酶联免疫吸附测试 Enzyme Linked Immunosorbent Assay
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植物乙烯(ETH)酶联免疫分析(ELISA)
试剂盒使用说明书
本试剂仅供科研使用,不得用于医学诊断
实验目的:
Experimental Purpose:
本试剂盒用于测定植物相关样本中乙烯(ETH)的含量。
This reagent box is used to determine the contents of ethylene (ETH) in plant-related samples.
实验原理:
experimental principle:
本试剂盒应用双抗体夹心法测定标本中植物乙烯(ETH)水平。用纯化的植物乙烯(ETH)捕获抗体包被微孔板,制成固相抗体,往包被的微孔中依次加入植物乙烯(ETH),再与HRP标记的检测抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的植物乙烯(ETH)呈正相关。用酶标仪在450nm波长下测定吸光度(OD值),通过标准曲线计算样品中植物乙烯(ETH)含量。
This reagent box should be used to determine plant ethylene (ETH) levels in the specimen using a double antibody kernel method. The antibody bag is captured by a pure plant ethylene (ETH) with a microhole, made into a solid phase antibody, inserted into the microhole of the package in turn into a plant ethylene (ETH), combined with the detector antibodies marked by HRP, forming an anti-antigen-enzyme antibody complex, and is thoroughly scrubbed with the TMB colour. The TMB transforms into blue under the catalyzing of the HRP enzyme and transforms it into final yellow under the acid. The plant ethylene (ETH) in the depth of the colour and in the sample is positively related.
注:标准品浓度依次为:240、120、 60、30、15、0 nmol/mL.
Note: Standard product concentrations: 240, 120, 60, 30, 15, 0 nmol/mL.
需自备的试剂和器材:
Reagents and devices to be prepared:
1. 酶标仪(450nm)
1. Enzyme marker (450nm)
2. 高精度加样器及枪头:0.5-10uL、2-20uL、20-200uL、200-1000uL
2. High accuracy samplers and gunheads: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
3. 37℃恒温箱
3. 37°C thermostats
4. 蒸馏水或去离子水
4. Distilled or deionated water
样本处理及要求:
Sample handling and requirements:
1. 血清:室温血液自然凝固10-20分钟,离心20分钟左右(2000-3000转/分)。仔细收集上清,保存过程中如出现沉淀,应再次离心。
1. Serolysis: room temperature blood is naturally condensed for 10-20 minutes, centrifugal for about 20 minutes (2000-3000 spins/minutes). Careful collection is carried out, and in the event of sedimentation during the preservation process, it should be severed again.
2. 血浆:应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂,混合10-20分钟后,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,应该再次离心。
2. Pulse: EDTA or sodium lemonate shall be selected as an anticondensant in accordance with the requirements of the specimen, and after a mixture of 10-20 minutes, the centrifugal shall be about 20 minutes (2000-3000 spin/point).
3. 尿液:用无菌管收集,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。胸腹水、脑脊液参照实行。
urinary fluids: collected with sterile tubes for about 20 minutes of centrifugal (2000-3000 spins/minutes).
4. 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。
4. Cell culture clean-up: When detection of the secretive components, the fungi are collected. Centrifugals are collected for about 20 minutes (2000-3000 spins/points). Carefully collected. When detection of the cell composition is done, the PBS (PH7.2-7.4) dilutes the cell suspension with a cell concentration of around 1 million/ml. By refrigerating to destroy the cell and release the cell contents. Centrifuges are collected for about 20 minutes (2000-3000 spins/s).
5. 组织标本:切割标本后,称取重量。加入一定量的PBS,PH7.4。用液氮迅速冷冻保存备用。标本融化后仍然保持2-8℃的温度。加入一定量的PBS(PH7.4),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。分装后一份待检测,其余冷冻备用。
5. tissue specimens: After cutting the specimens, they are weighed. Add a certain amount of PBS, PH7.4. Save the backup quickly by freezing liquid nitrogen. After melting, the temperature of 2-8°C is maintained. Add a certain amount of PBS (PH7.4), which is fully slurized by manual or slurry. Centrifugals (2000-3000 spin/s).
6. 标本采集后尽早进行提取,提取按相关文献进行,提取后应尽快进行实验。若不能马上进行试验,可将标本放于-20℃保存,但应避免反复冻融.
6. Samples shall be extracted as soon as possible after collection, shall be drawn according to the relevant literature, and shall be tested as soon as possible after extraction. If not immediately tested, samples may be kept at 20°C, but refrigerating shall be avoided.
7. 不能检测含NaN3的样品,因NaN3抑制辣根过氧化物酶的(HRP)活性。
7. Samples containing NaN3 could not be detected because of the inhibition of hot root peroxide enzyme (HRP) activity by NaN3.
注:标本溶血会影响最后检测结果,因此溶血标本不宜进行此项检测。
Note: The sample soluble blood affects the final test results and therefore the soluble blood sample is not suitable for this test.
操作步骤:
1. 标准品的加样:设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50μL;。
1. Sampling of standard products: setting of standard punctures and sample punctures with standard punctures with standard 50 μLs at various concentrations;
2. 加样:分别设空白孔(空白对照孔不加样品及酶标试剂,其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μl,然后再加待测样品10μl(样品最终稀释度为5倍)。加样将样品加于酶标板孔底部,尽量不触及孔壁,轻轻晃动混匀。
2. Sampling: separate blank holes (no samples and enzyme marker reagents, the rest of the steps are identical) and to be measured sample holes. A sample dilution fluid of 40 ml is added to the enzyme plate and then a sample of 10 ml is to be measured (the sample is eventually diluted by five times).
3. 加酶:每孔加入酶标试剂100μl,空白孔除外。
3. Enzymes added: 100 ml of enzyme marker reagent per hole, except for blank holes.
4. 温育:用封板膜封板后置37℃温育60分钟。
4. Temperature: For a period of 60 minutes, a 37°C temperature is set behind the tablet seal.
5. 配液:将20倍浓缩洗涤液用蒸馏水20倍稀释后备用。
5. Distribute: Retract 20 times the enriched scrubber with 20 times distilled water.
6. 洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30秒后弃去,如此重复5次,拍干。
6. Laundry: Carefully remove the membrane, discard the liquid, dump it dry, fill each hole with scrubber and leave it after 30 seconds, thus repeating it five times and drying it up.
7. 显色:每孔先加入显色剂A50μl,再加入显色剂B50μl,轻轻震荡混匀,37℃避光显色15分钟.
Highlighting: A50 ml per hole, B50 ml, light tremors, 37 °C for 15 minutes.
8. 终止:每孔加终止液50μl,终止反应(此时蓝色立转黄色)。
8. Termination: 50 ml of liquid per pore, termination of response (at this time blue-turned yellow).
9. 测定:以空白孔调零,450nm波长依序测量各孔的吸光度(OD值)。 测定应在加终止液后15分钟以内进行。
9. Determination: The desorption of the holes (OD value) shall be measured in a white hole with zero puncture and 450 nm wavelength in sequence, and shall be performed within 15 minutes of the termination of the liquid.
(此图仅供参考)
计算:
Calculate:
以标准物的浓度为横坐标,OD值为纵坐标,在坐标纸上绘出标准曲线,根据样品的OD值由标准曲线查出相应的浓度;再乘以稀释
Draw a standard curve on the coordinates sheet using the concentration of the standard material as a cross-coordinate, the OD value as a vertical coordinate, and the concentration is determined by the standard curve based on the OD value of the sample; multiplied by dilution
倍数;或用标准物的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式,计算出样品浓度,再乘以稀释
Multipliers; or a straight-line regression equation that calculates the standard curve using the concentration of the standard substance with the OD value, and replaces the sample's OD value with the equation, calculates the sample concentration and multiplys it by diluting it
倍数,即为样品的实际浓度。
A multiple, i.e. the actual concentration of the sample.
注意事项:
Attention:
1. 试剂盒从冷藏环境中取出应在室温平衡15-30分钟后方可使用,酶标包被板开封后如未用完,板 条应装入密封袋中保存。样本在使用前也要在室温平衡60分钟。
1. The reagent box shall be removed from the refrigerated environment only after 15 to 30 minutes for use, and if the enzyme bag is unspent, the plate shall be stored in a sealed bag. The sample shall also have a room temperature balance of 60 minutes before use.
2. 浓洗涤液可能会有结晶析出,稀释时可在水浴中加温助溶,洗涤时不影响结果。
2. Enriched scrubbers may be crystallized, which can be diluted with warm solubility in the bath, without prejudice to the result.
3. 各步加样均应使用加样器,并经常校对其准确性,以避免试验误差。一次加样时间最好控制在5分钟内,如标本数量多,推荐使用排枪加样。
3. Each step shall be sampled using a sampler and shall be checked for accuracy on a regular basis in order to avoid error in the test.
4. 请每次测定的同时做标准曲线,最好做复孔。如标本中待测物质含量过高(样本OD值大于标准品孔第一孔的OD值),请先用样品稀释液稀释一定倍数(n倍)后再测定,计算时请最后乘以总稀释倍数(×n×5)。
4. Please make a standard curve at the same time as each test, preferably with a double hole. If the content of the substance to be measured in the sample is too high (the sample OD value is greater than the OD value of the first hole in the standard puncture), then a multiple (n) of the sample dilution fluid should be used before it can be determined and the total dilution multiplier (xn x 5) should be applied to the calculation.
5. 封板膜只限一次性使用,以避免交叉污染。
5. The membrane is limited to one-time use only to avoid cross-contamination.
6. 底物请避光保存。
6. The substrate shall be saved from the light.
7. 严格按照说明书的操作进行,试验结果判定必须以酶标仪读数为准.
7. Strictly following the operation of the instructions, the test results are determined to be based on the enzyme reading.
8. 所有样品,洗涤液和各种废弃物都应按传染物处理。
8. All samples, detergents and wastes should be treated as infectious substances.
9. 本试剂不同批号组分不得混用。
9. This reagent shall not be commingled between batch numbers.
技术提示:
Technology tip:
1、混合蛋白溶液时,避免起泡。
1. Avoid blowing when mixing protein solutions.
2、加校准品与样本时,每个校准品浓度和样本都要更换移液枪头,公共组分应该悬臂加样,避免交叉污染。
2. When calibrated and sampled, each calibration concentration and sample should be replaced with a translucent gun head, and the public component should be extended to avoid cross-contamination.
3、合适的温育时间,和充分的洗涤步骤,是保证实验结果准确性的必要条件。
3. Appropriate timing and adequate washing steps are necessary to ensure the accuracy of the results of the experiment.
4、底物溶液为无色液体,保存过程中变为蓝色,代表底物溶液已经失效,不得使用。
4. The bottom solution is an innocuous liquid, which becomes blue during the preservation process and represents that the bottom solution is no longer valid and cannot be used.
5、终止液加样顺序与底物溶液加样顺序一致,加入终止液后,蓝色底物产物,会瞬间变为黄色。
5. The sequence of decomposing liquids corresponds to the order of sampling of the substrate solutions and, when added, the blue substrate products will immediately become yellow.
6、实验中,用剩的板条,应立即放回自封袋中,密封(低温干燥)保存。
6. In the experiment, the leftover plate shall immediately be returned to the self-contained bag and kept sealed (dry and cool).
7、所有液体组分,使用前充分摇匀,严格按照说明书标明的时间、加样量及加样顺序进行温育操作。
7. All liquid components, fully tangled prior to use, are performed in a warm-out manner in strict accordance with the time, volume and order of sampling indicated in the instructions.
8、检测必须符合实验室管理规范的规定,严格防止交叉污染,所有样品、洗弃液和各种废弃物都应按照传染物进行处置。
8. Testing must comply with laboratory regulations to strictly prevent cross-contamination, and all samples, discards and all kinds of waste should be disposed of infectively.
试剂盒性能:
reagent box performance:
1. 样品线性回归与预期浓度相关系数R值为0.95以上。
1. The linear regression of samples to the expected concentration factor R is above 0.95.
2. 批内变异系数与批间变异系数应分别小于10%和15% 。
2. The intra-basin variation factor and the inter-basin variation factor shall be less than 10 per cent and 15 per cent, respectively.
检测范围:
detection range:
7.5 nmol/mL – 240 nmol/mL
灵敏度:
Sensitivity:
最低检测浓度小于1 nmol/mL
Lowest detection concentration < 1 nmol/mL
保存条件及有效期:
Conservation conditions and validity:
1. 试剂盒保存: 2-8℃。
1. Reagent box preservation: 2-8°C.
2.有效期: 6个月
Duration: 6 months
风险说明:
risk statement:
由于现有条件及科学技术水平尚不能对所有供货商提供的所有原料进行全面的鉴定与分析,本产品可能存在一定的质量技术风险。
Since existing conditions and scientific and technical levels do not allow for the full identification and analysis of all raw materials supplied by all suppliers, there may be a certain quality technical risk for this product.
1. 最终的实验结果与试剂的有效性、实验者的相关操作以及当时的实验环境密切相关,请务必准备充足的标本备份。
1. The final results of the experiment are closely related to the effectiveness of the reagent, the relevant operation of the experimenter and the experimental environment at the time, and it is important that sufficient samples be prepared for backup.
2. 不同批次的同一产品可能会有少许差别,如:检测限、灵敏度以及显色时间等,请依据试剂盒内说明书进行实验操作,网站电子版说明书仅作参考。
2. There may be a few differences in the same product in different batches, e.g. detection limits, sensitivity and colouring times. Please use the instructions in the reagent box for experimental operations, which are used for reference purposes only in the electronic version of the website.
3. 只有全部使用本试剂盒配套试剂才能保证检测效果,不能混用其他制造商的产品。只有严格遵守本试剂盒的实验说明才会得到最佳的检测结果。
Only the full use of this reagent box is necessary to ensure that the test results are not mixed with the products of other manufacturers. The best test results are obtained only if the test description of this reagent box is strictly adhered to.
4. 本公司只对试剂盒本身负责,不对因使用该试剂盒所造成的样本消耗负责,请使用者使用前充分考虑到样本的可能使用量,预留充足的样本。
4. The company is responsible only for the reagent box itself and not for the consumption of the sample resulting from the use of the reagent box, and is requested to reserve sufficient samples to take full account of the potential use of the sample prior to its use.
5. 使用化学裂解液制备的组织匀浆或细胞提取液可能会由于某些化学物质的引入导致ELISA实验结果偏差。
5. The use of tissue slurry or cell extraction fluids prepared with chemical fission fluids may lead to deviations in ELISA experiments as a result of the introduction of certain chemical substances.
6. 若样本为细胞培养上清,因该类样本干扰因素较多,如:细胞状态、细胞数量、采样时间等,所以可能存在检测不出的情况。
6. If the sample is purified for cell culture, there may be undetectable conditions due to the fact that there are more disturbance factors in the sample, such as cell states, cell numbers, sampling times, etc.
7. 某些天然蛋白或重组蛋白,包括原核及真核重组蛋白,可能因为与本产品所使用的检测抗体及捕获抗体不匹配,而不被检测出。
7. Some natural proteins or recombinant proteins, including primary and true-nuclear recombinant proteins, may not be detected because they do not match the detector antibodies and capture antibodies used in the product.
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