癌胚抗原（CEA）描述了一组参与细胞粘附的高度相关的糖蛋白。它通常在胎儿发育过程中的胃肠组织中产生，但在出生前停止产生，所以 CEA 在健康成年人血液中的含量通常很低（约 2-4 纳克 / 毫升）。然而，在某些类型的癌症中血清水平会升高，这意味着它可以在临床试验中作为肿瘤标志物使用。重度吸烟者的血清水平也会升高。CEA 是糖基磷脂酰肌醇（GPI）细胞表面锚定的糖蛋白，其专门的硅藻糖基化糖型可作为功能性结肠癌 L - 选择素和 E - 选择素配体，可能对结肠癌细胞的转移扩散至关重要。从免疫学角度看，它们的特征是 CD66 分化簇的成员。这些蛋白包括 CD66a、CD66b、CD66c、CD66d、CD66e、CD66f。
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）。往预先包被有人癌胚抗原 (CEA) 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癌胚抗原 (CEA) 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。
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 150-200μL 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 1250pg/mL)，然后按照以下浓度：1250pg/mL、625pg/mL、312.5pg/mL、156.2pg/mL、78.1pg/mL、39.06pg/mL、19.53pg/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，1250pg/mL 的标准品工作液中吸取 500μL 到第一支 EP 管中混匀配成 625pg/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。
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 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) | 1250 | 625 | 312.5 | 156.2 | 78.1 | 39.06 | 19.53 | 0 |
|---|
| OD 值 | 2.05 | 1.12 | 0.57 | 0.35 | 0.25 | 0.2 | 0.18 | 0.08 | | 校正 OD 值 | 1.97 | 1.04 | 0.49 | 0.27 | 0.17 | 0.12 | 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 CEA，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 CEA，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 | 95 | | 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。
Carcinoembryonic Antigen (CEA) refers to a group of highly homologous glycoproteins involved in cell adhesion. It is normally synthesized in gastrointestinal tissues during fetal development and ceases production before birth, resulting in low serum CEA concentrations (approximately 2–4 ng/mL) in healthy adults. Nevertheless, serum CEA levels rise in certain malignancies, qualifying CEA as a tumor biomarker for clinical testing. Elevated CEA can also be detected in heavy smokers. As a GPI-anchored cell-surface glycoprotein, CEA bears specific fucosylated glycoforms that serve as functional ligands for L-selectin and E-selectin in colon carcinoma, playing a critical role in colon cancer cell migration and metastasis. Immunologically, CEA belongs to the CD66 cluster of differentiation family, comprising CD66a, CD66b, CD66c, CD66d, CD66e and CD66f.
Assay Principle
This kit adopts sandwich enzyme-linked immunosorbent assay (ELISA). Microwells pre-coated with anti-human CEA capture antibody are sequentially loaded with samples, standards, Biotin-antibody and Streptavidin-HRP following incubation and washing steps. Color development is triggered by TMB substrate: TMB turns blue under HRP catalysis and shifts to yellow upon acid termination. Color intensity is positively correlated with human CEA concentration in specimens. Absorbance (OD value) is measured at 450 nm via microplate reader to calculate sample analyte concentration.
Precautions
- Incubate strictly at specified time and temperature to ensure reliable test results. Equilibrate all reagents to ambient temperature (20–25℃) prior to use; refrigerate reagents promptly after usage.
- Improper plate washing causes erroneous readings. Remove residual liquid completely from wells before substrate addition; avoid prolonged desiccation of microwells throughout the assay.
- Wipe off residual liquid and fingerprints on the bottom of microplate to prevent OD interference.
- Fresh TMB substrate solution shall be colorless; discard blue-discolored substrate.
- Prevent cross-contamination between reagents and samples to avoid invalid experimental data.
- Shield reagents from direct intense light during storage and incubation.
- All assay reagents must avoid contact with bleach solution or volatile fumes from bleach, as bleach ingredients will inactivate biological activity of kit contents.
- Expired products are prohibited for use; components from different catalog numbers or batch lots cannot be mixed.
- Exogenous recombinant proteins from non-kit sources may fail antibody recognition due to epitope mismatch.
- All biohazardous specimens and testing consumables shall be managed and disposed of in accordance with institutional biosafety protocols.
Sample Pretreatment and Requirements
- The detection range of the kit is not equivalent to the analyte concentration range in samples. It is recommended to estimate the analyte concentration via relevant literature before experiment and confirm the actual concentration by pre-experiment. Properly dilute or concentrate samples with excessively high or low analyte concentrations.
- If the sample type is not listed in the manual, perform a pre-experiment to verify detection feasibility.
- Serum: Let whole blood collected in serum separator tubes stand at room temperature for 2 hours or at 2–8℃ overnight, then centrifuge at 1000×g for 20 min to collect supernatant. Store supernatant at -20℃ or -80℃ and avoid repeated freeze-thaw cycles.
- Plasma: Collect blood with EDTA or heparin as anticoagulant. Centrifuge specimens at 1000×g for 15 min within 30 min after collection under 2–8℃, collect supernatant for immediate testing or store at -20℃/-80℃ without repeated freeze-thaw.
- Tissue homogenate: Rinse fresh tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood as lysed red blood cells interfere detection. Weigh and mince tissue, add minced tissue into glass homogenizer with PBS at 1:9 weight-to-volume ratio (1 g tissue with 9 mL PBS; adjust volume as needed and record; protease inhibitor is recommended in PBS). Grind fully on ice or with homogenizer. Further lyse cells by sonication or repeated freeze-thaw, then centrifuge homogenate at 5000×g for 5~10 min and collect supernatant.
- Cell culture supernatant: Centrifuge at 1000×g for 20 min, collect supernatant for test or store at -20℃/-80℃ avoiding repeated freeze-thaw.
- Cell lysate: Wash adherent cells with pre-cooled PBS, digest with trypsin and harvest by 1000×g centrifugation for 5 min; collect suspension cells directly by centrifugation. Wash harvested cells 3 times with pre-cooled PBS, resuspend 1×10⁶ cells in 150-200μL PBS (protease inhibitor recommended; reduce PBS volume for low-abundance targets). Break cells via repeated freeze-thaw or sonication, centrifuge lysate at 1500×g for 10 min at 2–8℃ and collect supernatant.
- Other biological samples: Centrifuge at 1000×g for 20 min and retain supernatant.
- Sample appearance: Samples shall be clear; remove suspended sediments by centrifugation.
- Sample storage: Specimens can be kept at 4℃ within 7 days after collection. Aliquot unused samples and freeze at -20℃ (valid within 1 month) or -80℃ (valid within 6 months) to avoid repeated freeze-thaw. Hemolyzed samples are unsuitable for detection.
Sample Dilution Protocol
Estimate sample concentration in advance and dilute as required referring to below methods: 100-fold dilution: Single-step dilution, mix 5μL sample with 495μL Universal Diluent. 1000-fold dilution: Two-step dilution, first mix 5μL sample with 95μL Universal Diluent for 20× dilution, then take 5μL diluted solution into 245μL Universal Diluent for 50× dilution to reach total 1000-fold. 100000-fold dilution: Three-step dilution, first mix 5μL sample with 195μL Universal Diluent for 40× dilution; second take 5μL diluted liquid into 245μL diluent for 50× dilution; third take 5μL twice-diluted sample into 245μL diluent for another 50× dilution to reach total 100000-fold. Minimum pipetting volume per dilution ≥3μL and single dilution fold ≤100. Mix thoroughly without bubbles after each dilution.Required Laboratory Supplies
- Microplate reader (450nm)
- Precision pipettes and tips: 0.5-10μL, 5-50μL, 20-200μL, 200-1000μL
- 37℃ incubator
- Distilled or deionized water
Pre-Assay Reagent Preparation
1. Take kit out from refrigerator 10 min earlier and equilibrate to room temperature.
2. Preparation of serial standard working solution: Reconstitute lyophilized standard with 1mL Universal Diluent, leave for 15 min to fully dissolve and mix gently (stock concentration:1250pg/mL). Prepare serial concentrations:1250pg/mL,625pg/mL,312.5pg/mL,156.2pg/mL,78.1pg/mL,39.06pg/mL,19.53pg/mL,0pg/mL. Serial dilution operation: Prepare 7 EP tubes pre-filled with 500μL Universal Diluent each. Transfer 500μL 1250pg/mL standard into the first tube to get 625pg/mL solution, perform successive half-dilution sequentially. The last tube serves as blank control without adding standard liquid.
3. Preparation of Biotin-antibody working solution: Centrifuge 100× concentrated Biotin-antibody at 1000×g for 1 min 15 min before use. Dilute the concentrated stock to 1× working concentration with Universal Diluent (Example: 10 μL concentrate + 990 μL Universal Diluent). Prepare freshly before use.
4. Preparation of Streptavidin-HRP working solution: Centrifuge 100× concentrated Streptavidin-HRP at 1000×g for 1 min 15 min before use. Dilute the concentrated stock to 1× working concentration with Universal Diluent (Example: 10 μL concentrate + 990 μL Universal Diluent). Prepare freshly before use.
5. Preparation of 1× Wash Buffer: Add 10 mL of 20× concentrated Wash Buffer into 190 mL distilled water. Crystals may form in concentrated Wash Buffer stored under refrigeration, which is normal. Let the buffer stand at room temperature until crystals dissolve completely prior to dilution.
Assay Procedure
- Take required strips from the aluminum pouch equilibrated at room temperature for 10 min. Seal unused strips in zip lock bag and store at 4℃.
- Sample loading: Pipette 100 μL of samples or serially diluted standards into respective wells; add 100 μL Universal Diluent into blank well. Seal plate and incubate at 37℃ for 60 min. Recommendation: Dilute test samples no less than 1-fold with Universal Diluent to minimize matrix effect; multiply final concentration by corresponding dilution factor during calculation. Running duplicate wells is recommended for all samples and standards.
- Add Biotin-antibody: Discard well contents without washing. Add 100 μL prepared Biotin-antibody working solution per well, seal plate and incubate at 37℃ for 60 min.
- Plate washing: Decant liquid, fill each well with 300 μL 1× Wash Buffer, soak for 1 min, tap dry on absorbent paper. Repeat wash cycle for total 3 times; automatic plate washer is optional.
- Add Streptavidin-HRP working solution: Dispense 100 μL Streptavidin-HRP working solution per well, seal plate and incubate at 37℃ for 30 min.
- Plate washing: Remove liquid and repeat washing procedure described in step 4 for total 5 cycles.
- Substrate addition: Add 90 μL TMB substrate into each well, seal plate and incubate at 37℃ in dark for 15 min.
- Stop reaction: Add 50 μL Stop Solution to every well and immediately measure OD value at 450 nm.
Calculation of Test Results Result Interpretation
- Calculate average OD of duplicate wells for standards and samples, subtract blank OD to obtain corrected OD values. Plot 4-parameter logistic standard curve on log-log graph with concentration on X-axis and corrected OD on Y-axis.
- Retest samples with OD above upper limit of standard curve after appropriate dilution and correct final concentration with relevant dilution factor.
Typical Data & Reference Standard Curve
The listed data and curve are for reference only; each laboratory shall establish its own standard curve per assay run.
| Concentration (pg/mL) | 1250 | 625 | 312.5 | 156.2 | 78.1 | 39.06 | 19.53 | 0 |
|---|
| OD Value | 2.05 | 1.12 | 0.57 | 0.35 | 0.25 | 0.20 | 0.18 | 0.08 | | Corrected OD Value | 1.97 | 1.04 | 0.49 | 0.27 | 0.17 | 0.12 | 0.10 | - |
|
Note: This figure is for reference only; sample concentration shall be calculated based on the standard curve generated from experimental data of each individual test run.
Kit Performance
1. Precision: Intra-assay CV＜10%, Inter-assay CV＜10%.
2. Recovery: Three concentration levels of human CEA were spiked into healthy human serum, plasma and cell culture supernatant to calculate recovery rate.
| Sample Type | Range (%) | Average Recovery (%) |
|---|
| Serum (n=8) | 84-101 | 96 | | Plasma (n=8) | 92-105 | 102 | | Cell Culture Supernatant (n=8) | 96-108 | 105 |
|
3. Linearity on dilution: High-concentration human CEA was spiked into 4 batches of healthy human serum, plasma and cell culture supernatant respectively, followed by serial dilution within the dynamic range of standard curve to evaluate linearity.
| Dilution Ratio | Recovery (%) | Serum | Plasma | Cell Culture Supernatant |
|---|
| 1:2 | Range | 84-95 | 88-96 | 90-110 | | 1:2 | Average Recovery | 91 | 93 | 95 | | 1:4 | Range | 89-103 | 87-108 | 105-115 | | 1:4 | Average 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.
6. 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.
ZJ26F0636906
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