listed in this manual, perform a pre-test to verify assay compatibility.
3. Serum: Collect whole blood in serum separation tubes, incubate at room temperature for 2 hours or store overnight at 2–8°C, then centrifuge at 1000×g for 20 minutes. Harvest supernatant for immediate testing, or aliquot and store at -20°C / -80°C; avoid repeated freeze-thaw cycles.
4. Plasma: Collect blood with EDTA or heparin as anticoagulant. Centrifuge specimens within 30 minutes post-collection at 1000×g, 2–8°C for 15 minutes. Collect supernatant for immediate testing, or store at -20°C / -80°C; avoid repeated freeze-thaw cycles.
5 Tissue Homogenate: Rinse tissue with pre-cooled PBS (0.01 M, pH=7.4) to remove residual blood (lysed erythrocytes in homogenate interfere with detection results). Weigh and mince tissue, then mix tissue with PBS at a standard weight-to-volume ratio of 1:9 (e.g., 1 g tissue corresponds to 9 mL PBS; adjust volume as required and record details. Addition of protease inhibitor cocktail to PBS is recommended). Transfer the mixture to a glass homogenizer and grind thoroughly on ice, or use a mechanical homogenizer. For complete cell lysis, sonicate the homogenate or perform repeated freeze-thaw cycles. Finally, centrifuge the homogenate at 5000×g for 5–10 minutes and collect supernatant for testing.
6. Cell Culture Supernatant: Centrifuge culture medium at 1000×g for 20 minutes, collect supernatant for immediate testing, or store at -20°C / -80°C; avoid repeated freeze-thaw cycles.
7. Cell Lysate: For adherent cells, wash gently with pre-cooled PBS, digest with trypsin, then harvest cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be pelleted directly by centrifugation. Wash harvested cells 3 times with pre-cooled PBS, resuspend 1×10⁶ cells in 150–200 μL PBS (protease inhibitors are recommended; reduce PBS volume if analyte abundance is extremely low). Lyse cells via repeated freeze-thaw or sonication. Centrifuge the lysate at 1500×g, 2–8°C for 10 minutes and retain the supernatant for detection.
8. Other Biological Matrices: Centrifuge samples at 1000×g for 20 minutes and collect supernatant for testing.
9. Sample Appearance: Samples must be clear and transparent; all suspended particulates must be removed by centrifugation.
10. Sample Storage: Samples intended for testing within 1 week can be stored at 4°C. For delayed testing, aliquot samples into single-use volumes and freeze at -20°C (stable for 1 month) or -80°C (stable for 6 months). Avoid repeated freeze-thaw cycles. Hemolyzed samples are unsuitable for this assay, as hemolysis will distort final detection results.
Sample Activation
TGF‑β in biological samples generally exists in an inactive form. Activation treatment is required prior to TGF‑β detection.
Activation procedures are described below:
1. Serum and plasma: Add 355 μL Universal Diluent to 5 μL sample, mix well. Add 10 μL Activation Reagent ① (1 N HCl), mix and incubate at room temperature for 10 min. Add 10 μL Activation Reagent ② (1.2 N NaOH), mix thoroughly and test immediately.
Note: The sample is diluted. Multiply by the corresponding dilution factor when calculating sample concentration.
2. Tissue homogenate and cell culture supernatant: Add 20 μL Activation Reagent ① (1 N HCl) to 100 μL sample, mix well and incubate at room temperature for 10 min. Add 20 μL Activation Reagent ② (1.2 N NaOH), mix thoroughly and test immediately.
Note: The sample is diluted. Multiply by the corresponding dilution factor when calculating sample concentration.
Sample Dilution Protocols
Estimate analyte concentration in advance. Follow the dilution schemes below if sample dilution is required:
100-fold dilution (single step): Mix 5 μL sample with 495 μL Assay Diluent.
1000-fold dilution (two-step): Step 1: Dilute 5 μL sample into 95 μL diluent (20×). Step 2: Transfer 5 μL of the 20× diluted sample into 245 μL diluent (50×), achieving total 1000-fold dilution.
100000-fold dilution (three-step): Step 1: Mix 5 μL sample with 195 μL diluent (40×). Step 2: Transfer 5 μL 40× sample into 245 μL diluent (50×). Step 3: Transfer 5 μL 2000× sample into 245 μL diluent (50×), total dilution factor = 100000×.
Guidelines for dilution: Transfer volume per step ≥ 3 μL; single-step dilution factor ≤ 100×. Mix thoroughly after each dilution and avoid bubble formation.
Required Lab Equipment (User-Supplied)
1. Microplate reader (capable of 450 nm absorbance measurement)
2. High-precision pipettes and matching tips: 0.5–10 μL, 5–50 μL, 20–200 μL, 200–1000 μL
3. 37°C incubator
4. Distilled or deionized water
Pre-Assay Reagent Preparation
1. Remove the kit from refrigeration 10 minutes in advance and equilibrate to room temperature.
2. Preparation of Serial Standard Working Solution: Reconstitute the lyophilized standard with 1 mL assay diluent, leave it to stand for 15 minutes for complete dissolution, then mix gently (stock concentration =5000pg/mL). Prepare serial dilutions at concentrations of: 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.2pg/mL, 0pg/mL.
Serial dilution procedure: Prepare 7 EP tubes and add 500 μL assay diluent to each tube. Transfer 500 μL of the 2000pg/mL standard working solution into the first tube and mix well to obtain a 1000pg/mL standard working solution, then perform serial transfer and mixing in sequence for subsequent tubes. The last tube serves directly as the blank well; no liquid needs to be transferred from the penultimate tube. Schematic diagram shown below.
3. Preparation of Biotin-antibody Working Solution: Centrifuge the 100× concentrated Biotin-antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated Biotin-antibody to 1× working concentration with assay diluent (Example: 10 μL concentrate + 990 μL assay diluent). Prepare fresh right before use.
4. Preparation of Streptavidin-HRP Working Solution: Centrifuge the 100× concentrated Streptavidin-HRP at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated Streptavidin-HRP to 1× working concentration with assay diluent (Example: 10 μL concentrate + 990 μL assay diluent). Prepare fresh right before use.
5. Preparation of 1× Wash Buffer: Add 10 mL of 20× concentrated wash buffer into 190 mL distilled water. (Crystallization may occur in concentrated wash buffer stored in the refrigerator, which is a normal phenomenon. Place the buffer at room temperature and wait for all crystals to dissolve completely before preparation.)
Assay Procedure
1. Remove the required strips from the aluminum foil pouch equilibrated at room temperature for 10 min. Seal the remaining strips in a zip‑lock bag and store at 4 ℃.
1. Sample Loading: Add 100 μL of samples or standards of different concentrations into corresponding wells. Add 100 μL Universal Diluent to blank wells. Cover with Plate Sealer and incubate at 37 ℃ for 60 min. (Recommendation: Dilute test samples at least 1‑fold with Universal Diluent before loading onto the Pre‑coated Assay Plate to reduce matrix effect on test results. Multiply by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run all test samples and standards in replicate wells.)
3. Add Biotin‑antibody Working Solution: Take out the assay plate and discard liquid without washing. Directly add 100 μL Biotin‑antibody working solution to each well. Cover with Plate Sealer and incubate at 37 ℃ for 60 min.
4. Wash Plate: Discard liquid. Add 300 μL of 1× Wash Buffer to each well, let stand for 1 min, decant buffer and blot dry on absorbent paper. Repeat the washing step 3 times (an automated plate washer is also applicable).
5. Add Conjugate Working Solution: Add 100 μL Streptavidin‑HRP working solution to each well. Cover with Plate Sealer and incubate at 37 ℃ for 30 min.
6. Wash Plate: Discard liquid and perform washing 5 times following the procedure described in Step 4.
7. Add Substrate: Add 90 μL TMB Substrate to each well. Cover with Plate Sealer and incubate at 37 ℃ in the dark for 15 min.
8. Add Stop Solution: Take out the assay plate, add 50 μL Stop Solution directly into each well. Immediately measure the OD value of each well at 450 nm.
Data Analysis
1. Calculate the average OD value for replicate wells of standards and samples, then subtract the OD value of blank wells to obtain the corrected OD value. Plot a 4‑parameter logistic standard curve on log‑log graph paper with concentration on the x‑axis and OD value on the y‑axis.
2. If the sample OD value exceeds the upper limit of the standard curve, appropriately dilute the sample and re‑assay. Multiply by the corresponding dilution factor when calculating sample concentration.
Representative Data and Reference Curve
The data and curve shown below are for reference only. Operators shall generate the standard curve based on their own experimental data.
| Concentration (pg/mL) | 2000 | 1000 | 500 | 250 | 125 | 62.5 | 31.2 | 0 |
|---|
| OD Value | 2.22 | 1.56 | 0.92 | 0.63 | 0.42 | 0.25 | 0.18 | 0.08 |
| Corrected OD Value | 2.14 | 1.48 | 0.84 | 0.55 | 0.34 | 0.17 | 0.1 | - |
Note: This figure is for reference only. Sample concentrations shall be calculated using the standard curve generated from data of each individual experiment.
Kit Performance
1. Precision: Intra‑assay coefficient of variation ＜10%; inter‑assay coefficient of variation ＜10%.
2. Recovery: Three different levels of human TGF‑β were spiked into selected healthy human serum, plasma and cell culture supernatants for recovery calculation.
| Sample Type | Range (%) | Mean Recovery (%) |
|---|
| Serum (n=8) | 84‑101 | 96 |
| Plasma (n=8) | 92‑105 | 102 |
| Cell culture supernatant (n=8) | 96‑108 | 105 |
3. Linearity‑of‑dilution: High‑concentration human TGF‑β was spiked into 4 selected batches of healthy human serum, plasma and cell culture supernatants. Samples were diluted within the dynamic range of the standard curve to evaluate linear performance.
| Dilution Ratio | Recovery (%) | Serum | Plasma | Cell Culture Supernatant |
|---|
| 1:2 | Range | 84‑95 | 88‑96 | 90‑110 |
| 1:2 | Mean Recovery | 91 | 93 | 96 |
| 1:4 | Range | 89‑103 | 87‑108 | 105‑115 |
| 1:4 | Mean Recovery | 94 | 98 | 108 |
Troubleshooting Guide
If unsatisfactory assay results occur, take photos of color development, save experimental data, retain used microplate strips and unused reagents, then contact our technical support for troubleshooting. Refer to the troubleshooting items below:
1. Poor standard curve linearity
① Incorrect standard dilution: Reconstitute and dilute standard strictly following recommended protocol.
② Inaccurate pipetting: Calibrate pipettes regularly and verify tip tightness/sealing performance.
③ Evaporation of reaction solution: Seal microplate with plate sealer film.
④ Incomplete washing: Perform sufficient wash cycles with adequate volume of wash buffer.
⑤ Foreign residues at well bottom: Wipe microplate bottom prior to absorbance measurement.
2. Faint or absent color development
① Insufficient incubation duration: Adhere to specified incubation time.
② Incorrect incubation temperature: Incubate at recommended temperature.
③ Insufficient reagent addition volume: Verify pipette accuracy and follow operating procedure strictly.
④ Improper reagent dilution: Double-check dilution steps for all reagents.
⑤ Inactivated Streptavidin-HRP: Mix Streptavidin-HRP with substrate and verify activity via color reaction test.
3. Low OD readings
① Wrong microplate reader parameter: Confirm detection wavelength setting on instrument.
② Missing stop solution addition: Add appropriate volume of stop solution.
③ Overlong delay before plate reading: Measure absorbance immediately after termination.
④ Excessively high analyte concentration in sample: Determine optimal dilution factor via preliminary pre-test.
⑤ Too low analyte concentration in sample: Determine optimal dilution factor via preliminary pre-test.
4. High background signal
① Contaminated TMB substrate: Replace with fresh substrate solution.
② Excess substrate incubation time: Strictly control color development duration.
③ Wrong dilution for detection antibody or Streptavidin-HRP: Dilute reagents in accordance with official recommended protocol.
④ Incomplete plate washing: Carry out adequate wash cycles with sufficient wash buffer volume.
ZJ26F0939454
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