生长抑素，又称生长激素抑制素 (GHIH)等，是一种多肽激素，通过与G蛋白偶联的生长抑素受体相互作用和抑制众多次级激素的释放，调节内分泌系统，影响神经传导和细胞增殖。生长抑素抑制胰岛素和胰高血糖素的分泌。生长抑素有两种活性形式，由一个预蛋白的替代裂解产生：一种由14个氨基酸组成，另一种由28个氨基酸组成。在脊椎动物中，存在六种不同的体视肽基因，被命名为SS1、SS2、SS3、SS4、SS5和SS6。这六种不同的基因，加上五种不同的体视肽受体，使体视肽具有很大的功能范围。
实验原理：
本试剂盒采用竞争法酶联免疫吸附试验 (ELISA)。往预先包被有小鼠生长抑素 (SS)抗原的微孔中，依次加入样本、标准品、Biotin标记的抗体，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小鼠生长抑素 (SS)呈负相关。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150-200 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：
1. 稀释100倍：一步稀释。取5 μL样本到495 μL通用稀释液内，做100倍稀释；
2. 稀释1000倍：两步稀释。取5 μL样本到95 μL通用稀释液内，做20倍稀释，再取5 μL 20倍稀释样本到245 μL通用稀释液内，做50倍稀释，总共稀释1000倍；
3. 稀释100000倍：三步稀释。取5 μL样本到195 μL通用稀释液内，做40倍稀释，再取5 μL 40倍稀释样本到 245 μL通用稀释液内，做50倍稀释，最后取5 μL 2000倍稀释样本到245 μL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3 μL，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1 mL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500 pg/mL)，然后按照以下浓度：500 pg/mL、250 pg/mL、125 pg/mL、62.5 pg/mL、31.25 pg/mL、15.62 pg/mL、7.81 pg/mL、0 pg/mL进行稀释。
倍比稀释方法：取7支EP管，每管中加入500 μL通用稀释液，500 pg/mL的标准品工作液中吸取500 μL到第一支EP管中混匀配成250 pg/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. Biotin-抗体工作液配制：使用前15分钟将100×浓缩Biotin-抗体于1000× g离心1分钟，以通用稀释液将100×浓缩生物素化检测抗体稀释成1×工作浓度 (例：10 μL浓缩液+990 μL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：10 μL浓缩液+990 μL通用稀释液)，现配现用。
5. 1×洗涤液配制：取10 mL 20×洗涤液到190 mL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50 μL每孔加入相应孔中，空白孔加入50 μL通用稀释液，紧接着每孔加入50 μL Biotin-抗体工作液。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. 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)。
4. 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。
5. 洗板：弃去液体按步骤3洗涤方法，洗板5次。
6. 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。
7. 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。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 (作图时去掉空白组的值)。
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) | 500 | 250 | 125 | 62.5 | 31.25 | 15.62 | 7.81 | 0 | | OD 值 | 0.19 | 0.24 | 0.31 | 0.55 | 0.97 | 1.38 | 1.96 | 2.52 |
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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：在选取的健康小鼠血清、血浆和细胞培养上清中加入3个不同浓度水平的小鼠SS，计算回收率。
| 样本类型 | 范围 (%) | 平均回收率 (%) | | 血清 (n=8) | 81-101
| 96 | | 血浆 (n=8) | 92-105
| 101
| 细胞培养上清 (n=8)
| 97-108
| 103
|
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3. 线性稀释：分别在选取的4份健康小鼠血清、血浆和细胞培养上清中加入高浓度小鼠SS，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 | 83-95
| 88-96
| 90-111
| | 1: 2 | 平均回收率 | 91 | 93 | 97 | | 1: 4 | 范围 | 89-104
| 87-108
| 105-114
| | 1: 4 | 平均回收率 | 93 | 98 | 109 |
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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 | 相应对策 | | 标曲线性差 | 标准品稀释不正确 | 确保标准品按照推荐方法溶解和稀释 | | 标曲线性差 | 移液不准确 | 定期校准移液器并检查枪头密封性 | | 标曲线性差 | 反应液蒸发 | 用封板膜密封酶标板 | | 标曲线性差 | 洗板不彻底 | 足够的洗涤次数和加入足量洗涤液 | | 标曲线性差 | 孔底有异物 | 读数前清洁板底 | | 显色弱或无色 | 孵育时间不够 | 确保孵育时间 | | 显色弱或无色 | 孵育温度不正确 | 按推荐温度孵育 | | 显色弱或无色 | 试剂体积添加不足 | 检查移液器并严格按照操作步骤操作 | | 显色弱或无色 | 稀释不正确 | 检查试剂稀释步骤 | | 显色弱或无色 | 酶结合物失活 | 混合酶结合物和底物，通过显色反应检查 | | OD值低 | 酶标仪设置不正确 | 检查仪器波长 | | OD值低 | 没加终止液 | 加入适量终止液 | | OD值低 | 读板时等待时间太长 | 及时读板 | | OD值低 | 样本含量过高 | 通过预实验确定合适的稀释倍数 | | OD值低 | 样本含量过低 | 通过预实验确定合适的稀释倍数 | | 背景高 | 显色液被污染 | 更换显色液 | | 背景高 | 显色时间太长 | 控制显色时间 | | 背景高 | 检测抗体或酶结合物稀释错误 | 使用推荐稀释方法 | | 背景高 | 洗板不彻底 | 足够的洗涤次数和加入足量洗涤液 |
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Somatostatin, also known as growth hormone-inhibiting hormone (GHIH), is a polypeptide hormone. It regulates the endocrine system, affects neurotransmission and cell proliferation by interacting with G protein-coupled somatostatin receptors and inhibiting the release of numerous secondary hormones. Somatostatin inhibits the secretion of insulin and glucagon. There are two active forms of somatostatin, produced by alternative cleavage of a preproprotein: one consisting of 14 amino acids and the other consisting of 28 amino acids. In vertebrates, there are six distinct somatostatin genes, designated as SS1, SS2, SS3, SS4, SS5 and SS6. These six different genes, together with five distinct somatostatin receptors, confer a wide functional range to somatostatin.
Experimental Principle
This kit adopts a competitive enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled antibody and HRP conjugate are sequentially added to the microwells pre-coated with mouse somatostatin (SS) antigen. After incubation and washing, the substrate TMB is used for color development. TMB is converted to blue under the catalysis of horseradish peroxidase (HRP), and then converted to the final yellow color under the action of acid. The color intensity is inversely correlated with the level of mouse somatostatin (SS) in the sample. The absorbance (OD value) is measured at 450 nm wavelength using a microplate reader, and the sample concentration is calculated accordingly.
Precautions
1. Strictly follow the specified time and temperature for incubation to ensure accurate results. All reagents must be brought to room temperature (20-25 °C) before use. Store reagents in the refrigerator immediately after use.
2. Improper washing may lead to inaccurate results. Ensure wells are thoroughly drained before adding substrate. Avoid allowing microwells to dry for extended periods during the procedure.
3. Clean residual liquid and fingerprints from the well bottoms, as these can affect OD values.
4. Substrate developing solution should be colorless; blue-tinted substrate must not be used.
5. Avoid cross-contamination between reagents and samples to prevent erroneous results.
6. Protect from direct strong light during storage and incubation.
7. Do not allow any reaction reagents to come into contact with bleaching agents or their vapors. Any bleaching compounds will inactivate the biological activity of the kit reagents.
8. Do not use expired products. Components from different catalog or batch numbers must not be mixed.
9. Recombinant proteins from sources other than this kit may not be recognized by the kit antibodies.
10. All samples should be handled with care and processed and tested in accordance with established protocols if there is a risk of disease transmission.
Sample Handling and Requirements
1. The kit’s detection range does not equate to the concentration range of the analyte in the sample. It is recommended to estimate the analyte concentration in the sample using relevant literature and confirm the actual concentration via a preliminary experiment. If the analyte concentration is too high or too low, dilute or concentrate the sample appropriately.
2. If the sample type is not listed in the manual, a preliminary experiment is recommended to verify detection validity.
3. Serum: Allow whole blood collected in serum separation tubes to stand at room temperature for 2 hours or overnight at 2-8 °C, then centrifuge at 1000 × g for 20 minutes and collect the supernatant. Alternatively, store the supernatant at −20 °C or −80 °C, avoiding repeated freeze-thaw cycles.
4. Plasma: Collect samples using EDTA or heparin as anticoagulant, centrifuge at 1000 × g for 15 minutes at 2-8 °C within 30 minutes of collection, and use the supernatant for testing. Alternatively, store the supernatant at −20 °C or −80 °C, avoiding repeated freeze-thaw cycles.
5. Tissue homogenate: Rinse tissue with pre-chilled PBS (0.01 M, pH=7.4) to remove residual blood (lysed red blood cells in homogenate will interfere with results). Weigh and mince the tissue. Place minced tissue and an appropriate volume of PBS (typically 1:9 w/v, e.g., 1 g tissue + 9 mL PBS; volume can be adjusted as needed and recorded) into a glass homogenizer, and grind on ice or using a homogenizer. For further cell lysis, sonicate or subject the homogenate to repeated freeze-thaw cycles. Centrifuge the homogenate at 5000 × g for 5-10 minutes and use the supernatant for testing.
6. Cell culture supernatant: Centrifuge at 1000 × g for 20 minutes and use the supernatant for testing. Alternatively, store the supernatant at −20 °C or −80 °C, avoiding repeated freeze-thaw cycles.
7. Cell lysate: Gently wash adherent cells with pre-chilled PBS, then digest with trypsin and collect cells by centrifugation at 1000 × g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with pre-chilled PBS, resuspend in 150-200 μL PBS per 1× 10⁶ cells (add protease inhibitors to PBS if recommended; reduce PBS volume if analyte is low), and lyse cells by repeated freeze-thaw or sonication. Centrifuge the extract at 1500 × g for 10 minutes at 2-8 °C and use the supernatant for testing.
8. Other biological samples: Centrifuge at 1000 × g for 20 minutes and use the supernatant for testing.
9. Sample appearance: Samples should be clear and transparent; remove any suspended matter by centrifugation.
10. Sample storage: If testing within 1 week, store samples at 4 °C. If testing is delayed, aliquot and store at −20 °C (for testing within 1 month) or −80 °C (for testing within 6 months), avoiding repeated freeze-thaw cycles. Hemolyzed samples should not be used as hemolysis interferes with results.
Sample Dilution Protocol
Estimate the sample concentration range in advance. If sample dilution is required, follow the protocol below:
1. 100-fold dilution: One-step dilution. Add 5 μL sample to 495 μL general diluent.
2. 1000-fold dilution: Two-step dilution. Add 5 μL sample to 95 μL general diluent (20-fold), then add 5 μL of the 20-fold dilution to 245 μL general diluent (50-fold); total dilution=1000-fold.
3. 100,000-fold dilution: Three-step dilution. Add 5 μL sample to 195 μL general diluent (40-fold), then add 5 μL of the 40-fold dilution to 245 μL general diluent (50-fold), then add 5 μL of the 2000-fold dilution to 245 μL general diluent (50-fold); total dilution = 100,000-fold.
In each step, use at least 3 μL for pipetting and do not exceed 100-fold dilution per step. Mix thoroughly after each dilution and avoid bubbles.
Required Self‑Provided Equipment
1. Microplate reader (450 nm)
2. High-precision pipettes and 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. Remove the kit from the refrigerator 10 minutes in advance and equilibrate it to room temperature.
2. Preparation of standard serial working solutions: Add 1 mL of universal diluent to the lyophilized standard, allow it to stand for 15 minutes until fully dissolved, then mix gently (concentration: 500 pg/mL). Subsequently, perform serial dilutions to the following concentrations: 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.62 pg/mL, 7.81 pg/mL, and 0 pg/mL.
Serial dilution method: Take 7 EP tubes, add 500 μL of universal diluent to each tube. Pipette 500 μL from the 500 pg/mL standard working solution into the first EP tube, mix well to prepare 250 pg/mL standard working solution. Repeat this pipetting and mixing step sequentially for subsequent tubes. The last tube is directly used as the blank well, and no liquid needs to be transferred from the penultimate tube, as shown in the figure 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 universal diluent (e.g., 10 μL concentrated solution + 990 μL universal diluent). Prepare freshly before use.
4. Preparation of enzyme conjugate working solution: Centrifuge the 100× concentrated enzyme conjugate at 1000 × g for 1 minute 15 minutes before use. Dilute the 100× concentrate to 1× working concentration with general diluent (e.g., 10 μL concentrate + 990 μL diluent). Prepare fresh.
5. Preparation of 1× washing solution: Add 10 mL of 20× washing solution to 190 mL distilled water (Crystals may form in the concentrated washing solution stored in the refrigerator; this is normal. Allow to reach room temperature and dissolve completely before preparation).
Procedure
1. Remove required strips from the aluminum foil bag that has been equilibrated at room temperature for 10 minutes. Seal the remaining strips in a self-sealing bag and return to 4 ℃.
2. Sample addition: Add 50 μL of samples or standards of different concentrations to corresponding wells respectively. Add 50 μL of universal diluent to the blank well. Then add 50 μL of Biotin‑antibody working solution to each well. Cover with sealing film and incubate at 37 ℃ for 60 minutes. (Suggestion: Dilute test samples at least 1‑fold with universal diluent before adding to the microplate to reduce matrix effect on results. Multiply by the corresponding dilution factor when calculating final concentration. Duplicate wells are recommended for all test samples and standards.)
3. Washing: Discard the liquid, add 300 μL of 1× washing solution to each well, let stand for 1 minute, discard the washing solution, and pat dry on absorbent paper. Repeat washing 3 times (or use an automatic plate washer).
4. Addition of enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well. Cover with sealing film and incubate at 37 ℃ for 30 minutes.
5. Washing: Discard the liquid and wash 5 times according to the method in step 3.
6. Substrate addition: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37 ℃ in the dark for 15 minutes.
7. Stop solution addition: Take out the microplate, add 50 μL of stop solution directly to each well, and measure the OD value of each well at 450 nm immediately.
Calculation of Experimental Results
Result Judgment:
1. Calculate the average OD value of duplicate wells for standards and samples. Using concentration as the abscissa and OD value as the ordinate, draw a standard curve of four-parameter logistic function on a log-log coordinate paper (exclude the blank group value during plotting).
2. If the sample OD value is higher than the upper limit of the standard curve, the sample should be properly diluted and re-measured, and the corresponding dilution factor shall be multiplied when calculating the sample concentration.
Typical Data and Reference Curve
The following data and curve are for reference only. The experimenter should establish a standard curve based on their own experiment.
| Concentration (pg/mL) | 500 | 250 | 125 | 62.5 | 31.25 | 15.62 | 7.81 | 0 | | OD Value | 0.19 | 0.24 | 0.31 | 0.55 | 0.97 | 1.38 | 1.96 | 2.52 |
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Note: This figure is for reference only. The sample content should be calculated using the standard curve plotted from the experimental data of each run.
Kit Performance
1. Repeatability: Intra‑assay CV< 10%; inter‑assay CV< 10%.
2. Recovery rate: Mouse SS at three different concentration levels was spiked into selected healthy mouse serum, plasma and cell culture supernatant, and the recovery rate was calculated.
Sample Type
| Range (%) | Average Recovery (%) | | Serum (n=8) | 81-101
| 96 | | Plasma (n=8) | 92-105
| 101 | | Cell culture supernatant (n=8) | 97-108
| 103 |
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3. Linear dilution: High-concentration mouse SS was spiked into 4 selected healthy mouse serum, plasma and cell culture supernatant samples respectively. Serial dilutions were performed within the dynamic range of the standard curve to evaluate linearity.
Dilution Ratio
| Recovery (%) | Serum
| Plasma
| Cell culture supernatant
| | 1: 2 | Range
| 83-95
| 88-96 | 90-111
| | 1: 2 | Average recovery
| 91 | 93 | 97
| | 1: 4 | Range
| 89-104
| 87-108 | 105-114
| | 1: 4 | Average recovery
| 93 | 98 | 109 |
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Troubleshooting
If experimental results are unsatisfactory, photograph the color development, save experimental data, retain the used strips and unused reagents, and contact our technical support for assistance. You may also refer to the following:
| Problem Description | Possible Cause | Recommended Solution | Poor standard curve linearity
| Incorrect standard dilution
| Ensure standards are dissolved and diluted as recommended
| Poor standard curve linearity
| Inaccurate pipetting | Calibrate pipettes regularly and check tip sealing
| Poor standard curve linearity
| Evaporation of reaction solution
| Seal the plate with a film
| Poor standard curve linearity
| Inadequate washing
| Adequate washing times and sufficient washing solution volume
| Poor standard curve linearity
| Foreign matter on well bottoms
| Clean well bottoms before reading
| | Weak or no color development | Weak or no color development
| Insufficient incubation time Ensure full incubation time
| | Weak or no color development | Incorrect incubation temperature
| Incubate at the recommended temperature
| | Weak or no color development | Insufficient reagent volume
| Check pipettes and follow the protocol strictly
| | Weak or no color development | Incorrect dilution
| Verify reagent dilution steps
| | Weak or no color development | Inactivated enzyme conjugate
| Mix conjugate and substrate and check for color development
| Low OD values
| Incorrect microplate reader settings | Check instrument wavelength | Low OD values
| No stop solution added
| Add appropriate amount of stop solution
| Low OD values
| Too long a wait before reading
| Read the plate promptly
| Low OD values
| Sample concentration too high | Determine appropriate dilution via preliminary experiment
| Low OD values
| Sample concentration too low | Determine appropriate dilution via preliminary experiment
| | High background | Contaminated Substrate solution
| Replace Substrate solution
| | High background | Excessive color development time
| Control color development time
| | High background | Incorrect dilution of detection antibody or enzyme conjugate | Use recommended dilution method | | High background | Inadequate washing
| Adequate washing times and sufficient washing solution volume
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ZJ26F0333722
危险品化学品经营许可证（带存储）